Benjamin Pinsky

All Publications

• Targeted plasma metabolomics combined with machine learning for the diagnosis of severe acute respiratory syndrome virus type 2. Frontiers in microbiology Le, A. T., Wu, M., Khan, A., Phillips, N., Rajpurkar, P., Garland, M., Magid, K., Sibai, M., Huang, C., Sahoo, M. K., Bowen, R., Cowan, T. M., Pinsky, B. A., Hogan, C. A. 2022; 13: 1059289

  Abstract

  The routine clinical diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely restricted to real-time reverse transcription quantitative PCR (RT-qPCR), and tests that detect SARS-CoV-2 nucleocapsid antigen. Given the diagnostic delay and suboptimal sensitivity associated with these respective methods, alternative diagnostic strategies are needed for acute infection.We studied the use of a clinically validated liquid chromatography triple quadrupole method (LC/MS-MS) for detection of amino acids from plasma specimens. We applied machine learning models to distinguish between SARS-CoV-2-positive and negative samples and analyzed amino acid feature importance.A total of 200 samples were tested, including 70 from individuals with COVID-19, and 130 from negative controls. The top performing model overall allowed discrimination between SARS-CoV-2-positive and negative control samples with an area under the receiver operating characteristic curve (AUC) of 0.96 (95%CI 0.91, 1.00), overall sensitivity of 0.99 (95%CI 0.92, 1.00), and specificity of 0.92 (95%CI 0.85, 0.95).This approach holds potential as an alternative to existing methods for the rapid and accurate diagnosis of acute SARS-CoV-2 infection.

  View details for DOI 10.3389/fmicb.2022.1059289

  View details for PubMedID 37063449

  View details for PubMedCentralID PMC10092816

• Infection and Vaccine-Induced Neutralizing-Antibody Responses to the SARS-CoV-2 B.1.617 Variants. The New England journal of medicine Edara, V., Pinsky, B. A., Suthar, M. S., Lai, L., Davis-Gardner, M. E., Floyd, K., Flowers, M. W., Wrammert, J., Hussaini, L., Ciric, C. R., Bechnak, S., Stephens, K., Graham, B. S., Bayat Mokhtari, E., Mudvari, P., Boritz, E., Creanga, A., Pegu, A., Derrien-Colemyn, A., Henry, A. R., Gagne, M., Douek, D. C., Sahoo, M. K., Sibai, M., Solis, D., Webby, R. J., Jeevan, T., Fabrizio, T. P. 2021

  View details for DOI 10.1056/NEJMc2107799

  View details for PubMedID 34233096

• Identification of a SARS-CoV-2 Variant with L452R and E484Q Neutralization Resistance Mutations. Journal of clinical microbiology Verghese, M., Jiang, B., Iwai, N., Mar, M., Sahoo, M. K., Yamamoto, F., Mfuh, K. O., Miller, J., Wang, H., Zehnder, J., Pinsky, B. A. 2021

  Abstract

  The emergence of SARS-CoV-2 variants t 23 hat reduce antibody neutralization and vaccine efficacy is of significant global concern..

  View details for DOI 10.1128/JCM.00741-21

  View details for PubMedID 33952596

• Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2. JAMA Hogan, C. A., Sahoo, M. K., Pinsky, B. A. 2020

  View details for DOI 10.1001/jama.2020.5445

  View details for PubMedID 32250394

• B cell clonal expansion and convergent antibody responses to SARS-CoV-2. Research square Nielsen, S. C., Yang, F. n., Hoh, R. A., Jackson, K. J., Roeltgen, K. n., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Kim, P. S., Wang, T. T., Pinsky, B. n., Blish, C. A., Boyd, S. D. 2020

  Abstract

  During virus infection B cells are critical for the production of antibodies and protective immunity. Establishment of a diverse antibody repertoire occurs by rearrangement of germline DNA at the immunoglobulin heavy and light chain loci to encode the membrane-bound form of antibodies, the B cell antigen receptor. Little is known about the B cells and antigen receptors stimulated by the novel human coronavirus SARS-CoV-2. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of V genes, and extensive activation of IgG and IgA subclasses without significant somatic mutation. We detect expansion of B cell clones as well as convergent antibodies with highly similar sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. A shared convergent B cell clonotype in SARS-CoV-2 infected patients was previously seen in patients with SARS. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

  View details for DOI 10.21203/rs.3.rs-27220/v1

  View details for PubMedID 32702737

  View details for PubMedCentralID PMC7336706

• SARS-CoV-2 RNAemia in a Healthy Blood Donor 40 Days After Respiratory Illness Resolution. Annals of internal medicine Pham, T. D., Huang, C. n., Wirz, O. F., Röltgen, K. n., Sahoo, M. K., Layon, A. n., Pandey, S. n., Foung, S. K., Boyd, S. D., Pinsky, B. A. 2020

  View details for DOI 10.7326/L20-0725

  View details for PubMedID 32678685

• Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Long, D. R., Gombar, S. n., Hogan, C. A., Greninger, A. L., Shah, V. O., Bryson-Cahn, C. n., Stevens, B. n., Rustagi, A. n., Jerome, K. R., Kong, C. S., Zehnder, J. n., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. n. 2020

  Abstract

  Using data for 20,912 patients from two large academic health systems, we analyzed the frequency of SARS-CoV-2 RT-PCR test-discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and similar across institutions.

  View details for DOI 10.1093/cid/ciaa722

  View details for PubMedID 32506118

• Report from the American Society for Microbiology COVID-19 International Summit, 23 March 2020: Value of Diagnostic Testing for SARS-CoV-2/COVID-19. mBio Patel, R. n., Babady, E. n., Theel, E. S., Storch, G. A., Pinsky, B. A., St George, K. n., Smith, T. C., Bertuzzi, S. n. 2020; 11 (2)

  View details for DOI 10.1128/mBio.00722-20

  View details for PubMedID 32217609

• Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Science immunology Röltgen, K. n., Powell, A. E., Wirz, O. F., Stevens, B. A., Hogan, C. A., Najeeb, J. n., Hunter, M. n., Wang, H. n., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Manohar, M. n., Manalac, J. n., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A. n., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Narasimhan, B. n., Gombar, S. n., Tibshirani, R. n., Nadeau, K. C., Kim, P. S., Pinsky, B. A., Boyd, S. D. 2020; 5 (54)

  Abstract

  SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

  View details for DOI 10.1126/sciimmunol.abe0240

  View details for PubMedID 33288645

• hrHPV prevalence and type distribution in rural Zimbabwe: A community-based self-collection study using near-point-of-care GeneXpert HPV testing INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES Fitzpatrick, M. B., Mandishora, R., Katzenstein, D. A., McCarty, K., Weber, J., Sahoo, M. K., Manasa, J., Chirenje, Z., Pinsky, B. A. 2019; 82: 21–29

  View details for DOI 10.1016/j.ijid.2019.02.022

  View details for Web of Science ID 000466424400007

• Persistence of Human Immunodeficiency Virus-1 Drug Resistance Mutations in Proviral Deoxyribonucleic Acid After Virologic Failure of Efavirenz-Containing Antiretroviral Regimens. Open forum infectious diseases De La Cruz, J., Vardhanbhuti, S., Sahoo, M. K., Rovner, R., Bosch, R. J., Manasa, J., Katzenstein, D. A., Pinsky, B. A. 2019; 6 (3): ofz034

  Abstract

  Background: Efavirenz (EFV)-based regimens select broad drug resistance to nonnucleoside reverse-transcriptase inhibitors (NNRTIs), limiting the effectiveness of EFV and other NNRTIs. The duration, persistence, and decay of drug resistance mutations (DRMs) in the proviral reservoir is not well defined.Methods: Participants with virologic failure of EFV-based regimens and drug-resistant viremia with the K103N mutation in plasma ribonucleic acid (RNA) were identified from AIDS Clinical Trials Group (ACTG) studies A364 and A5095. These individuals received a second-line, boosted protease inhibitor-based regimen with suppression of viremia for up to10 years during long-term follow-up (median = 3.6 years; interquartile range, 2.1-6.9 years). Proviral deoxyribonucleic acid (DNA) from cryopreserved peripheral blood mononuclear cells was sequenced to identify the persistence of DRM.Results: Twenty-eight participants from ACTG 364 and ACTG 5095 were evaluated. Sanger sequencing of proviral DNA detected K103N as well as additional reverse-transcriptase inhibitor (RTI) mutations. Ultradeep sequencing confirmed persistence of K103N in 71% of participants with minimal decay over time. In an adjusted model including years since suppression, persistent proviral K103N was 2.6 times more likely (95% confidence interval, 1.0-6.4) per log10 higher human immunodeficiency virus RNA at EFV failure.Conclusions: Persistence of RTI mutations in proviral DNA after virologic failure has implications for the effectiveness of future drug regimens and the recycling of RTI drugs.

  View details for PubMedID 30863788

• Yellow Fever Virus: Diagnostics for a Persistent Arboviral Threat JOURNAL OF CLINICAL MICROBIOLOGY Waggoner, J. J., Rojas, A., Pinsky, B. A. 2018; 56 (10)

  Abstract

  Yellow fever (YF) is the prototypical hemorrhagic fever and results from infection with yellow fever virus (YFV), which is endemic to regions of Africa and South America. Despite the availability of an effective vaccine, YFV continues to cause disease throughout regions where it is endemic, including intermittent large outbreaks among undervaccinated populations. A number of diagnostic methods and assays have been described for the detection of YFV infection, including viral culture, molecular testing, serology, and antigen detection. Commercial diagnostics are not widely available, and testing is generally performed at a small number of reference laboratories. The goal of this article, therefore, is to review available clinical diagnostics for YFV, which may not be familiar to many practitioners outside areas where it is endemic. Additionally, we identify gaps in our current knowledge about YF that pertain to diagnosis and describe interventions that may improve YFV detection.

  View details for PubMedID 30021822

  View details for PubMedCentralID PMC6156298

• Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip NATURE BIOTECHNOLOGY Hassibi, A., Manickam, A., Singh, R., Bolouki, S., Sinha, R., Jirage, K. B., McDermott, M. W., Hassibi, B., Vikalo, H., Mazarei, G., Pei, L., Bousse, L., Miller, M., Heshami, M., Savage, M. P., Taylor, M. T., Gamini, N., Wood, N., Mantina, P., Grogan, P., Kuimelis, P., Savalia, P., Conradson, S., Li, Y., Meyer, R. B., Ku, E., Ebert, J., Pinsky, B. A., Dolganov, G., Van, T., Johnson, K. A., Naraghi-Arani, P., Kuimelis, R. G., Schoolnik, G. 2018; 36 (8): 738–45

  Abstract

  The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

  View details for PubMedID 30010676

• Characterization of Dengue Virus Infections Among Febrile Children Clinically Diagnosed With a Non-Dengue Illness, Managua, Nicaragua JOURNAL OF INFECTIOUS DISEASES Waggoner, J. J., Gresh, L., Mohamed-Hadley, A., Balmaseda, A., Soda, K., Abeynayake, J., Sahoo, M. K., Liu, Y., Kuan, G., Harris, E., Pinsky, B. A. 2017; 215 (12): 1816–23

  Abstract

  We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases").DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013.One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P < .001). Humoral immunity was associated with viral load at presentation: 40 of 43 patients (93.0%) with a viral load ≥7.0 log10 copies/mL serum developed the expected rise in anti-DENV antibodies in annual samples versus 13 of 68 (19.1%) patients with a viral load below this level (P < .001).Antibody responses to DENV-positive C cases differ from responses to classic symptomatic dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.

  View details for PubMedID 28863466

  View details for PubMedCentralID PMC5853235

• Diagnosis of Zika virus infection on a nanotechnology platform. Nature medicine Zhang, B., Pinsky, B. A., Ananta, J. S., Zhao, S., Arulkumar, S., Wan, H., Sahoo, M. K., Abeynayake, J., Waggoner, J. J., Hopes, C., Tang, M., Dai, H. 2017

  Abstract

  We developed a multiplexed assay on a plasmonic-gold platform for measuring IgG and IgA antibodies and IgG avidity against both Zika virus (ZIKV) and dengue virus (DENV) infections. In contrast to IgM cross-reactivity, IgG and IgA antibodies against ZIKV nonstructural protein 1 (NS1) antigen were specific to ZIKV infection, and IgG avidity revealed recent ZIKV infection and past DENV-2 infection in patients in dengue-endemic regions. This assay could enable specific diagnosis of ZIKV infection over other flaviviral infections.

  View details for DOI 10.1038/nm.4302

  View details for PubMedID 28263312

• Viremia and Clinical Presentation in Nicaraguan Patients Infected with Zika Virus, Chikungunya Virus, and Dengue Virus. Clinical infectious diseases Waggoner, J. J., Gresh, L., Vargas, M. J., Ballesteros, G., Tellez, Y., Soda, K. J., Sahoo, M. K., Nuñez, A., Balmaseda, A., Harris, E., Pinsky, B. A. 2016

  Abstract

   Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses. Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded. A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001). ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance.

  View details for PubMedID 27578819

• Zika Virus: Diagnostics for an Emerging Pandemic Threat. Journal of clinical microbiology Waggoner, J. J., Pinsky, B. A. 2016; 54 (4): 860-867

  Abstract

  Zika virus (ZIKV) is anAedesmosquito-borne flavivirus that emerged in Brazil in 2015 and then rapidly spread throughout the tropical and subtropical Americas. Based on clinical criteria alone, ZIKV cannot be reliably distinguished from infections with other pathogens that cause an undifferentiated systemic febrile illness, including infections with two common arboviruses, dengue virus and chikungunya virus. This minireview details the methods that are available to diagnose ZIKV infection.

  View details for DOI 10.1128/JCM.00279-16

  View details for PubMedID 26888897

  View details for PubMedCentralID PMC4809954

• The Histopathologic Features of Early COVID Pneumonia in a Pediatric Patient: New Insight into the Role of Macrophages. International journal of surgical pathology Bulterys, P. L., Xu, G., Pinsky, B. A., Troxell, M. L., Menke, J. R., Berry, G. J., Fernandez-Pol, S., Hazard, F. K. 2024; 32 (8): 1595-1601

  Abstract

  A life-threatening complication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is acute respiratory distress syndrome. Our understanding of the pathologic changes in coronavirus disease 2019 (COVID-19) is based almost exclusively on post-mortem analyses of adults. These studies established several hallmarks of SARS-CoV-2 lung infection, including diffuse alveolar damage, microvascular thrombi, and acute bronchopneumonia. We describe a fatal example of COVID pneumonia in a 9-year-old girl who presented with fever 10 months following the diagnosis of ALK-positive anaplastic large cell lymphoma (ALCL). A chest computed tomography scan revealed left upper lobe lung consolidation and nodular airspace disease, and an initial SARS-CoV-2 nasopharyngeal swab (RT-PCR) was negative. A subsequent lung biopsy performed due to concern for relapsed ALCL demonstrated sheets of intra-alveolar and interstitial macrophages, and macrophage-rich fibrinous exudates. Immunohistochemical and in-situ hybridization stains confirmed these macrophages as the predominant SARS-CoV-2-infected cell type. Subsequent RT-PCR testing of upper and lower respiratory tract samples was positive for SARS-CoV-2 infection. Whole genome sequencing confirmed the presence of the B.1.617.2 (Delta) variant. This biopsy illustrates the histopathologic features of early COVID pneumonia in antemortem lung tissue from a pediatric patient, and establishes macrophages as a potential source of SARS-CoV-2 amplification.

  View details for DOI 10.1177/10668969241236704

  View details for PubMedID 39435671

• Nucleic acid amplification testing using dried blood spots to confirm the diagnosis of HIV-1 in adults. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Pinsky, B. A., Sahoo, M. K., Manasa, J., Makadzange, T., Wallis, C. L., Marins, E. G., Kumarasamy, N., Bartlett, J. A., Bosch, R. J., Israelski, D., Katzenstein, D. A. 2024; 175: 105746

  Abstract

  The WHO HIV testing algorithm for high prevalence populations recommends the use of three different serologic assays, though this approach may lead to diagnostic misclassification. The study objective was to compare dried blood spot (DBS)-based HIV-1 nucleic acid detection methods to determine their suitability to confirm the diagnosis of HIV-1 in adults generally with suppressed or low-level plasma HIV-1 RNA.Four methods were evaluated: Cepheid Xpert HIV-1 Qual Assay (Xpert), Hologic Aptima HIV-1 Quant Dx assay (Aptima), Roche Cobas Ampliprep/Cobas TaqMan HIV-1 test, v.2.0 (CAP/CTM) with guanidinium-based sample pre-extraction buffer (SPEX), or CAP/CTM with phosphate-buffered saline (PBS). Testing was performed on 163 DBS samples collected from participants with HIV-1 in the AIDS Clinical Trial Group (ACTG) A5230 study (73 samples) and the Peninsula AIDS Research Cohort (PARC) study (90 samples).Xpert and SPEX CAP/CTM [96.9 % (158/163):95.7 % (156/163); P = 0.75) showed similar sensitivity. However, PBS CAP/CTM and Aptima demonstrated significantly lower sensitivity, 68.2 % (107/157) and 69.2 % (99/143), respectively, compared to Xpert and SPEX CAP/CTM (P < 0.0001 for all comparisons). Overall agreement between Xpert and SPEX CAP/CTM was 93.9 % (153/163), including 152 DBS samples in which both methods detected HIV-1 nucleic acids.Xpert and SPEX CAP/CTM provide sensitive performance for the detection of HIV-1 nucleic acids using DBS collected from adults living with HIV-1, including those with suppressed virus loads. Given the cost and side-effects associated with inappropriate life-long antiretroviral therapy, these assays may play a role in diagnosing HIV-1 infection in individuals with suspected false-positive serologic testing.

  View details for DOI 10.1016/j.jcv.2024.105746

  View details for PubMedID 39566166

• Opportunities and challenges for the U.S. laboratory response to highly pathogenic avian influenza A(H5N1). Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Pinsky, B. A., Bradley, B. T. 2024; 174: 105723

  Abstract

  On March 25, 2024 an outbreak of highly pathogenic avian influenza (HPAI) A H5N1 was identified in dairy cows across multiple farms in the United States. Zoonotic cases originating in individuals with close contact to infected herds and poultry flocks have been subsequently identified. Spillover events such as this raise the specter of recent pandemics including COVID-19 and Mpox and may lead clinical laboratories to assess their capacity for diagnosis of HPAI H5N1. In this review, we detail the origins of the H5N1 clade 2.3.4.4b outbreak as well as the existing capacity to identify HPAI H5N1 as influenza A virus by commercially available assays. Furthermore, we highlight the absence of commercially available influenza A H5 subtyping assays and limitations associated with the current 510(k)-cleared assay. This outbreak also serves as an early opportunity to assess the new and unknown regulatory challenges faced by laboratory-developed tests in light of the FDA's final rule on in vitro diagnostic devices. National agencies along with public health and clinical laboratories all serve an essential role in the response to HPAI H5N1. To most effectively utilize each group's strength requires open communication and willingness to embrace novel approaches.

  View details for DOI 10.1016/j.jcv.2024.105723

  View details for PubMedID 39213758

• Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus. Emerging infectious diseases Sahoo, M. K., Morante, I. E., Huang, C., Solis, D., Yamamoto, F., Ohiri, U. C., Romero, D., Pinsky, B. A. 2024; 30 (8): 1710-1713

  Abstract

  An increased risk for human infection with avian influenza A(H5N1) viruses is of concern. We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping. This test could be used to detect influenza A(H5) in clinical samples.

  View details for DOI 10.3201/eid3008.240785

  View details for PubMedID 38986151

• Natural history of shedding and household transmission of severe acute respiratory syndrome coronavirus 2 using intensive high-resolution sampling. PloS one Altamirano, J., Govindarajan, P., Blomkalns, A. L., Leary, S., Robinson, I., Chun, L. X., Shaikh, N. J., Robinson, M. L., Lopez, M., Tam, G. K., Carrington, Y. J., De Araujo, M. B., Walter, K. S., Andrews, J. R., Burns, J., Hogan, C., Pinsky, B. A., Maldonado, Y. 2024; 19 (7): e0305300

  Abstract

  The COVID-19 pandemic has led to 775 million documented cases and over 7 million deaths worldwide as of March 2024 and is an ongoing health crisis. To limit viral spread within households and in the community, public health officials have recommended self-isolation, self-quarantine of exposed household contacts, and mask use. Yet, risk of household transmission (HHT) may be underestimated due to low frequency of sampling, and risk factors for HHT are not well understood.To estimate the secondary attack rate of SARS-CoV-2 within households and to define the risk factors for new infections in household members who are in close contact with the index case.In this prospective cohort study, from March 2020-December 2021 we enrolled 60 households with index cases who tested positive for SARS-CoV-2. All household contacts and index cases were tested daily for SARS-CoV-2 via reverse transcription polymerase chain reaction (RT-PCR) using self-collected anterior nares specimens. Households were followed until all study participants in the household tested negative for SARS-CoV-2 for seven consecutive days. We collected sex, age, race/ethnicity, comorbidities, and relationship to index case for secondary contacts, household level characteristics including primary income, household density, and square feet per person on property. We compared the sociodemographic variables between COVID-19 positive and negative household members and between households where secondary transmission did and did not occur.Daily anterior nares swabs were tested for SARS-CoV-2 using RT-PCR, in order to assess duration of nasal shedding of SARS-CoV-2, as well as risk of transmission to secondary household contacts.Of the 163 participants in this study, 84 (51.5%) were women; median age (IQR) was 36.0 (17.0-54.0) years of age; 78 (47.8%) were white and 48 (29.5%) were Hispanic/LatinX. Of the fifty households with household contacts, at least one secondary case occurred in twenty-six households (52.0%) and forty-five household contacts (43.7%) were infected. Secondary attack rate was lowest among children of index cases (6/23, 26.1%). Modified Poisson regression identified that the risk of transmission to household contacts increases significantly with age (Risk ratio for each increase in years of age = 1.01, 95% CI = 1.00-1.02). Mixed effects regression models identified that participants with chronic diseases, such as asthma, diabetes, cancer, or cardiac disease, had higher Cts at baseline when compared to participants without chronic diseases (6.62, 95% CI: 1.46-11.77, p = 0.02) and show a slower rate of increase in Ct over time (-0.43, 95% CI: -0.77 to -0.09, p = 0.02).This study suggests that HHT represents a key source of community-based infection of SARS-CoV-2. Allocation of resources for contact investigations and prevention interventions should focus on the individuals at highest risk of infection in households, especially those with higher density homes.

  View details for DOI 10.1371/journal.pone.0305300

  View details for PubMedID 39052659

• Epstein-Barr virus qPCR testing on bronchoalveolar lavage fluid from immunocompromised patients. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Liang, B., Mah, J., Sahoo, M. K., Pinsky, B. A. 2024; 174: 105705

  Abstract

  Epstein-Barr Virus (EBV) is associated with lung disease in immunocompromised patients, particularly transplant recipients. EBV DNA testing of lower respiratory tract specimens may have diagnostic utility.This was a retrospective, observational study of all patients with bronchoalveolar lavage (BAL) fluids submitted for EBV qPCR testing from February 2016 to June 2022 at the Stanford Clinical Virology Laboratory.There were 140 patients that underwent 251 EBV qPCR BAL tests (median 1; range 1 - 10). These patients had a mean age of 15.9 years (standard deviation, 15.1 years) and 50 % were female. Transplant recipients accounted for 67.1 % (94/140) of patients, including 67.0 % (63/94) solid organ transplant (SOT) and 33.0 % (31/94) hematopoietic cell transplant. Diagnostic testing was performed more commonly than surveillance testing [57.0 % (143/251) v. 43.0 % (108/251)]; 96.2 % (104/108) of surveillance samples were from lung transplant recipients. Excluding internal control failures, 34.7 % (83/239) of BAL had detectable EBV DNA, encompassing a wide range of viral loads (median=3.03 log10 IU/mL, range 1.44 to 6.06). Overall agreement of EBV DNA in BAL compared to plasma was 74.1 % [117/158; 95 % confidence interval (CI): 66.5 % to 80.7 %], with a kappa coefficient of 0.44 (95 % CI: 0.30 to 0.57). Only 20.1 % (48/239) of results were discussed in a subsequent clinical note, and one result (0.4 %; 1/239) changed clinical management.EBV qPCR testing on BAL offers limited clinical impact. Additional biomarkers are required to improve the diagnosis of EBV-associated lung diseases.

  View details for DOI 10.1016/j.jcv.2024.105705

  View details for PubMedID 39002309

• Spatial and temporal variation in respiratory syncytial virus (RSV) subtype RNA in wastewater and relation to clinical specimens. mSphere Zambrana, W., Huang, C., Solis, D., Sahoo, M. K., Pinsky, B. A., Boehm, A. B. 2024: e0022424

  Abstract

  Respiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. Using molecular methods, we quantified RSV A and RSV B RNA in wastewater solids across multiple seasons and metropolitan areas to gain insight into the predominance of RSV subtypes. We determined the predominant subtype for each group using the proportion of RSV A to total RSV (RSV A + RSV B) in each wastewater sample (PA,WW) and conducted a comparative analysis temporally, spatially, and against clinical specimens. A median PA,WW of 0.00 in the first season and 0.58 in the second season indicated a temporal shift in the predominant subtype. Spatially, while we observed dominance of the same subtype, PA,WW was higher in some areas (PA,WW = 0.58-0.88). The same subtype predominated in wastewater and clinical samples, but clinical samples showed higher levels of RSV A (RSV A positivity in clinical samples = 0.79, median PA,WW = 0.58). These results suggest that wastewater, alongside clinical data, holds promise for enhanced subtype surveillance.IMPORTANCERespiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. The study illustrates that information on subtype predominance can be gleaned from wastewater. As a biological composite sample from the entire contributing population, wastewater monitoring of RSV A and B can complement clinical surveillance of RSV.

  View details for DOI 10.1128/msphere.00224-24

  View details for PubMedID 38926903

• Expanding Understanding of Urban Rift Valley Fever Risk and Associated Vector Ecology at Slaughterhouses in Kisumu, Kenya. Pathogens (Basel, Switzerland) Gerken, K. N., Owuor, K. O., Ndenga, B., Wambua, S., Winter, C., Chemutai, S., Omukuti, R., Arabu, D., Miring'u, I., Wilson, W. C., Mutuku, F., Waggoner, J. J., Pinsky, B., Bosire, C., LaBeaud, A. D. 2024; 13 (6)

  Abstract

  Rift Valley fever virus (RVFV) is an adaptable arbovirus that can be transmitted by a wide variety of arthropods. Widespread urban transmission of RVFV has not yet occurred, but peri-urban outbreaks of RVFV have recently been documented in East Africa. We previously reported low-level exposure in urban communities and highlighted the risk of introduction via live animal influx. We deployed a slaughtered animal testing framework in response to an early warning system at two urban slaughterhouses and tested animals entering the meat value chain for anti-RVFV IgG and IgM antibodies. We simultaneously trapped mosquitoes for RVFV and bloodmeal testing. Out of 923 animals tested, an 8.5% IgG seroprevalence was identified but no evidence of recent livestock exposure was detected. Mosquito species abundance varied greatly by slaughterhouse site, which explained 52% of the variance in blood meals. We captured many Culex spp., a known RVFV amplifying vector, at one of the sites (p < 0.001), and this species had the most diverse blood meals. No mosquito pools tested positive for RVFV antigen using a rapid VecTOR test. These results expand understanding of potential RVF urban disease ecology, and highlight that slaughterhouses are key locations for future surveillance, modelling, and monitoring efforts.

  View details for DOI 10.3390/pathogens13060488

  View details for PubMedID 38921786

• Optimization and Local Cost-Effectiveness of Nasopharyngeal Carcinoma Screening Strategies in Southern China: Secondary Analysis of the Guangdong Randomized Trial. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology Miller, J. A., Liu, Z., Pinsky, B. A., Le, Q., Li, T., Yu, K. J., Hildesheim, A., Cao, S. 2024

  Abstract

  BACKGROUND: Screening with anti-Epstein-Barr Virus (EBV) serology and endoscopy decreased nasopharyngeal carcinoma (NPC) mortality in Guangdong in a randomized trial. We conducted a secondary analysis of this trial using local incidence and cost data to optimize screening programs, hypothesizing that screening could be cost-effective in southern China.METHODS: Screening costs and life-years after NPC diagnosis were obtained from the Guangdong trial's intent-to-screen population (men and women age 30-69). Seropositive subjects were rescreened annually for five years. Thereafter, we evaluated 12 screening strategies in Guangdong and Guangxi using a validated model. Strategies used combinations of serology, nasopharyngeal swab PCR (NP PCR), endoscopy, and MRI from trial sub-cohorts. Incidence data and costs were obtained from local cancer registries and the provincial healthcare system.RESULTS: In the intent-to-screen population, screening with serology and endoscopy was cost-effective (42,366/life-year, 0.52 GDP per-capita). Screening for 5-15 years between ages 35-59 met a willingness-to-pay threshold of 1.5 GDP/QALY in all modeled populations. Despite doubling costs, adding MRI could be cost-effective via improved sensitivity. NP PCR triage reduced endoscopy/MRI referrals by 37%. One lifetime screen could reduce NPC mortality by pproximately 20%.CONCLUSIONS: EBV-based serologic screening for NPC is likely to be cost-effective in southern China. Among seropositive subjects, the preferred strategies use endoscopy alone or selective endoscopy triaged by MRI with or without NP PCR. These data may aid the design of screening programs in this region.IMPACT: These findings support population-based screening in southern China by defining the target population, cost effectiveness, and optimized screening approach.

  View details for DOI 10.1158/1055-9965.EPI-23-1486

  View details for PubMedID 38695706

• PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2. bioRxiv : the preprint server for biology Karim, M., Mishra, M., Lo, C. W., Saul, S., Cagirici, H. B., Tran, D. H., Agrawal, A., Ghita, L., Ojha, A., East, M. P., Gammeltoft, K. A., Sahoo, M. K., Johnson, G. L., Das, S., Jochmans, D., Cohen, C. A., Gottwein, J., Dye, J., Neff, N., Pinsky, B. A., Laitinen, T., Pantsar, T., Poso, A., Zanini, F., Jonghe, S. D., Asquith, C. R., Einav, S. 2024

  Abstract

  In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

  View details for DOI 10.1101/2024.04.15.589676

  View details for PubMedID 38659941

  View details for PubMedCentralID PMC11042293

• The Histopathologic Features of Early COVID Pneumonia in a Pediatric Patient: New Insight into the Role of Macrophages INTERNATIONAL JOURNAL OF SURGICAL PATHOLOGY Bulterys, P. L., Xu, G., Pinsky, B. A., Troxell, M. L., Menke, J. R., Berry, G. J., Fernandez-Pol, S., Hazard, F. K. 2024

  View details for DOI 10.1177/10668969241236704

  View details for Web of Science ID 001196092100001

• Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs. Microbiology spectrum Relich, R. F., Van Benten, K., Lei, G., Robinson, C. M., Carozza, M., Sahoo, M. K., Huang, C., Solis, D., Sibai, M., Myers, C. A., Sikorski, C., Balagot, C., Yang, D., Pinsky, B. A., Loeffelholz, M. J. 2024: e0390823

  Abstract

  Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

  View details for DOI 10.1128/spectrum.03908-23

  View details for PubMedID 38466093

• Characterization of Dengue Virus 4 Cases in Paraguay, 2019-2020. Viruses Rojas, A., Shen, J., Cardozo, F., Bernal, C., Caballero, O., Ping, S., Key, A., Haider, A., de Guillén, Y., Langjahr, P., Acosta, M. E., Aria, L., Mendoza, L., Páez, M., Von-Horoch, M., Luraschi, P., Cabral, S., Sánchez, M. C., Torres, A., Pinsky, B. A., Piantadosi, A., Waggoner, J. J. 2024; 16 (2)

  Abstract

  In 2019-2020, dengue virus (DENV) type 4 emerged to cause the largest DENV outbreak in Paraguay's history. This study sought to characterize dengue relative to other acute illness cases and use phylogenetic analysis to understand the outbreak's origin. Individuals with an acute illness (≤7 days) were enrolled and tested for DENV nonstructural protein 1 (NS1) and viral RNA by real-time RT-PCR. Near-complete genome sequences were obtained from 62 DENV-4 positive samples. From January 2019 to March 2020, 799 participants were enrolled: 253 dengue (14 severe dengue, 5.5%) and 546 other acute illness cases. DENV-4 was detected in 238 dengue cases (94.1%). NS1 detection by rapid test was 52.5% sensitive (53/101) and 96.5% specific (387/401) for dengue compared to rRT-PCR. DENV-4 sequences were grouped into two clades within genotype II. No clustering was observed based on dengue severity, location, or date. Sequences obtained here were most closely related to 2018 DENV-4 sequences from Paraguay, followed by a 2013 sequence from southern Brazil. DENV-4 can result in large outbreaks, including severe cases, and is poorly detected with available rapid diagnostics. Outbreak strains seem to have been circulating in Paraguay and Brazil prior to 2018, highlighting the importance of sustained DENV genomic surveillance.

  View details for DOI 10.3390/v16020181

  View details for PubMedID 38399957

  View details for PubMedCentralID PMC10892180

• BA.5 bivalent booster vaccination enhances neutralization of XBB.1.5, XBB.1.16 and XBB.1.9 variants in patients with lung cancer. NPJ vaccines Valanparambil, R. M., Lai, L., Johns, M. A., Davis-Gardner, M., Linderman, S. L., McPherson, T. O., Chang, A., Akhtar, A., Gamarra, E. L., Matia, H., McCook-Veal, A. A., Switchenko, J., Nasti, T. H., Green, F., Saini, M., Wieland, A., Pinsky, B. A., Solis, D., Dhodapkar, M. V., Carlisle, J., Ramalingam, S., Ahmed, R., Suthar, M. S. 2023; 8 (1): 179

  Abstract

  This study reports that most patients with NSCLC had a significant increase in the nAb response to the currently circulating Omicron variants after bivalent booster vaccination and had Ab titers comparable to healthy participants. Interestingly, though the durability of the nAb response persisted in most of the healthy participants, patients with NSCLC had significantly reduced nAb titers after 4-6 months of vaccination. Our data highlight the importance of COVID-19 bivalent booster vaccination as the standard of care for patients with NSCLC given the evolution of new variants of concern.

  View details for DOI 10.1038/s41541-023-00779-8

  View details for PubMedID 37990024

  View details for PubMedCentralID PMC10663480

• Global and cell type-specific immunological hallmarks of severe dengue progression identified via a systems immunology approach. Nature immunology Ghita, L., Yao, Z., Xie, Y., Duran, V., Cagirici, H. B., Samir, J., Osman, I., Rebellón-Sánchez, D. E., Agudelo-Rojas, O. L., Sanz, A. M., Sahoo, M. K., Robinson, M. L., Gelvez-Ramirez, R. M., Bueno, N., Luciani, F., Pinsky, B. A., Montoya, J. G., Estupiñan-Cardenas, M. I., Villar-Centeno, L. A., Rojas-Garrido, E. M., Rosso, F., Quake, S. R., Zanini, F., Einav, S. 2023

  Abstract

  Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

  View details for DOI 10.1038/s41590-023-01654-3

  View details for PubMedID 37872316

  View details for PubMedCentralID 3651993

• Human immunodeficiency virus 1 5'-leader mutations in plasma viruses before and after the development of reverse transcriptase inhibitor-resistance mutations. The Journal of general virology Nouhin, J., Tzou, P. L., Rhee, S., Sahoo, M. K., Pinsky, B. A., Krupkin, M., Puglisi, J. D., Puglisi, E. V., Shafer, R. W. 2023; 104 (10)

  Abstract

  Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) initiation depends on interaction between viral 5'-leader RNA, RT and host tRNA3Lys. Therefore, we sought to identify co-evolutionary changes between the 5'-leader and RT in viruses developing RT-inhibitor resistance mutations. We sequenced 5'-leader positions 37-356 of paired plasma virus samples from 29 individuals developing the nucleoside RT inhibitor (NRTI)-resistance mutation M184V, 19 developing a non-nucleoside RT inhibitor (NNRTI)-resistance mutation and 32 untreated controls. 5'-Leader variants were defined as positions where ≥20 % of next-generation sequencing (NGS) reads differed from the HXB2 sequence. Emergent mutations were defined as nucleotides undergoing a ≥4-fold change in proportion between baseline and follow-up. Mixtures were defined as positions containing ≥2 nucleotides each present in ≥20 % of NGS reads. Among 80 baseline sequences, 87 positions (27.2 %) contained a variant; 52 contained a mixture. Position 201 was the only position more likely to develop a mutation in the M184V (9/29 vs 0/32; P=0.0006) or NNRTI-resistance (4/19 vs 0/32; P=0.02; Fisher's exact test) groups than the control group. Mixtures at positions 200 and 201occurred in 45.0 and 28.8 %, respectively, of baseline samples. Because of the high proportion of mixtures at these positions, we analysed 5'-leader mixture frequencies in two additional datasets: five publications reporting 294 dideoxyterminator clonal GenBank sequences from 42 individuals and six National Center for Biotechnology Information (NCBI) BioProjects reporting NGS datasets from 295 individuals. These analyses demonstrated position 200 and 201 mixtures at proportions similar to those in our samples and at frequencies several times higher than at all other 5'-leader positions. Although we did not convincingly document co-evolutionary changes between RT and 5'-leader sequences, we identified a novel phenomenon, wherein positions 200 and 201 immediately downstream of the HIV-1 primer binding site exhibited an extraordinarily high likelihood of containing a nucleotide mixture. Possible explanations for the high mixture rates are that these positions are particularly error-prone or provide a viral fitness advantage.

  View details for DOI 10.1099/jgv.0.001898

  View details for PubMedID 37801004

• Low infectivity among asymptomatic patients with a positive severe acute respiratory coronavirus virus 2 (SARS-CoV-2) admission test at a tertiary care center, 2020-2022. Infection control and hospital epidemiology Tayyar, R., Kiener, M. A., Liang, J. W., Contreras, G., Rodriguez-Nava, G., Zimmet, A. N., Contag, C. A., Srinivasan, K., McIntyre, K., Subramanian, A., Shepard, J., Tompkins, L. S., Pinsky, B. A., Salinas, J. L. 2023: 1-3

  Abstract

  We used a strand-specific RT-qPCR to evaluate viral replication as a surrogate for infectiousness among 242 asymptomatic inpatients with a positive severe acute respiratory coronavirus virus 2 (SARS-CoV-2) admission test. Only 21 patients (9%) had detectable SARS-CoV-2 minus-strand RNA. Because most patients were found to be noninfectious, our findings support the suspension of asymptomatic admission testing.

  View details for DOI 10.1017/ice.2023.210

  View details for PubMedID 37746805

• Tools Needed to Support Same-day Diagnosis and Treatment of Current Hepatitis C Infection. The Journal of infectious diseases Fricker, G. P., Ghany, M. G., Mera, J., Pinsky, B. A., Ward, J. W., Chung, R. T. 2023

  Abstract

  Current HCV prevention efforts and treatment rates must improve for the United States (U.S.) to achieve WHO global elimination targets by 2030[1]. The current multi-day diagnosis and treatment paradigm for hepatitis C (HCV) infection leads to significant loss in the cascade of care, resulting in far fewer patients receiving treatment with direct acting antiviral agents (DAAs) than those diagnosed with HCV infection [2,3]. To achieve HCV elimination, a paradigm shift in access to HCV treatment is needed from current multi-day testing and treatment algorithms to same day diagnosis and treatment. This shift will require new tools, such as FDA-approved, CLIA-waived point-of-care (POC) antigen or nucleic acid tests (NAT) for HCV and HBV and NAT for HIV that do not require venous blood. Such a shift will also require better utilization of existing resources, expanding access to HCV treatment through availability of onsite treatment, removal of payer barriers to approval, adoption of minimal monitoring approaches during treatment, expanded access to available POC tests, and available specialist referral networks for patients who fail initial therapy, have advanced liver fibrosis, or have co-incident HIV or HBV infection. A same-day diagnosis and treatment paradigm will substantially contribute to HCV elimination by improving treatment rates for those diagnosed with HCV infection and expanding access to treatment in settings where patients have brief encounters with healthcare.

  View details for DOI 10.1093/infdis/jiad177

  View details for PubMedID 37739799

• Assessment of an automated Cytomegalovirus nucleic acid amplification test using clinical plasma, bronchoalveolar lavage, and tissue specimens. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Huang, C., Solis, D., Sahoo, M. K., Pinsky, B. A. 2023; 168: 105582

  Abstract

  Cytomegalovirus (CMV) causes significant morbidity and mortality in immunocompromised patients, particularly transplant recipients. Quantitation of CMV DNA in peripheral blood is used to monitor prophylactic and pre-emptive approaches to prevent CMV disease, whereas CMV DNA testing of non-plasma specimens may aid in the diagnosis of end-organ disease.The analytical performance of the FDA-approved Aptima CMV Quant Assay was evaluated using reference CMV (SeraCare) diluted in defibrinated human plasma, as well as negative bronchoalveolar lavage fluid and tissue. Agreement was determined using 100 clinical acid-citrate-dextrose (ACD) plasma specimens, 77 bronchoalveolar lavage (BAL) fluids, and 101 tissues previously tested using artus CMV qPCR.Aptima CMV lower limit of detection (LLOD) was 169 IU/mL for ACD plasma, 100 IU/mL for BAL, and 50 IU/mL for tissue. Positive percent agreement (PPA) was 100.0% (50/50; 95% CI: 92.9% - 100.0%) and negative percent agreement (NPA) was 94.0% (47/50; 95% CI: 83.5% - 98.8%) for ACD plasma. Bland-Altman analysis revealed a bias of 0.20 log10 IU/mL (Aptima - artus) with 95% limits of agreement of -0.53 to 0.93. For BAL fluids, PPA was 70.0% (14/20; 95% CI: 45.7% - 88.1%) and NPA was 82.4% (43/51; 95% CI: 69.1% - 91.6%). For tissues, PPA was 90.0% (45/50; 95% CI: 78.2% - 96.7%) and NPA was 94.0% (47/50; 95% CI: 83.5% - 98.8%).The Aptima CMV Quant Assay demonstrates high analytical sensitivity and good overall agreement using clinical plasma and tissue specimens.

  View details for DOI 10.1016/j.jcv.2023.105582

  View details for PubMedID 37788527

• Two cases of MPXV infection during pregnancy in heterosexual cisgender women without classic cutaneous lesions, Northern California, 2022. IDCases Renfro, Z. T., Contag, C. A., Lu, J., Solis, D., Huang, C., Sahoo, M. K., Yamamoto, F., Mah, J., Jones, M. S., Lin, J., Levy, V., Pinsky, B. A. 2023; 33: e01881

  Abstract

  As part of an epidemiologic survey, we screened remnant samples collected for STI testing for mpox virus. We identified two cases of presumed MPXV infection in pregnant, heterosexual cisgender women. Here, we describe their pregnancy and birth outcomes. Both patients required induction of labor and experienced labor complicated by chorioamnionitis.

  View details for DOI 10.1016/j.idcr.2023.e01881

  View details for PubMedID 37680215

  View details for PubMedCentralID PMC10480306

• Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects. The Journal of clinical investigation Saul, S., Karim, M., Ghita, L., Huang, P. T., Chiu, W., Durán, V., Lo, C. W., Kumar, S., Bhalla, N., Leyssen, P., Alem, F., Boghdeh, N. A., Tran, D. H., Cohen, C. A., Brown, J. A., Huie, K. E., Tindle, C., Sibai, M., Ye, C., Khalil, A. M., Chiem, K., Martinez-Sobrido, L., Dye, J. M., Pinsky, B. A., Ghosh, P., Das, S., Solow-Cordero, D. E., Jin, J., Wikswo, J. P., Jochmans, D., Neyts, J., De Jonghe, S., Narayanan, A., Einav, S. 2023

  Abstract

  Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

  View details for DOI 10.1172/JCI169510

  View details for PubMedID 37581931

• Evaluation of an automated system for the quantitation of human Herpesvirus-6 DNA from clinical specimens. Practical laboratory medicine Mah, J., Huang, C., Sahoo, M. K., Pinsky, B. A. 2023; 36: e00329

  Abstract

  Quantitation of human herpesvirus-6 (HHV-6) DNA in clinical specimens is important for the diagnosis and management of HHV-6-associated infection and reactivation in immunocompromised patients, particularly transplant recipients.The analytical performance of the Altona RealStar ASR HHV-6 qPCR on the semi-automated AltoStar AM16 system was assessed using HHV-6 reference material in plasma and cerebral spinal fluid (CSF). Qualitative and quantitative agreement was determined using 123 clinical EDTA plasma specimens tested using a laboratory-developed HHV-6 qPCR.The 95% Lower Limit of Detection was 20 IU/mL [95% confidence interval (CI): 10 to 29] in plasma and 78 IU/mL (95% CI: 55 to 146) in CSF. The assay was linear from 7.0 to 2.0 log10 IU/mL in both matrices. Overall agreement of the RealStar ASR HHV-6 qPCR on the AltoStar AM16 with a laboratory-developed test was 95.9% (95% CI: 90.8 to 98.7). Passing-Bablok analysis of specimens quantifiable by both methods and at levels >1000 copies/mL revealed a regression line of Y = 1.00*X-0.20, with neither systematic (95% CI Y-intercept: -0.66 to 0.26) nor proportional (95% CI slope: 0.89 to 1.10) bias compared to the reference.The RealStar ASR HHV-6 qPCR on the AltoStar AM16 provides accurate quantitation for clinical monitoring of HHV-6 in immunocompromised hosts.

  View details for DOI 10.1016/j.plabm.2023.e00329

  View details for PubMedID 37649537

  View details for PubMedCentralID PMC10462668

• Rapid genetic screening with high quality factor metasurfaces. Nature communications Hu, J., Safir, F., Chang, K., Dagli, S., Balch, H. B., Abendroth, J. M., Dixon, J., Moradifar, P., Dolia, V., Sahoo, M. K., Pinsky, B. A., Jeffrey, S. S., Lawrence, M., Dionne, J. A. 2023; 14 (1): 4486

  Abstract

  Genetic analysis methods are foundational to advancing personalized medicine, accelerating disease diagnostics, and monitoring the health of organisms and ecosystems. Current nucleic acid technologies such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) rely on sample amplification and can suffer from inhibition. Here, we introduce a label-free genetic screening platform based on high quality (high-Q) factor silicon nanoantennas functionalized with nucleic acid fragments. Each high-Q nanoantenna exhibits average resonant quality factors of 2,200 in physiological buffer. We quantitatively detect two gene fragments, SARS-CoV-2 envelope (E) and open reading frame 1b (ORF1b), with high-specificity via DNA hybridization. We also demonstrate femtomolar sensitivity in buffer and nanomolar sensitivity in spiked nasopharyngeal eluates within 5 minutes. Nanoantennas are patterned at densities of 160,000 devices per cm2, enabling future work on highly-multiplexed detection. Combined with advances in complex sample processing, our work provides a foundation for rapid, compact, and amplification-free molecular assays.

  View details for DOI 10.1038/s41467-023-39721-w

  View details for PubMedID 37495593

  View details for PubMedCentralID PMC10372074

• Postpandemic Effects of COVID-19 Shelter-in-Place Orders on the Gastrointestinal Pathogen Landscape. Journal of clinical microbiology Bulterys, P. L., Leung, N. Y., Saleem, A., Budvytiene, I., Pinsky, B. A., Banaei, N. 2023: e0038523

  View details for DOI 10.1128/jcm.00385-23

  View details for PubMedID 37466426

• Identification and confirmation via in situ hybridization of Merkel cell polyomavirus in rare cases of posttransplant cutaneous T-cell lymphoma. Journal of cutaneous pathology Lawrence, L., Wang, A., Charville, G., Liu, C. L., Garofalo, A., Alizadeh, A., Jangam, D., Pinsky, B. A., Sahoo, M., Gratzinger, D., Khodadoust, M., Kim, Y., Novoa, R., Stehr, H. 2023

  Abstract

  Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens.Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip.Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes.Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.

  View details for DOI 10.1111/cup.14486

  View details for PubMedID 37394808

• Use of a severe acute respiratory coronavirus virus 2 (SARS-CoV-2) strand-specific assay to evaluate for prolonged viral replication >20 days from illness onset. Infection control and hospital epidemiology Ferguson, J. D., Tayyar, R., Contreras, G., Kiener, M., Zimmet, A. N., Contag, C. A., Rodriguez Nava, G., Tompkins, L. S., Shepard, J., Rosenthal, A., Subramanian, A. K., Pinsky, B. A., Salinas, J. L. 2023: 1-3

  Abstract

  Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse-transcription polymerase chain reaction (rRT-PCR) strand-specific assay can be used to identify active SARS-CoV-2 viral replication. We describe the characteristics of 337 hospitalized patients with at least 1 minus-strand SARS-CoV-2 assay performed >20 days after illness onset. This test is a novel tool to identify high-risk hospitalized patients with prolonged SARS-CoV-2 replication.

  View details for DOI 10.1017/ice.2023.105

  View details for PubMedID 37381726

• Hepatitis E virus seropositivity in an ethnically diverse community blood donor population. Vox sanguinis Mah, J. K., Keck, M., Chu, D. Y., Sooryanarain, H., Sahoo, M. K., Lau, P., Huang, C., Weber, J., Belanger, G. A., Keck, Z., Shan, H., Meng, X., Foung, S. K., Pinsky, B. A., Pham, T. D. 2023

  Abstract

  BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is an underrecognized and emerging infectious disease that may threaten the safety of donor blood supply in many parts of the world. We sought to elucidate whether our local community blood supply is at increased susceptibility for transmission of transfusion-associated HEV infections.MATERIALS AND METHODS: We screened 10,002 randomly selected donations over an 8-month period between 2017 and 2018 at the Stanford Blood Center for markers of HEV infection using commercial IgM/IgG serological tests and reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR). Donor demographic information, including gender, age, self-identified ethnicity, location of residence and recent travel, were obtained from the donor database and used to generate multivariate binary logistic regressions for risk factors of IgG seropositivity.RESULTS: A total of 10,002 blood donations from 7507 unique donors were screened, and there was no detectable HEV RNA by RT-qPCR. The overall seropositivity rate was 12.1% for IgG and 0.56% for IgM. Multivariate analysis of unique donors revealed a significantly higher risk of IgG seropositivity with increasing age, White/Asian ethnicities and residence in certain local counties.CONCLUSION: Although HEV IgG seroprevalence in the San Francisco Bay Area is consistent with ongoing infection, the screening of a large donor population did not identify any viraemic blood donors. While HEV is an underrecognized and emerging infection in other regions, there is no evidence to support routine blood screening for HEV in our local blood supply currently; however, periodic monitoring may still be required to assess the ongoing risk.

  View details for DOI 10.1111/vox.13487

  View details for PubMedID 37366233

• HIV-1 5'-Leader Mutations in Plasma Viruses Before and After the Development of Reverse Transcriptase Inhibitor-Resistance Mutations. medRxiv : the preprint server for health sciences Nouhin, J., Tzou, P., Rhee, S., Sahoo, M. K., Pinsky, B. A., Krupkin, M. A., Puglisi, J. D., Puglisi, E. V., Shafer, R. W. 2023

  Abstract

  Background: HIV-1 RT initiation depends on interaction between viral 5'-leader RNA, RT, and host tRNA3 Lys . We therefore sought to identify co-evolutionary changes between the 5'-leader and RT in viruses developing RT-inhibitor resistance mutations.Methods: We sequenced 5'-leader positions 37-356 of paired plasma virus samples from 29 individuals developing the NRTI-resistance mutation M184V, 19 developing an NNRTI-resistance mutation, and 32 untreated controls. 5'-leader variants were defined as positions where ≥20% of NGS reads differed from the HXB2 sequence. Emergent mutations were defined as nucleotides undergoing ≥4-fold change in proportion between baseline and follow-up. Mixtures were defined as positions containing ≥2 nucleotides each present in ≥20% of NGS reads.Results: Among 80 baseline sequences, 87 positions (27.2%) contained a variant; 52 contained a mixture. Position 201 was the only position more likely to develop a mutation in the M184V (9/29 vs. 0/32; p=0.0006) or NNRTI-resistance (4/19 vs. 0/32; p=0.02; Fisher's Exact Test) groups than the control group. Mixtures at positions 200 and 201 occurred in 45.0% and 28.8%, respectively, of baseline samples. Because of the high proportion of mixtures at these positions, we analyzed 5'-leader mixture frequencies in two additional datasets: five publications reporting 294 dideoxyterminator clonal GenBank sequences from 42 individuals and six NCBI BioProjects reporting NGS datasets from 295 individuals. These analyses demonstrated position 200 and 201 mixtures at proportions similar to those in our samples and at frequencies several times higher than at all other 5'-leader positions.Conclusions: Although we did not convincingly document co-evolutionary changes between RT and 5'-leader sequences, we identified a novel phenomenon, wherein positions 200 and 201, immediately downstream of the HIV-1 primer binding site exhibited an extraordinarily high likelihood of containing a nucleotide mixture. Possible explanations for the high mixture rates are that these positions are particularly error-prone or provide a viral fitness advantage.

  View details for DOI 10.1101/2023.06.04.23290942

  View details for PubMedID 37333388

• Prevalence of Mpox (Monkeypox) in patients undergoing STI screening in northern California, April-September 2022. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Contag, C. A., Renfro, Z. T., Lu, J., Shen, S., Karan, A., Solis, D., Huang, C., Sahoo, M. K., Yamamoto, F., Jones, M. S., Lin, J., Levy, V., Pinsky, B. A. 2023; 164: 105493

  Abstract

  Despite the sharp increase in mpox (formerly monkeypox) incidence and the wide geographic spread of mpox during the 2022 outbreak, the community prevalence of infection remains poorly characterized. This study is a retrospective epidemiologic survey to estimate mpox prevalence.Samples obtained for sexually transmitted infection (STI) testing from April to September 2022 in the public hospital and clinic system of San Mateo County, California were screened for mpox virus (MPXV) using polymerase chain reaction.16/1,848 samples from 11/1,645 individuals were positive for MPXV by qPCR. 4/11 individuals with positive MPXV testing were cisgender women, 2 of whom were pregnant at the time of sample collection. Both deliveries were complicated by chorioamnionitis. Anorectal and oropharyngeal samples were the most likely to be positive for MPXV (4/60 anorectal samples and 4/66 oropharyngeal samples compared with 5/1,264 urine samples and 3/445 vaginal samples).Our study is one of the first epidemiologic surveys for MPXV infection outside of sexual health/STI clinic settings. Relatively high rates of MPXV from oropharyngeal and anorectal samples reinforces the importance of MPXV testing at various anatomic sites, particularly if patients are presenting with non-lesional symptoms (pharyngitis, proctitis). However, the United States Food and Drug Administration (FDA) has not yet authorized non-lesional MPXV testing. The identification of MPXV in women in our cohort suggests that the rates of mpox in women may have previously been underestimated and highlights the risk of pregnancy complications associated with mpox.

  View details for DOI 10.1016/j.jcv.2023.105493

  View details for PubMedID 37220710

  View details for PubMedCentralID PMC10184869

• Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection. Research square Choi, S. S., van Unen, V., Zhang, H., Rustagi, A., Alwahabi, S. A., Santos, A. J., Chan, J. E., Lam, B., Solis, D., Mah, J., Röltgen, K., Trope, W., Guh-Siesel, A., Lin, Z., Beck, A., Edwards, C., Mallajosyula, V., Martin, B. A., Dunn, J. C., Shrager, J., Baric, R. A., Pinsky, B., Boyd, S. D., Blish, C. A., Davis, M. M., Kuo, C. J. 2023

  Abstract

  Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

  View details for DOI 10.21203/rs.3.rs-2870695/v1

  View details for PubMedID 37205380

  View details for PubMedCentralID PMC10187413

• Comparison of nucleocapsid antigen with strand-specific reverse-transcription PCR for monitoring SARS-CoV-2 infection. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Chang-Graham, A. L., Sahoo, M. K., Huang, C., Solis, D., Sibai, M., August, G., Calayag, L., Kenji, O. M., Shi, R. Z., Mostafa, H. H., Lei, G. S., Relich, R. F., Pinsky, B. A. 2023; 164: 105468

  Abstract

  Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA.Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard.Overall agreement was 92.0% [95% confidence interval (CI): 89.0 - 94.5], positive percent agreement was 90.6% (95% CI: 84.4 - 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 - 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 - 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log10 IU/mL; resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0).Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management.

  View details for DOI 10.1016/j.jcv.2023.105468

  View details for PubMedID 37119583

  View details for PubMedCentralID PMC10124094

• Comparison of Real-Time PCR and Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma. The Journal of molecular diagnostics : JMD Miller, J. A., Huang, C., Yamamoto, F., Sahoo, M. K., Le, Q., Pinsky, B. A. 2023

  Abstract

  Plasma Epstein-Barr Virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma (NPC). However, existing real-time PCR (qPCR) assays are limited by poor inter-laboratory reproducibility. This is a barrier to biomarker integration into staging systems and management. It was hypothesized that EBV digital PCR (dPCR) would have similar sensitivity but improved precision relative to qPCR. Using the WHO EBV standard and patient specimens, the NRG BamHI-W qPCR, two commercial EBNA-1 qPCR assays, and two laboratory-developed dPCR assays amplifying the BamHI-W, EBNA-1, and EBER targets were compared. Testing was conducted in the North American reference laboratory for the NRG-HN001 randomized trial. The EBV dPCR assays achieved similar performance compared with qPCR. Although dPCR does not require quantitation standards, different dPCR thresholding algorithms yielded significant qualitative and quantitative variation. This was most evident with low levels of EBV DNA. No-template control-informed thresholding (ddpcRquant) mitigated false positives/negatives. The NRG BamHI-W qPCR and laboratory-developed BamHI-W droplet dPCR offered higher sensitivity, lower limit of blank, higher precision at low plasma EBV DNA levels (≤1500 IU/mL), and higher overall agreement with clinical specimens compared to single-copy qPCR/dPCR targets (EBNA-1/EBER). These data confirm the rationale for the use of the BamHI-W target to define prognostic thresholds, and indicate that both qPCR and dPCR methods harmonized to the WHO standard can provide the necessary analytical performance.

  View details for DOI 10.1016/j.jmoldx.2023.03.007

  View details for PubMedID 37068736

• Evaluation of a multiplexed oligonucleotide ligation assay for SARS-CoV-2 variant identification. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Solis, D., Sibai, M., Kung, F., Break, T. J., Harkins, S. B., Huang, C., Yamamoto, F., Sahoo, M. K., Wohlstadter, J. N., Sigal, G. B., Pinsky, B. A. 2023; 162: 105444

  Abstract

  SARS-CoV-2 variant surveillance informs vaccine composition and decisions to de-authorize antibody therapies. Though detailed genetic characterization requires whole-genome sequencing, targeted mutation analysis may complement pandemic surveillance efforts.This study investigated the qualitative performance of a multiplex oligonucleotide ligation assay targeting 19 spike mutations using 192 whole genome sequenced upper respiratory samples representing SARS-CoV-2 variants of concern.Initial valid results were obtained from 95.8% [95% confidence interval (CI): 92.0 - 98.2; 184/192] of samples. All eight invalid samples were valid on repeat testing. When comparing SARS-CoV-2 oligonucleotide ligase assay SARS-CoV-2 variant calls with whole genome sequencing, overall positive percent agreement was 100% (95% CI: 98.1 - 100.0; 192/192), as was the positive and negative percent agreement for each of the tested variants; Gamma, Delta, Omicron BA.1, BA.2, and BA.4/BA.5.This multiplexed oligonucleotide ligation assays demonstrated accurate SARS-CoV-2 variant typing compared to whole genome sequencing. Such an approach has the potential to provide improved turnaround compared to sequencing and more detailed mutation coverage than RT-qPCR.

  View details for DOI 10.1016/j.jcv.2023.105444

  View details for PubMedID 37043903

• Retrospective Screening of Clinical Samples for Monkeypox Virus DNA, California, USA, 2022. Emerging infectious diseases Contag, C. A., Lu, J., Renfro, Z. T., Karan, A., Salinas, J. L., Khan, M., Solis, D., Sahoo, M. K., Yamamoto, F., Pinsky, B. A. 2023; 29 (4): 848-850

  Abstract

  We retrospectively screened oropharyngeal and rectal swab samples originally collected in California, USA, for Chlamydia trachomatis and Neisseria gonorrhoeae testing for the presence of monkeypox virus DNA. Among 206 patients screened, 17 (8%) had samples with detectable viral DNA. Monkeypox virus testing from mucosal sites should be considered for at-risk patients.

  View details for DOI 10.3201/eid2904.221576

  View details for PubMedID 36918374

  View details for PubMedCentralID PMC10045697

• Evaluation of acebilustat, a selective inhibitor of leukotriene B4 biosynthesis, for treatment of outpatients with mild-moderate COVID-19 disease: A randomized, double-blind, placebo- controlled Phase 2 trial. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Levitt, J. E., Hedlin, H., Duong, S., Lu, D., Lee, J., Bunning, B., Elkarra, N., Pinsky, B. A., Heffernan, E., Springman, E., Moss, R. B., Bonilla, H. F., Parsonnet, J., Zamanian, R. T., Langguth, J. J., Bollyky, J., Khosla, C., Nicolls, M. R., Desai, M., Rogers, A. J. 2023

  Abstract

  The vast majority of COVID-19 disease occurs in outpatients where treatment is limited to anti-virals for high-risk subgroups. Acebilustat, a leukotriene B4 (LTB4) inhibitor, has potential to reduce inflammation and symptom duration.In a single-center trial spanning Delta and Omicron variants, outpatients were randomized to 100 mg of oral acebilustat or placebo for 28 days. Patients reported daily symptoms via electronic query through Day 28 with phone follow-up on Day 120 and collected nasal swabs on Days 1-10. The primary outcome was sustained symptom resolution to Day 28. Secondary 28-day outcomes included time to first symptom resolution, area under the curve (AUC) of longitudinal daily symptom scores; duration of viral shedding through Day 10; and symptoms on Day 120.Sixty participants were randomized to each study arm. At enrollment, median duration and number of symptoms were 4 (IQR 3-5) days and 9 (IQR 7-11) symptoms. Most patients (90%) were vaccinated with 73% having neutralizing antibodies. A minority (44%) of participants (35% in the acebilustat arm and 53% in placebo) had sustained symptom resolution at Day 28 (HR 0.6, 95% CI 0.34-1.04, p = 0.07 favoring placebo). There was no difference in mean AUC of symptom scores over 28 days (difference in mean of AUC 9.4, 95% CI -42.1-60.9, p=0.72). Acebilustat did not impact viral shedding or symptoms at Day 120.Sustained symptoms through Day 28 were common in this low-risk population. Despite this, LTB4 antagonism with acebilustat did not shorten symptom duration in outpatients with COVID-19.

  View details for DOI 10.1093/cid/ciad187

  View details for PubMedID 36996150

• Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics. Science advances Robinson, M. L., Glass, D. R., Duran, V., Agudelo Rojas, O. L., Sanz, A. M., Consuegra, M., Sahoo, M. K., Hartmann, F. J., Bosse, M., Gelvez, R. M., Bueno, N., Pinsky, B. A., Montoya, J. G., Maecker, H., Estupiñan Cardenas, M. I., Villar Centeno, L. A., Garrido, E. M., Rosso, F., Bendall, S. C., Einav, S. 2023; 9 (12): eade7702

  Abstract

  Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

  View details for DOI 10.1126/sciadv.ade7702

  View details for PubMedID 36961888

• Detection of Adenovirus in Formalin-Fixed Paraffin-Embedded Tissue by qPCR Compared to Immunohistochemistry Chang-Graham, A., Pepper, M., Sahoo, M., Huang, C., Garamani, N., Higgins, J., Pinsky, B. ELSEVIER SCIENCE INC. 2023: S1276

  View details for Web of Science ID 000990969802371

• Evaluation of a Semiautomated System for the Quantitation of Human Adenovirus DNA from Clinical Samples. Microbiology spectrum Mah, J., Huang, C. H., Sahoo, M. K., Pinsky, B. A. 2023: e0501022

  Abstract

  Human adenoviruses (HAdVs) cause severe disease in immunocompromised patients. Quantitation of HAdV DNA in peripheral blood is used to assess the risk of disseminated disease and to monitor response to therapy. The lower limit of detection, precision, and linearity of the semiautomated AltoStar adenovirus quantitative PCR (qPCR) was evaluated using reference HAdV-E4 in EDTA plasma and respiratory virus matrix. Qualitative and quantitative agreement was determined using 122 clinical EDTA plasma specimens previously tested using a laboratory-developed HAdV qPCR. The 95% lower limit of detection (LLOD) was 33IU/mL (95% confidence interval [CI], 10 to 56) for EDTA plasma and 188IU/mL (95% CI, 145 to 304) for respiratory swab matrix. In both matrices, the AltoStar HAdV qPCR was linear from 7.0 to 2.0 log10 IU/mL. For the clinical specimens, overall agreement was 96.7% (95% CI, 91.8 to 99.1), positive percent agreement was 95.5% (95% CI, 87.6 to 98.5), and negative percent agreement was 98.2% (95% CI, 88.5 to 99.7). Passing-Bablok analysis of specimens quantifiable by both methods revealed a regression line of Y=1.11 · X+0.00; there was positive proportional bias (95% CI of the slope, 1.05 to 1.22) but no systematic bias (95% CI of the Y-intercept, -0.43 to 0.23) compared to the reference. The AltoStar platform provides accurate quantitation of HAdV DNA and provides a semiautomated option for the clinical monitoring of HAdV following transplantation. IMPORTANCE Accurate quantification of human adenovirus DNA in the peripheral blood plays a critical role in the management of adenovirus infections in transplant recipients. Many laboratories utilize in-house laboratory-based PCR assays for the quantification of human adenovirus, as there are few commercial options available. Here, we describe the analytical and clinical performance of the semiautomated AltoStar adenovirus quantitative PCR (Altona Diagnostics). This platform provides sensitive, precise, and accurate quantification of adenovirus DNA that is well suited for virological testing following transplantation. Prior to implementing a new quantitative test in the clinical laboratory, a rigorous evaluation is required to determine assay performance characteristics and to correlate results to current in-house methods of quantitation.

  View details for DOI 10.1128/spectrum.05010-22

  View details for PubMedID 36847504

• Serum biomarkers and anti-flavivirus antibodies at presentation as indicators of severe dengue. PLoS neglected tropical diseases Bernal, C., Ping, S., Rojas, A., Caballero, O., Stittleburg, V., de Guillen, Y., Langjahr, P., Pinsky, B. A., Von-Horoch, M., Luraschi, P., Cabral, S., Sanchez, M. C., Torres, A., Cardozo, F., Waggoner, J. J. 2023; 17 (2): e0010750

  Abstract

  BACKGROUND: Dengue is the most common vector-borne viral disease worldwide. Most cases are mild, but some evolve into severe dengue (SD), with high lethality. Therefore, it is important to identify biomarkers of severe disease to improve outcomes and judiciously utilize resources.METHODS/PRINCIPAL FINDINGS: One hundred forty-five confirmed dengue cases (median age, 42; range <1-91 years), enrolled from February 2018 to March 2020, were selected from an ongoing study of suspected arboviral infections in metropolitan Asuncion, Paraguay. Cases included dengue virus types 1, 2, and 4, and severity was categorized according to the 2009 World Health Organization guidelines. Testing for anti-dengue virus IgM and IgG and serum biomarkers (lipopolysaccharide binding protein and chymase) was performed on acute-phase sera in plate-based ELISAs; in addition, a multiplex ELISA platform was used to measure anti-dengue virus and anti-Zika virus IgM and IgG. Complete blood counts and chemistries were performed at the discretion of the care team. Age, gender, and pre-existing comorbidities were associated with SD vs. dengue with/without warning signs in logistic regression with odds ratios (ORs) of 1.07 (per year; 95% confidence interval, 1.03, 1.11), 0.20 (female; 0.05,0.77), and 2.09 (presence; 1.26, 3.48) respectively. In binary logistic regression, for every unit increase in anti-DENV IgG in the multiplex platform, odds of SD increased by 2.54 (1.19-5.42). Platelet count, lymphocyte percent, and elevated chymase were associated with SD in a combined logistic regression model with ORs of 0.99 (1,000/muL; 0.98,0.999), 0.92 (%; 0.86,0.98), and 1.17 (mg/mL; 1.03,1.33) respectively.CONCLUSIONS: Multiple, readily available factors were associated with SD in this population. These findings will aid in the early detection of potentially severe dengue cases and inform the development of new prognostics for use in acute-phase and serial samples from dengue cases.

  View details for DOI 10.1371/journal.pntd.0010750

  View details for PubMedID 36848385

• Clinicopathologic Features of Human Monkeypox Lymphadenitis. Histopathology Qian, Z. J., Gong, R., Mann, D. S., Walding, K., Miller, T., Nicholas, V., Kappagoda, S., Pinsky, B. A., Silva, O., Lau, H. D. 2023

  View details for DOI 10.1111/his.14878

  View details for PubMedID 36734592

• Recommendations for Epstein-Barr virus-based screening for nasopharyngeal cancer in high- and intermediate-risk regions. Journal of the National Cancer Institute Lam, W. K., King, A. D., Miller, J. A., Liu, Z., Yu, K. J., Chua, M. L., Ma, B. B., Chen, M. Y., Pinsky, B. A., Lou, P. J., Woo, J. K., Hsu, W. L., Simon, J., Doolan, D. L., Waterboer, T., Hui, E. P., Li, H., Tsang, R. K., Wong, K. C., Goh, J. P., Vlantis, A. C., Ai, Q. Y., Wong, L. M., Abdullah, V., Lin, J. C., Chen, C. J., Pfeiffer, R. M., Le, Q. T., Lee, A. W., Ji, M., Cao, S., Ma, J., Chan, A. T., Chan, K. C., Hildesheim, A. 2023

  Abstract

  A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early-stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program.

  View details for DOI 10.1093/jnci/djad012

  View details for PubMedID 36723440

• Laboratory Preparedness for the Current Monkeypox Outbreak. Clinical chemistry Rodino, K. G., Babady, N. E., Bearman, G., Binnicker, M. J., Isaacs, S. N., Pinsky, B. A., Weber, D. J. 2022

  View details for DOI 10.1093/clinchem/hvac198

  View details for PubMedID 36544358

• Neutralization against BA.2.75.2, BQ.1.1, and XBB from mRNA Bivalent Booster. The New England journal of medicine Davis-Gardner, M. E., Lai, L., Wali, B., Samaha, H., Solis, D., Lee, M., Porter-Morrison, A., Hentenaar, I. T., Yamamoto, F., Godbole, S., Liu, Y., Douek, D. C., Lee, F. E., Rouphael, N., Moreno, A., Pinsky, B. A., Suthar, M. S. 2022

  View details for DOI 10.1056/NEJMc2214293

  View details for PubMedID 36546661

• Sierra SARS-CoV-2 sequence and antiviral resistance analysis program. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Tzou, P. L., Tao, K., Sahoo, M. K., Kosakovsky Pond, S. L., Pinsky, B. A., Shafer, R. W. 2022; 157: 105323

  Abstract

  INTRODUCTION: Although most laboratories are capable of employing established protocols to perform full-genome SARS-CoV-2 sequencing, many are unable to assess sequence quality, select appropriate mutation-detection thresholds, or report on the potential clinical significance of mutations in the targets of antiviral therapy METHODS: We describe the technical aspects and benchmark the performance of Sierra SARS-CoV-2, a program designed to perform these functions on user-submitted FASTQ and FASTA sequence files and lists of Spike mutations. Sierra SARS-CoV-2 indicates which sequences contain an unexpectedly large number of unusual mutations and which mutations are associated with reduced susceptibility to clinical stage mAbs, the RdRP inhibitor remdesivir, or the Mpro inhibitor nirmatrelvir RESULTS: To assess the performance of Sierra SARS-CoV-2 on FASTQ files, we applied it to 600 representative FASTQ sequences and compared the results to the COVID-19 EDGE program. To assess its performance on FASTA files, we applied it to nearly one million representative FASTA sequences and compared the results to the GISAID mutation annotation. To assess its performance on mutations lists, we applied it to 13,578 distinct Spike RBD mutation patterns and showed that exactly or partially matching annotations were available for 88% of patterns CONCLUSION: Sierra SARS-CoV-2 leverages previously published data to improve the quality control of submitted viral genomic data and to provide functional annotation on the impact of mutations in the targets of antiviral SARS-CoV-2 therapy. The program can be found at https://covdb.stanford.edu/sierra/sars2/ and its source code at https://github.com/hivdb/sierra-sars2.

  View details for DOI 10.1016/j.jcv.2022.105323

  View details for PubMedID 36334368

• Deep sequencing analysis of clinical samples from patients with acute infectious conjunctivitis during the COVID-19 delta surge in Madurai, India. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Lalitha, P., Prajna, N. V., Gunasekaran, R., Teja, G. V., Sharma, S. S., Hinterwirth, A., Ruder, K., Zhong, L., Chen, C., Deiner, M., Huang, C., Pinsky, B. A., Lietman, T. M., Doan, T., Seitzman, G. D. 2022; 157: 105318

  Abstract

  Seasonal outbreaks of infectious conjunctivitis remain a public health issue. Determination of outbreak etiologies in the context of a worldwide pandemic may provide useful information to guide public health strategies. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge.This prospective study was conducted from April 2021 to September 2021. All outpatients presenting to the Aravind Eye Center (Madurai, India) with signs and symptoms consistent with acute infectious conjunctivitis were eligible. Three swabs were obtained from each participant: one from each conjunctiva and one from the anterior nares. Samples were processed for metagenomic RNA deep sequencing (RNA-seq).Samples from 106 study participants were sequenced. The most common presenting symptoms were tearing (86%) and itching (71%). Preauricular lymphadenopathy was present in 38% of participants. 20% of participants had close contacts with similar symptoms. Systemic symptoms such as coughing, runny nose, vomiting or diarrhea were uncommonly reported. 60% of all participants used some medicated eye drops upon enrollment. 75% of study participants demonstrated infection with human adenovirus D (HAdV-D). 11% of conjunctivitis was associated with SARS-CoV-2. 15% had no definitive pathogen detected. 8% of all participants had codetection of more than one pathogen on RNA-seq.During the COVID-19 Delta surge in India, HAdV-D was the most common pathogen associated with infectious conjunctivitis. SARS-CoV-2 was the second most common associated pathogen. Seasonal surveillance may be necessary for the determination of emerging and reemerging pathogens responsible for infectious conjunctivitis.

  View details for DOI 10.1016/j.jcv.2022.105318

  View details for PubMedID 36242841

• Outpatient human coronavirus associated conjunctivitis in India. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Prajna, N. V., Lalitha, P., Teja, G. V., Gunasekaran, R., Sharma, S. S., Hinterwirth, A., Ruder, K., Zhong, L., Chen, C., Deiner, M., Huang, C., Pinsky, B. A., Lietman, T. M., Seitzman, G. D., Doan, T., SCORPIO (Seasonal Conjunctivitis Outbreak Reporting for Prevention and Improved Outcomes) Study Group 2022; 157: 105300

  Abstract

  BACKGROUND: Viral conjunctivitis (pink eye) can be highly contagious and is of public health importance. There remains significant debate whether SARS-CoV-2 can present as a primary conjunctivitis. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge.METHODS: This prospective study was conducted in the spring and summer months of 2021. 106 patients with acute conjunctivitis who presented to the Aravind Eye Center in Madurai, India were included. One anterior nasal swab and one conjunctival swab of each eye were obtained for each enrolled patient. Samples were subsequently processed for unbiased metagenomic RNA deep sequencing (RNA-seq). Outcomes included clinical findings and codetection of other pathogens with SARS-CoV-2 in patients with conjunctivitis.RESULTS: Among the 13 patients identified with human coronavirus RNA fragments in their swabs, 6 patients had SARS-CoV-2 infection, 5 patients had coinfections of SARS-CoV-2 and human adenovirus (HAdV), 1 patient had a coinfection with human coronavirus OC43 and HAdV, and 1 patient had a coinfection of Vittaforma corneae and SARS-CoV-2. 30% had bilateral disease and symptoms on presentation. Petechial hemorrhage was noted in 33% of patients with SARS-CoV-2 infection. No patients with SARS-CoV-2 or SARS-CoV-2 and HAdV infections had subepithelial infiltrates on presentation. All patients denied systemic symptoms.CONCLUSIONS: Among the patients presented with conjunctivitis associated with human coronavirus infection, over 50% of the patients had co-infections with other circulating pathogens, suggesting the public-health importance of broad pathogen testing and surveillance in the outpatient conjunctivitis population.

  View details for DOI 10.1016/j.jcv.2022.105300

  View details for PubMedID 36209621

• Trichodysplasia Spinulosa Polyomavirus Endothelial Infection, California, USA. Emerging infectious diseases Lawrence, L., Wang, A., Charville, G., Toland, A., Pinsky, B., Natkunam, Y., Younes, S., Stehr, H., Gratzinger, D. 2022; 28 (9): 1935-1937

  Abstract

  We describe 3 patients in California, USA, with trichodysplasia spinulosa polyomavirus (TSPyV) infection of endothelium after steroid administration. We detected TSPyV RNA in tissue specimens by in situ hybridization, which revealed localization to endothelial cells. These cases suggest that diseases associated with endothelial inflammation could be associated with TSPyV infection.

  View details for DOI 10.3201/eid2809.220856

  View details for PubMedID 35997483

• Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy. Nature communications Parikh, V. N., Ioannidis, A. G., Jimenez-Morales, D., Gorzynski, J. E., De Jong, H. N., Liu, X., Roque, J., Cepeda-Espinoza, V. P., Osoegawa, K., Hughes, C., Sutton, S. C., Youlton, N., Joshi, R., Amar, D., Tanigawa, Y., Russo, D., Wong, J., Lauzon, J. T., Edelson, J., Mas Montserrat, D., Kwon, Y., Rubinacci, S., Delaneau, O., Cappello, L., Kim, J., Shoura, M. J., Raja, A. N., Watson, N., Hammond, N., Spiteri, E., Mallempati, K. C., Montero-Martín, G., Christle, J., Kim, J., Kirillova, A., Seo, K., Huang, Y., Zhao, C., Moreno-Grau, S., Hershman, S. G., Dalton, K. P., Zhen, J., Kamm, J., Bhatt, K. D., Isakova, A., Morri, M., Ranganath, T., Blish, C. A., Rogers, A. J., Nadeau, K., Yang, S., Blomkalns, A., O'Hara, R., Neff, N. F., DeBoever, C., Szalma, S., Wheeler, M. T., Gates, C. M., Farh, K., Schroth, G. P., Febbo, P., deSouza, F., Cornejo, O. E., Fernandez-Vina, M., Kistler, A., Palacios, J. A., Pinsky, B. A., Bustamante, C. D., Rivas, M. A., Ashley, E. A. 2022; 13 (1): 5107

  Abstract

  The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

  View details for DOI 10.1038/s41467-022-32397-8

  View details for PubMedID 36042219

• Human Monkeypox without Viral Prodrome or Sexual Exposure, California, USA, 2022. Emerging infectious diseases Karan, A., Styczynski, A. R., Huang, C., Sahoo, M. K., Srinivasan, K., Pinsky, B. A., Salinas, J. L. 2022; 28 (10)

  Abstract

  We report human monkeypox in a man who returned to the United States from the United Kingdom and reported no sexual contact. He had vesicular and pustular skin lesions but no anogenital involvement. The potential modes of transmission may have implications for the risk of spread and for epidemic control.

  View details for DOI 10.3201/eid2810.221191

  View details for PubMedID 35971952

• An intranasal ASO therapeutic targeting SARS-CoV-2. Nature communications Zhu, C., Lee, J. Y., Woo, J. Z., Xu, L., Nguyenla, X., Yamashiro, L. H., Ji, F., Biering, S. B., Van Dis, E., Gonzalez, F., Fox, D., Wehri, E., Rustagi, A., Pinsky, B. A., Schaletzky, J., Blish, C. A., Chiu, C., Harris, E., Sadreyev, R. I., Stanley, S., Kauppinen, S., Rouskin, S., Näär, A. M. 2022; 13 (1): 4503

  Abstract

  The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

  View details for DOI 10.1038/s41467-022-32216-0

  View details for PubMedID 35922434

• Multiplex Epstein-Barr virus BALF2 genotyping detects high-risk variants in plasma for population screening of nasopharyngeal carcinoma. Molecular cancer Miller, J. A., Sahoo, M. K., Yamamoto, F., Huang, C., Wang, H., Zehnder, J. L., Le, Q. T., Pinsky, B. A. 2022; 21 (1): 154

  Abstract

  Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) exhibits unusual geographic restriction despite ubiquitous lifelong infection. Screening programs can detect most NPC cases at an early stage, but existing EBV diagnostics are limited by false positives and low positive predictive value (PPV), leading to excess screening endoscopies, MRIs, and repeated testing. Recent EBV genome-wide association studies (GWAS) suggest that EBV BALF2 variants account for more than 80% of attributable NPC risk. We therefore hypothesized that high-risk BALF2 variants could be readily detected in plasma for once-lifetime screening triage.We designed and validated a multiplex genotyping assay to detect EBV BALF2 polymorphisms in human plasma. Targeted next-generation sequencing was used to validate this assay, conduct association studies with clinical phenotype, and longitudinally genotype plasma to assess within-host haplotype stability. We examined the association between NPC and BALF2 haplotypes in a large non-endemic population and three prior EBV GWAS. Finally, we estimated NPC mortality reduction, resource utilization, and cost-effectiveness of BALF2 variant-informed screening using a previously-validated cohort model.Following analytical validation, the BALF2 genotyping assay had 99.3% concordance with sequencing in a cohort of 24 NPC cases and 155 non-NPC controls. BALF2 haplotype was highly associated with NPC in this non-endemic population (I613V: odds ratio [OR] 7.9; V317M: OR 178.8). No other candidate BALF2 polymorphisms were significantly associated with NPC or hematologic disorders. Longitudinal genotyping revealed 97.8% within-host haplotype concordance, indicative of lifelong latent infection. In a meta-analysis of 755 NPC cases and 981 non-NPC controls, BALF2 I613V and V317M were significantly associated with NPC in both endemic and non-endemic populations. Modeled variant-informed screening strategies achieved a 46% relative increase in PPV with 7% decrease in effective screening sensitivity, thereby averting nearly half of screening endoscopies/MRIs among endemic populations in east/southeast Asia.EBV BALF2 haplotypes are temporally stable within hosts and can be readily detected in plasma via an inexpensive multiplex genotyping assay that offers near-perfect sequencing concordance. In endemic and non-endemic populations, I613V and V317M were highly associated with NPC and could be leveraged to develop variant-informed screening programs that mitigate false positives with small reductions in screening sensitivity.

  View details for DOI 10.1186/s12943-022-01625-6

  View details for PubMedID 35902864

• Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection. Nature genetics Biering, S. B., Sarnik, S. A., Wang, E., Zengel, J. R., Leist, S. R., Schafer, A., Sathyan, V., Hawkins, P., Okuda, K., Tau, C., Jangid, A. R., Duffy, C. V., Wei, J., Gilmore, R. C., Alfajaro, M. M., Strine, M. S., Nguyenla, X., Van Dis, E., Catamura, C., Yamashiro, L. H., Belk, J. A., Begeman, A., Stark, J. C., Shon, D. J., Fox, D. M., Ezzatpour, S., Huang, E., Olegario, N., Rustagi, A., Volmer, A. S., Livraghi-Butrico, A., Wehri, E., Behringer, R. R., Cheon, D., Schaletzky, J., Aguilar, H. C., Puschnik, A. S., Button, B., Pinsky, B. A., Blish, C. A., Baric, R. S., O'Neal, W. K., Bertozzi, C. R., Wilen, C. B., Boucher, R. C., Carette, J. E., Stanley, S. A., Harris, E., Konermann, S., Hsu, P. D. 2022

  Abstract

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

  View details for DOI 10.1038/s41588-022-01131-x

  View details for PubMedID 35879412

• Harmonization of SARS-CoV-2 reverse transcription quantitative PCR tests to the first WHO international standard for SARS-CoV-2 RNA. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Sahoo, M. K., Huang, C., Sibai, M., Solis, D., Pinsky, B. A. 2022; 154: 105242

  Abstract

  BACKGROUND: Cycle threshold (Ct) values from SARS-CoV-2 reverse transcription quantitative PCR (RT-qPCR) tests are used to measure viral burden. Calibration to the First WHO International Standard for SARS-CoV-2 RNA may improve quantitative inter-assay agreement.METHODS: WHO standard was tested using four emergency use authorized RT-qPCRs to generate calibration curves and evaluate Ct value differences. Harmonization of two assays, Cepheid Xpert Xpress SARS-CoV-2 targeting E and nucleocapsid (N2) [Xpert (E) and Xpert (N2)] and a laboratory-developed test targeting E [LDT (E)], was assessed using 93 positive upper respiratory samples. Platform (target) pairs were compared via Bland-Altman analysis and Passing-Bablok regression.RESULTS: Ct values with the WHO standard were comparable across platforms and targets, except Xpert (N2) for which the mean difference was a median of 3.68 cycles (Interquartile Range, IQR=3.23 to 3.76 cycles) greater than other platform (target) pairs. Using clinical samples, the mean difference of Xpert (N2) to LDT (E) was 3.64 cycles (95% Confidence Interval, CI =1.51 to 5.76). After calibration, the mean difference of Xpert (N2) to LDT (E) was 0.08 log10 IU/mL (95% CI=-0.56 to 0.71) and the regression was y=1.00x * 0.08 (95% CI slope=0.93 to 1.07, 95% CI intercept=0.28 to 0.42).CONCLUSIONS: Calibration to the WHO standard resulted in the harmonization of two RT-qPCR tests, whereas analysis by Ct value alone may have led to erroneous quantitation. Harmonization to the WHO standard has the potential to improve the generalizability of clinical associations with SARS-CoV-2 RNA levels.

  View details for DOI 10.1016/j.jcv.2022.105242

  View details for PubMedID 35944343

• SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19. Journal of neuropathology and experimental neurology Serrano, G. E., Walker, J. E., Tremblay, C., Piras, I. S., Huentelman, M. J., Belden, C. M., Goldfarb, D., Shprecher, D., Atri, A., Adler, C. H., Shill, H. A., Driver-Dunckley, E., Mehta, S. H., Caselli, R., Woodruff, B. K., Haarer, C. F., Ruhlen, T., Torres, M., Nguyen, S., Schmitt, D., Rapscak, S. Z., Bime, C., Peters, J. L., Alevritis, E., Arce, R. A., Glass, M. J., Vargas, D., Sue, L. I., Intorcia, A. J., Nelson, C. M., Oliver, J., Russell, A., Suszczewicz, K. E., Borja, C. I., Cline, M. P., Hemmingsen, S. J., Qiji, S., Hobgood, H. M., Mizgerd, J. P., Sahoo, M. K., Zhang, H., Solis, D., Montine, T. J., Berry, G. J., Reiman, E. M., Roltgen, K., Boyd, S. D., Pinsky, B. A., Zehnder, J. L., Talbot, P., Desforges, M., DeTure, M., Dickson, D. W., Beach, T. G. 2022

  Abstract

  Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

  View details for DOI 10.1093/jnen/nlac056

  View details for PubMedID 35818336

• Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome. JCI insight Jia, X., Cao, S., Lee, A. S., Manohar, M., Sindher, S. B., Ahuja, N., Artandi, M., Blish, C. A., Blomkalns, A. L., Chang, I., Collins, W. J., Desai, M., Din, H. N., Do, E., Fernandes, A., Geng, L. N., Rosenberg-Hasson, Y., Mahoney, M. R., Glascock, A. L., Chan, L. Y., Fong, S. Y., Phelps, M., Raeber, O., Purington, N., Röltgen, K., Rogers, A. J., Snow, T., Wang, T. T., Solis, D., Vaughan, L., Verghese, M., Maecker, H., Wittman, R., Puri, R., Kistler, A., Yang, S., Boyd, S. D., Pinsky, B. A., Chinthrajah, S., Nadeau, K. C. 2022; 7 (13)

  Abstract

  BACKGROUNDProlonged symptoms after SARS-CoV-2 infection are well documented. However, which factors influence development of long-term symptoms, how symptoms vary across ethnic groups, and whether long-term symptoms correlate with biomarkers are points that remain elusive.METHODSAdult SARS-CoV-2 reverse transcription PCR-positive (RT-PCR-positive) patients were recruited at Stanford from March 2020 to February 2021. Study participants were seen for in-person visits at diagnosis and every 1-3 months for up to 1 year after diagnosis; they completed symptom surveys and underwent blood draws and nasal swab collections at each visit.RESULTSOur cohort (n = 617) ranged from asymptomatic to critical COVID-19 infections. In total, 40% of participants reported at least 1 symptom associated with COVID-19 six months after diagnosis. Median time from diagnosis to first resolution of all symptoms was 44 days; median time from diagnosis to sustained symptom resolution with no recurring symptoms for 1 month or longer was 214 days. Anti-nucleocapsid IgG level in the first week after positive RT-PCR test and history of lung disease were associated with time to sustained symptom resolution. COVID-19 disease severity, ethnicity, age, sex, and remdesivir use did not affect time to sustained symptom resolution.CONCLUSIONWe found that all disease severities had a similar risk of developing post-COVID-19 syndrome in an ethnically diverse population. Comorbid lung disease and lower levels of initial IgG response to SARS-CoV-2 nucleocapsid antigen were associated with longer symptom duration.TRIAL REGISTRATIONClinicalTrials.gov, NCT04373148.FUNDINGNIH UL1TR003142 CTSA grant, NIH U54CA260517 grant, NIEHS R21 ES03304901, Sean N Parker Center for Allergy and Asthma Research at Stanford University, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative, Sunshine Foundation, Crown Foundation, and Parker Foundation.

  View details for DOI 10.1172/jci.insight.156713

  View details for PubMedID 35801588

• Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19. Cell reports. Medicine Feyaerts, D., Hédou, J., Gillard, J., Chen, H., Tsai, E. S., Peterson, L. S., Ando, K., Manohar, M., Do, E., Dhondalay, G. K., Fitzpatrick, J., Artandi, M., Chang, I., Snow, T. T., Chinthrajah, R. S., Warren, C. M., Wittman, R., Meyerowitz, J. G., Ganio, E. A., Stelzer, I. A., Han, X., Verdonk, F., Gaudillière, D. K., Mukherjee, N., Tsai, A. S., Rumer, K. K., Jacobsen, D. R., Bjornson-Hooper, Z. B., Jiang, S., Saavedra, S. F., Valdés Ferrer, S. I., Kelly, J. D., Furman, D., Aghaeepour, N., Angst, M. S., Boyd, S. D., Pinsky, B. A., Nolan, G. P., Nadeau, K. C., Gaudillière, B., McIlwain, D. R. 2022: 100680

  Abstract

  The biological determinants underlying the range of coronavirus 2019 (COVID-19) clinical manifestations are not fully understood. Here, over 1,400 plasma proteins and 2,600 single-cell immune features comprising cell phenotype, endogenous signaling activity, and signaling responses to inflammatory ligands are cross-sectionally assessed in peripheral blood from 97 patients with mild, moderate, and severe COVID-19 and 40 uninfected patients. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identify and independently validate a multi-variate model classifying COVID-19 severity (multi-class area under the curve [AUC]training = 0.799, p = 4.2e-6; multi-class AUCvalidation = 0.773, p = 7.7e-6). Examination of informative model features reveals biological signatures of COVID-19 severity, including the dysregulation of JAK/STAT, MAPK/mTOR, and nuclear factor κB (NF-κB) immune signaling networks in addition to recapitulating known hallmarks of COVID-19. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for prevention and/or treatment of COVID-19 progression.

  View details for DOI 10.1016/j.xcrm.2022.100680

  View details for PubMedID 35839768

• Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies. Antiviral research Karim, M., Saul, S., Ghita, L., Sahoo, M. K., Ye, C., Bhalla, N., Lo, C. W., Jin, J., Park, J., Martinez-Gualda, B., East, M. P., Johnson, G. L., Pinsky, B. A., Martinez-Sobrido, L., Asquith, C. R., Narayanan, A., De Jonghe, S., Einav, S. 2022: 105367

  Abstract

  The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

  View details for DOI 10.1016/j.antiviral.2022.105367

  View details for PubMedID 35738348

• Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Yang, L. M., Costales, C., Ramanathan, M., Bulterys, P. L., Murugesan, K., Schroers-Martin, J., Alizadeh, A. A., Boyd, S. D., Brown, J. M., Nadeau, K. C., Nadimpalli, S. S., Wang, A. X., Busque, S., Pinsky, B. A., Banaei, N. 2022; 153: 105217

  Abstract

  BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response.METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naive individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination.RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P<0.001), sphingosine 1-phsophate (S1P) receptor modulators (P<0.001), mycophenolate (P=0.002), and B cell lymphoma (P=0.004); those associated with low cellular response rates included S1P receptor modulators (P<0.001) and mycophenolate (P<0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination.CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.

  View details for DOI 10.1016/j.jcv.2022.105217

  View details for PubMedID 35714462

• Accuracy of Rapid Antigen vs Reverse Transcriptase-Polymerase Chain Reaction Testing for SARS-CoV-2 Infection in College Athletes During Prevalence of the Omicron Variant. JAMA network open Tsao, J., Kussman, A. L., Costales, C., Pinsky, B. A., Abrams, G. D., Hwang, C. E. 2022; 5 (6): e2217234

  View details for DOI 10.1001/jamanetworkopen.2022.17234

  View details for PubMedID 35704320

• Characterizing the Severity of SARS-CoV-2 Variants at a Single Pediatric Center FRONTIERS IN MEDICINE Khan, A., Ichura, C., Wang, H., Rezende, I., Sahoo, M. K., Huang, C., Solis, D., Sibai, M., Yamamoto, F., Nyathi, S., Bayrau, B., Pinsky, B. A., LaBeaud, A. 2022; 9: 896352

  Abstract

  Since March 2020, SARS-CoV-2 has plagued the world with COVID-19 and individuals of all ages have experienced varying symptoms of disease. Older adults were experiencing more severe disease compared to children and were prioritized by vaccination efforts. While biologic therapies and vaccinations were implemented, there were changes in public health restrictions with subsequent surges resulting in more infected children. During these surges there was a rise of different SARS-CoV-2 variants with the dominant variant initially alpha (B.1.1.7 and other Pango lineages) and epsilon (B.1.427/B.1.429) in early 2021 and a dramatic shift to delta (B.1.617.2 and other Pango lineages) by mid-summer 2021. In this study we aimed to characterize the clinical severity and host factors associated with disease by SARS-CoV-2 variant and evaluate if there are differences in disease severity by circulating variant. We retrospectively included all individuals 0-25 years of age who presented to our center and had a positive SARS-CoV-2 RT-PCR, SARS-CoV-2 variant mutation testing, and documented clinical notes from 1 January 2021 through 31 December 2021. We identified 745 individuals who met inclusion criteria and found the delta variant was associated with severe/critical disease compared to the other variants studied. The results of the model showed that underlying respiratory disease and diabetes were risk factors for progression to severe disease. These insights are important when evaluating public health measures and treatment options for children as more variants arise.

  View details for DOI 10.3389/fmed.2022.896352

  View details for Web of Science ID 000807127000001

  View details for PubMedID 35677819

  View details for PubMedCentralID PMC9168367

• Dysregulation of Brain inflammation in COVID-19: An autopsy series Piras, I., Huentelman, M., Walker, J., Arce, R., Tremblay, C., Glass, M., Sue, L., Nelson, C., Suszczewicz, K., Borja, C., Cline, M., Montine, T., Pinsky, B., Zehnder, J., DeTuree, M., Dickson, D., Beach, T. OXFORD UNIV PRESS INC. 2022: 476

  View details for Web of Science ID 000798368400137

• Acute Infarction, Hemorrhage and White Matter Beta-Amyloid Precursor Protein Immunostaining in COVID-19 and Control Subjects DeTure, M., Walker, J., Arce, R., Glass, M., Sue, L., Intorcia, A., Nelson, C., Suszczewicz, K., Borja, C., Montine, T., Pinsky, B., Zehnder, J., Serrano, G., Dickson, D. OXFORD UNIV PRESS INC. 2022: 476

  View details for Web of Science ID 000798368400135

• Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature communications Zeng, L., Liu, Y., Nguyenla, X. H., Abbott, T. R., Han, M., Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang, H., Rane, D. A., Spatz, J. M., Jain, S., Rustagi, A., Pinsky, B., Zepeda, A. E., Kadina, A. P., Walker, J. A., Holden, K., Temperton, N., Cochran, J. R., Barron, A. E., Connolly, M. D., Blish, C. A., Lewis, D. B., Stanley, S. A., La Russa, M. F., Qi, L. S. 2022; 13 (1): 2766

  Abstract

  A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

  View details for DOI 10.1038/s41467-022-30546-7

  View details for PubMedID 35589813

• SARS-CoV-2 RNA and N Antigen Quantification via Wastewater at the Campus Level, Building Cluster Level, and Individual-Building Level ACS ES&T WATER Zambrana, W., Catoe, D., Coffman, M. M., Kim, S., Anand, A., Solis, D., Sahoo, M. K., Pinsky, B. A., Bhatt, A. S., Boehm, A. B., Wolfe, M. K. 2022

  View details for DOI 10.1021/acsestwater.2c00050

  View details for Web of Science ID 000820376800001

• SARS-CoV-2 RNA and N Antigen Quantification via Wastewater at the Campus Level, Building Cluster Level, and Individual-Building Level. ACS ES&T water Zambrana, W., Catoe, D., Coffman, M. M., Kim, S., Anand, A., Solis, D., Sahoo, M. K., Pinsky, B. A., Bhatt, A. S., Boehm, A. B., Wolfe, M. K. 2022; 2 (11): 2025-33

  Abstract

  Monitoring wastewater for SARS-CoV-2 from populations smaller than those served by wastewater treatment plants may help identify small spatial areas (subsewersheds) where COVID-19 infections are present. We sampled wastewater from three nested locations with different sized populations within the same sewer network at a university campus and quantified SARS-CoV-2 RNA using reverse transcriptase droplet digital polymerase chain reaction (PCR). SARS-CoV-2 RNA concentrations and/or concentrations normalized by PMMoV were positively associated with laboratory-confirmed COVID-19 cases for both the sewershed level and the subsewershed level. We also used an antigen-based assay to detect the nucleocapsid (N) antigen from SARS-CoV-2 in wastewater samples at the sewershed level. The N antigen was regularly detected at the sewershed level, but the results were not associated with either laboratory-confirmed COVID-19 cases or SARS-CoV-2 RNA concentrations. The results of this study indicate that wastewater monitoring based on quantification of SARS-CoV-2 RNA using PCR-based methods is associated with COVID-19 cases at multiple geographic scales within the subsewershed level and can serve to aid the public health response.

  View details for DOI 10.1021/acsestwater.2c00050

  View details for PubMedID 37552722

  View details for PubMedCentralID PMC9128006

• A Spurious Positive Result on the Abbott Architect 4th generation HIV Ag/Ab Combo Assay in a Low-Risk Patient. Clinica chimica acta; international journal of clinical chemistry Pepper, M. A., Wiredja, D. D., Patel, P. M., Stevens, B. A., Pinsky, B. A., Shi, R. Z. 2022

  View details for DOI 10.1016/j.cca.2022.05.004

  View details for PubMedID 35568208

• Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification. Journal of clinical microbiology Yeung, P. S., Wang, H., Sibai, M., Solis, D., Yamamoto, F., Iwai, N., Jiang, B., Hammond, N., Truong, B., Bihon, S., Santos, S., Mar, M., Mai, C., Mfuh, K. O., Miller, J. A., Huang, C., Sahoo, M. K., Zehnder, J. L., Pinsky, B. A. 2022: e0017822

  Abstract

  The ability to distinguish between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, responses to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription-quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in reaction 1 and del69-70, K417N, and T478K in reaction 2. This assay had 95 to 100% agreement with WGS for 502 upper respiratory tract swab samples collected between 26 April 2021 and 1 August 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

  View details for DOI 10.1128/jcm.00178-22

  View details for PubMedID 35465708

• Interepidemic Respiratory Syncytial Virus during the COVID-19 Pandemic. Microbiology spectrum Huang, C., Sahoo, M. K., Verghese, M., Sibai, M., Solis, D., Mfuh, K. O., Kurzer, J., Hogan, C. A., Doan, T. A., Pinsky, B. A. 2022: e0094722

  View details for DOI 10.1128/spectrum.00947-22

  View details for PubMedID 35467362

• Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection. Med (New York, N.Y.) Natarajan, A., Zlitni, S., Brooks, E. F., Vance, S. E., Dahlen, A., Hedlin, H., Park, R. M., Han, A., Schmidtke, D. T., Verma, R., Jacobson, K. B., Parsonnet, J., Bonilla, H. F., Singh, U., Pinsky, B. A., Andrews, J. R., Jagannathan, P., Bhatt, A. S. 2022

  Abstract

  COVID-19 manifests with respiratory, systemic, and gastrointestinal (GI) symptoms.1,2 SARS-CoV-2 RNA is detected in respiratory and fecal samples, and recent reports demonstrate viral replication in both the lung and intestinal tissue.3-5 Although much is known about early fecal RNA shedding, little is known about the long term shedding, especially in those with mild COVID-19. Furthermore, most reports of fecal RNA shedding do not correlate these findings with GI symptoms.6.We analyze the dynamics of fecal RNA shedding up to 10 months after COVID-19 diagnosis in 113 individuals with mild to moderate disease. We also correlate shedding with disease symptoms.Fecal SARS-CoV-2 RNA is detected in 49.2% [95% Confidence interval = 38.2%-60.3%] of participants within the first week after diagnosis. Whereas there was no ongoing oropharyngeal SARS-CoV-2 RNA shedding in subjects at and after 4 months, 12.7% [8.5%-18.4%] of participants continued to shed SARS-CoV-2 RNA in the feces at 4 months after diagnosis and 3.8% [2.0%-7.3%] shed at 7 months. Finally, we find that GI symptoms (abdominal pain, nausea, vomiting) are associated with fecal shedding of SARS-CoV-2 RNA.The extended presence of viral RNA in feces, but not respiratory samples, along with the association of fecal viral RNA shedding with GI symptoms suggest that SARS-CoV-2 infects the GI tract, and that this infection can be prolonged in a subset of individuals with COVID-19.

  View details for DOI 10.1016/j.medj.2022.04.001

  View details for PubMedID 35434682

  View details for PubMedCentralID PMC9005383

• Detailed characterization of hospitalized patients infected with the Omicron variant of SARS-CoV-2. Journal of internal medicine Ozdalga, E., Ahuja, N., Sehgal, N., Hom, J., Weng, Y., Pinsky, B., Schulman, K. A., Collins, W. 2022

  View details for DOI 10.1111/joim.13501

  View details for PubMedID 35417053

• An 8-gene machine learning model improves clinical prediction of severe dengue progression. Genome medicine Liu, Y. E., Saul, S., Rao, A. M., Robinson, M. L., Agudelo Rojas, O. L., Sanz, A. M., Verghese, M., Solis, D., Sibai, M., Huang, C. H., Sahoo, M. K., Gelvez, R. M., Bueno, N., Estupinan Cardenas, M. I., Villar Centeno, L. A., Rojas Garrido, E. M., Rosso, F., Donato, M., Pinsky, B. A., Einav, S., Khatri, P. 2022; 14 (1): 33

  Abstract

  BACKGROUND: Each year 3-6 million people develop life-threatening severe dengue (SD). Clinical warning signs for SD manifest late in the disease course and are nonspecific, leading to missed cases and excess hospital burden. Better SD prognostics are urgently needed.METHODS: We integrated 11 public datasets profiling the blood transcriptome of 365 dengue patients of all ages and from seven countries, encompassing biological, clinical, and technical heterogeneity. We performed an iterative multi-cohort analysis to identify differentially expressed genes (DEGs) between non-severe patients and SD progressors. Using only these DEGs, we trained an XGBoost machine learning model on public data to predict progression to SD. All model parameters were "locked" prior to validation in an independent, prospectively enrolled cohort of 377 dengue patients in Colombia. We measured expression of the DEGs in whole blood samples collected upon presentation, prior to SD progression. We then compared the accuracy of the locked XGBoost model and clinical warning signs in predicting SD.RESULTS: We identified eight SD-associated DEGs in the public datasets and built an 8-gene XGBoost model that accurately predicted SD progression in the independent validation cohort with 86.4% (95% CI 68.2-100) sensitivity and 79.7% (95% CI 75.5-83.9) specificity. Given the 5.8% proportion of SD cases in this cohort, the 8-gene model had a positive and negative predictive value (PPV and NPV) of 20.9% (95% CI 16.7-25.6) and 99.0% (95% CI 97.7-100.0), respectively. Compared to clinical warning signs at presentation, which had 77.3% (95% CI 58.3-94.1) sensitivity and 39.7% (95% CI 34.7-44.9) specificity, the 8-gene model led to an 80% reduction in the number needed to predict (NNP) from 25.4 to 5.0. Importantly, the 8-gene model accurately predicted subsequent SD in the first three days post-fever onset and up to three days prior to SD progression.CONCLUSIONS: The 8-gene XGBoost model, trained on heterogeneous public datasets, accurately predicted progression to SD in a large, independent, prospective cohort, including during the early febrile stage when SD prediction remains clinically difficult. The model has potential to be translated to a point-of-care prognostic assay to reduce dengue morbidity and mortality without overwhelming limited healthcare resources.

  View details for DOI 10.1186/s13073-022-01034-w

  View details for PubMedID 35346346

• Novel Utilization of Strand-Specific Reverse Transcription Polymerase Chain Reaction in Perioperative Clinical Decision Making for SARS-CoV-2 Polymerase Chain Reaction Positive Patients. Paediatric anaesthesia Jette, C. G., Wang, T., Wang, E., Man, J. Y., Mireles, S., Maass, B., Mathew, R., Pinsky, B. A., Claure, R., D'Souza, G. 2022

  Abstract

  In order to prevent in-hospital transmission and potential complications related to SARS-CoV-2 in the perioperative patient, most healthcare institutions require preoperative testing for SARS-CoV-2 prior to proceeding with elective surgery. The Centers for Disease Control and Prevention (CDC) recommends a time and symptom-based duration of isolation for the presumed infectious period. The guidance to avoid retesting of asymptomatic patients in the 90days following a positive reverse transcription polymerase chain reaction (RT-PCR) test is because of the possibility of detection of non-infectious viral shedding. When to reschedule asymptomatic patients who test RT-PCR positive for SARS-CoV-2 preoperatively is of considerable debate, both from the perspective of ensuring a patient's full preoperative fitness, as well as reducing the risk of viral transmission within the hospital. We describe the novel perioperative use of a strand-specific assay to detect minus strand ribonucleic acid (RNA) in a clinical decision-making algorithm to determine optimal timing of elective surgery after a patient tests RT-PCR positive for SARS-CoV-2. This is the first description in the literature of an attempt to further stratify patients who repeatedly test positive for SARS-CoV-2 into infectious versus non-infectious for perioperative planning.

  View details for DOI 10.1111/pan.14448

  View details for PubMedID 35338765

• Incidence and prevalence of COVID-19 within a healthcare worker cohort during the first year of the SARS-CoV-2 pandemic. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Doernberg, S. B., Holubar, M., Jain, V., Weng, Y., Lu, D., Bollyky, J. B., Sample, H., Huang, B., Craik, C. S., Desai, M., Rutherford, G. W., Maldonado, Y., CHART Study Consortium 2022

  Abstract

  BACKGROUND: Preventing SARS-CoV2 infections in healthcare workers (HCWs) is critical for healthcare delivery. We aimed to estimate and characterize the prevalence and incidence of COVID-19 in a US HCW cohort and to identify risk factors associated with infection.METHODS: We conducted a longitudinal cohort study of HCWs at 3 Bay Area medical centers using serial surveys and SARS-CoV-2 viral and orthogonal serological testing, including measurement of neutralizing antibodies. We estimated baseline prevalence and cumulative incidence of COVID-19. We performed multivariable Cox proportional hazards models to estimate associations of baseline factors with incident infections and evaluated the impact of time-varying exposures on time to COVID-19 using marginal structural models.RESULTS: 2435 HCWs contributed 768 person years of follow-up time. We identified 21/2435 individuals with prevalent infection, resulting in a baseline prevalence of 0.86% (95% CI, 0.53% to 1.32%). We identified 70/2414 (2.9%) incident infections yielding a cumulative incidence rate of 9.11 cases per 100 person years (95% CI 7.11 to 11.52). Community contact with a known COVID-19 case most strongly correlated with increased hazard for infection (HR 8.1, 95% CI, 3.8, 17.5). High-risk work-related exposures (i.e., breach in protective measures) drove an association between work exposure and infection (HR 2.5, 95% CI, 1.3-4.8). More cases were identified in HCW when community case rates were high.CONCLUSION: We observed modest COVID-19 incidence despite consistent exposure at work. Community contact was strongly associated with infections but contact at work was not unless accompanied by high-risk exposure.

  View details for DOI 10.1093/cid/ciac210

  View details for PubMedID 35279023

• SARS-CoV-2 Neutralizing Monoclonal Antibodies for the Treatment of COVID-19 in Kidney Transplant Recipients. Kidney360 Wang, A. X., Busque, S., Kuo, J., Singh, U., Roeltgen, K., Pinsky, B. A., Chertow, G. M., Scandling, J. D., Lenihan, C. R. 2022; 3 (1): 133-143

  Abstract

  Background: Morbidity and mortality associated with coronavirus disease 2019 (COVID-19) infection in kidney transplant recipients are high and early outpatient interventions to prevent progression to severe disease are needed. SARS-CoV-2 neutralizing mAbs, including bamlanivimab and casirivimab-imdevimab, received emergency use authorization in the United States in November 2020 for treatment of mild to moderate COVID-19 disease.Methods: We performed a retrospective analysis of 27 kidney transplant recipients diagnosed with COVID-19 between July 2020 and February 2021 who were treated with bamlanivimab or casirivimab-imdevimab and immunosuppression reduction. We additionally identified 13 kidney transplant recipients with COVID-19 who had mild to moderate disease at presentation, who did not receive mAbs, and had SARS-CoV-2 serology testing available.Results: There were no deaths or graft failures in either group. Both infusions were well tolerated. Four of the 27 patients treated with mAbs required hospitalization due to COVID-19. Four of 13 patients who did not receive mAbs required hospitalization due to COVID-19. Patients who received mAbs demonstrated measurable anti-SARS-CoV-2 IgG with angiotensin-converting enzyme 2 (ACE2) receptor blocking activity at the highest level detectable at 90 days postinfusion, whereas ACE2 blocking activity acquired from natural immunity in the mAb-untreated group was weak.Conclusions: Bamlanivimab and casirivimab-imdevimab combined with immunosuppression reduction were well tolerated and associated with favorable clinical outcomes in kidney transplant recipients diagnosed with mild to moderate COVID-19.

  View details for DOI 10.34067/KID.0005732021

  View details for PubMedID 35368573

• Feasibility of Specimen Self-collection in Young Children Undergoing SARS-CoV-2 Surveillance for In-Person Learning. JAMA network open Altamirano, J., Lopez, M., Robinson, I. G., Chun, L. X., Tam, G. K., Shaikh, N. J., Hoyte, E. G., Carrington, Y. J., Jani, S. G., Toomarian, E. Y., Hsing, J. C., Ma, J., Pulendran, U., Govindarajan, P., Blomkalns, A. L., Pinsky, B. A., Wang, C. J., Maldonado, Y. 2022; 5 (2): e2148988

  Abstract

  There is an urgent need to assess the feasibility of COVID-19 surveillance measures in educational settings.To assess whether young children can feasibly self-collect SARS-CoV-2 samples for surveillance testing over the course of an academic year.This prospective pilot cohort study was conducted from September 10, 2020, to June 10, 2021, at a K-8 school in San Mateo County, California. The research consisted of quantitative data collection efforts: (1) demographic data collected, (2) student sample self-collection error rates, and (3) student sample self-collection time durations. Students were enrolled in a hybrid learning model, a teaching model in which students were taught in person and online, with students having the option to attend virtually as needed. Data were collected under waiver of consent from students participating in weekly SARS-CoV-2 testing.Errors over time for self-collection of nasal swabs such as contaminated swabs and inadequate or shallow swabbing; time taken for sample collection.Of 296 participants, 148 (50.0%) were boys and 148 (50.0%) were girls. A total of 87 participants (29.2%) identified as Asian; 2 (0.6%), Black or African American; 13 (4.4%), Hispanic/Latinx; 103 (34.6%), non-Hispanic White; 87 (29.2%), multiracial; and 6 (2.0%), other. The median school grade was fourth grade. From September 2020 to March 2021, a total of 4203 samples were obtained from 221 students on a weekly basis, while data on error rates were collected. Errors occurred in 2.7% (n = 107; 95% CI, 2.2%-3.2%) of student encounters, with the highest rate occurring on the first day of testing (20 [10.2%]). There was an overall decrease in error rates over time. From April to June 2021, a total of 2021 samples were obtained from 296 students on a weekly basis while data on encounter lengths were collected. Between April and June 2021, 193 encounters were timed. The mean duration of each encounter was 70 seconds (95% CI, 66.4-73.7 seconds).Mastery of self-collected lower nasal swabs is possible for children 5 years and older. Testing duration can be condensed once students gain proficiency in testing procedures. Scalability for larger schools is possible if consideration is given to the resource-intensive nature of the testing and the setting's weather patterns.

  View details for DOI 10.1001/jamanetworkopen.2021.48988

  View details for PubMedID 35175340

• Development and evaluation of an RT-qPCR for the identification of the SARS-CoV-2 Omicron variant. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Sibai, M., Wang, H., Yeung, P. S., Sahoo, M. K., Solis, D., Mfuh, K. O., Huang, C., Yamamoto, F., Pinsky, B. A. 2022; 148: 105101

  View details for DOI 10.1016/j.jcv.2022.105101

  View details for PubMedID 35151048

• Vaccine-Associated Measles Encephalitis in Immunocompromised Child, California, USA. Emerging infectious diseases Costales, C., Sahoo, M. K., Huang, C., Guimaraes, C. V., Born, D., Kushner, L., Gans, H. A., Doan, T. A., Pinsky, B. A. 2022; 28 (4): 906-908

  Abstract

  We report a fatal case of vaccine-associated measles encephalitis in an immunocompromised child in California, USA. The infection was confirmed by whole-genome RNA sequencing of measles virus from brain tissue. We observed biased matrix-gene hypermutation consistent with persistent measles virus central nervous system infection.

  View details for DOI 10.3201/eid2804.212357

  View details for PubMedID 35318930

• Long Term Accuracy of SARS-CoV-2 Interferon-γ Release Assay and its Application in Household Investigation. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K., Jagannathan, P., Altamirano, J., Maldonado, Y. A., Bonilla, H. F., Jacobson, K. B., Parsonnet, J., Andrews, J. R., Shi, R. Z., Boyd, S., Pinsky, B. A., Singh, U., Banaei, N. 2022

  Abstract

  An immunodiagnostic assay that sensitively detects a cell-mediated immune response to SARS-CoV-2 is needed for epidemiological investigation and for clinical assessment of T cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses.The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in COVID-19 convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen.The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI] 79.0-89.0) and 86.6% (123/142; 95% CI;80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI 64.4-89.2) at 10 months post-infection (P<0.01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs. 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of +50% (95% CI +25.4-+74.6) and +21.7% (95% CI, +9.23-+42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts.The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

  View details for DOI 10.1093/cid/ciac045

  View details for PubMedID 35079772

• Immunogenicity of a Third COVID-19 mRNA Vaccine Dose in PID Patients with Functional B-cell Defects. The journal of allergy and clinical immunology. In practice Gernez, Y., Murugesan, K., Cortales, C. R., Banaei, N., Hoyte, L., Pinsky, B. A., Lewis, D. B., Pham, M. 2022

  View details for DOI 10.1016/j.jaip.2022.02.030

  View details for PubMedID 35259538

• Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination. Cell Röltgen, K., Nielsen, S. C., Silva, O., Younes, S. F., Zaslavsky, M., Costales, C., Yang, F., Wirz, O. F., Solis, D., Hoh, R. A., Wang, A., Arunachalam, P. S., Colburg, D., Zhao, S., Haraguchi, E., Lee, A. S., Shah, M. M., Manohar, M., Chang, I., Gao, F., Mallajosyula, V., Li, C., Liu, J., Shoura, M. J., Sindher, S. B., Parsons, E., Dashdorj, N. J., Dashdorj, N. D., Monroe, R., Serrano, G. E., Beach, T. G., Chinthrajah, R. S., Charville, G. W., Wilbur, J. L., Wohlstadter, J. N., Davis, M. M., Pulendran, B., Troxell, M. L., Sigal, G. B., Natkunam, Y., Pinsky, B. A., Nadeau, K. C., Boyd, S. D. 2022

  Abstract

  During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

  View details for DOI 10.1016/j.cell.2022.01.018

  View details for PubMedID 35148837

• Immunogenicity and Tolerability of COVID-19 mRNA Vaccines in PID patients with functional B-cell defects. The Journal of allergy and clinical immunology Pham, M. N., Murugesan, K., Banaei, N., Pinsky, B. A., Tang, M., Hoyte, E., Lewis, D. B., Gernez, Y. 1800

  Abstract

  BACKGROUND: Data on the safety and efficacy of COVID-19 vaccination in people with a range of primary immune deficiencies are lacking since these patients were excluded from COVID-19 vaccine trials. This information may help in clinical management of this vulnerable patient group.OBJECTIVE: To assess humoral and T-cell immune responses after two doses of SARS-CoV-2 mRNA vaccines in patients with PIDs and functional B-cell defects.METHODS: A double-center retrospective review of patients with PID who completed COVID-19 mRNA vaccination and who had humoral responses assessed through SARS-CoV-2 spike protein receptor binding domain (RBD) IgG antibody levels with reflex assessment of the antibody to block RBD binding to ACE2 (hereafter referred to as ACE2 receptor blocking activity, as a surrogate test for neutralization) and T-cell response evaluated by an interferon-gamma release assay (IGRA). Immunization reactogenicity was also reviewed.RESULTS: A total of 33 patients with humoral defect were evaluated. 69.6% received BNT162b2 vaccine (Pfizer-BioNTech) and 30.3% received mRNA-1273 (Moderna). The mRNA vaccines were generally well tolerated without severe reactions. The IGRA was positive in 77.4% of our patients (24 of 31). About half of our subjects (16 of 33) had detectable RBD-specific IgG responses but only two of these 16 subjects had an ACE2 receptor blocking activity level of >50%.CONCLUSION: Vaccination of this cohort of PID patients with COVID-19 mRNA vaccines was safe and cellular immunity was stimulated in a majority. However, antibody responses to the spike protein RBD were less consistent, and, when detected, was not effective at ACE2 blocking.CLINICAL IMPLICATION: mRNA vaccination may be less effective at preventing acquisition of SARS-CoV-2 in our cohort of PID patients with functional B-cell defects. The Induction of SARS-CoV-2 spike protein-specific T-cell immunity by vaccination might help reduce the severity of disease in these patients.

  View details for DOI 10.1016/j.jaci.2021.11.022

  View details for PubMedID 34952033

• Direct comparison of antibody responses to four SARS-CoV-2 vaccines in Mongolia. Cell host & microbe Dashdorj, N. J., Wirz, O. F., Roltgen, K., Haraguchi, E., Buzzanco, A. S., Sibai, M., Wang, H., Miller, J. A., Solis, D., Sahoo, M. K., Arunachalam, P. S., Lee, A. S., Shah, M. M., Liu, J., Byambabaatar, S., Bat-Ulzii, P., Enkhbat, A., Batbold, E., Zulkhuu, D., Ochirsum, B., Khurelsukh, T., Dalantai, G., Burged, N., Baatarsuren, U., Ariungerel, N., Oidovsambuu, O., Bungert, A. S., Genden, Z., Yagaanbuyant, D., Mordorj, A., Pulendran, B., Chinthrajah, S., Nadeau, K. C., Jardetzky, T., Wilbur, J. L., Wohlstadter, J. N., Sigal, G. B., Pinsky, B. A., Boyd, S. D., Dashdorj, N. D. 2021

  Abstract

  Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V, and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins revealed marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide.

  View details for DOI 10.1016/j.chom.2021.11.004

  View details for PubMedID 34861167

• The Effect of Povidone-Iodine Nasal Spray on COVID-19 Nasopharyngeal Viral Load in Patients: A Randomized Control Trial. The Laryngoscope Zarabanda, D., Vukkadala, N., Phillips, K. M., Qian, Z. J., Mfuh, K. O., Hatter, M., Lee, I. T., Rao, V. K., Hwang, P. H., Domb, G., Patel, Z. M., Pinsky, B. A., Nayak, J. V. 2021

  Abstract

  OBJECTIVES: To determine the effect of povidone-iodine (PVP-I) nasal sprays on nasopharyngeal (NP) viral load as assessed by cycle threshold on quantitative polymerase chain reaction (qPCR) of SARS-CoV-2 in outpatients.STUDY DESIGN: Three arm, triple blinded, randomized, placebo-controlled clinical trial.METHODS: Participants were randomized within 5days of testing positive for COVID-19 to receive nasal sprays containing either placebo (0.9% saline), 0.5% PVP-I, or 2.0% PVP-I. NP swabs for qPCR analysis were taken at baseline, 1-hour post-PVP-I spray (2 sprays/nostril), and 3days post-PVP-I spray (20 sprays/nostril). Symptom and adverse event questionnaires were completed at baseline, day 3, and day 5. University of Pennsylvania Smell Identification Tests (UPSIT) were completed at baseline and day 30.RESULTS: Mean cycle threshold (Ct) values increased over time in all groups, indicating declining viral loads, with no statistically significant difference noted in the rate of change between placebo and PVP-I groups. 2.0% PVP-I group showed statistically significant improvement in all symptom categories, however also reported a high rate of nasal burning. Olfaction via UPSIT showed improvement by at least one category in all groups. There were no hospitalizations or mortalities within 30days of study enrollment.CONCLUSION: Saline and low concentration PVP-I nasal sprays are well tolerated. Similar reductions in SARS-CoV-2 nasopharyngeal viral load were seen over time in all groups. All treatment groups showed improvement in olfaction over 30days. These data suggest that dilute versions of PVP-I nasal spray are safe for topical use in the nasal cavity, but that PVP-I does not demonstrate virucidal activity in COVID-19 positive outpatients. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/lary.29935

  View details for PubMedID 34724213

• Rapid genetic screening with high quality factor metasurfaces. ArXiv Hu, J., Safir, F., Chang, K., Dagli, S., Balch, H. B., Abendroth, J. M., Dixon, J., Moradifar, P., Dolia, V., Sahoo, M. K., Pinsky, B. A., Jeffrey, S. S., Lawrence, M., Dionne, J. A. 2021

  Abstract

  Genetic analysis methods are foundational to advancing personalized and preventative medicine, accelerating disease diagnostics, and monitoring the health of organisms and ecosystems. Current nucleic acid technologies such as polymerase chain reaction (PCR), next-generation sequencing (NGS), and DNA microarrays rely on fluorescence and absorbance, necessitating sample amplification or replication and leading to increased processing time and cost. Here, we introduce a label-free genetic screening platform based on high quality (high-Q) factor silicon nanoantennas functionalized with monolayers of nucleic acid fragments. Each nanoantenna exhibits substantial electromagnetic field enhancements with sufficiently localized fields to ensure isolation from neighboring resonators, enabling dense biosensor integration. We quantitatively detect complementary target sequences using DNA hybridization simultaneously for arrays of sensing elements patterned at densities of 160,000 pixels per cm$^2$. In physiological buffer, our nanoantennas exhibit average resonant quality factors of 2,200, allowing detection of two gene fragments, SARS-CoV-2 envelope (E) and open reading frame 1b (ORF1b), down to femtomolar concentrations. We also demonstrate high specificity sensing in clinical nasopharyngeal eluates within 5 minutes of sample introduction. Combined with advances in biomarker isolation from complex samples (e.g., mucus, blood, wastewater), our work provides a foundation for rapid, compact, amplification-free and high throughput multiplexed genetic screening assays spanning medical diagnostics to environmental monitoring.

  View details for PubMedID 34671699

  View details for PubMedCentralID PMC8528080

• SARS-CoV-2 Nucleocapsid Plasma Antigen for Diagnosis and Monitoring of COVID-19. Clinical chemistry Wang, H., Hogan, C. A., Verghese, M., Solis, D., Sibai, M., Huang, C., Roltgen, K., Stevens, B. A., Yamamoto, F., Sahoo, M. K., Zehnder, J., Boyd, S. D., Pinsky, B. A. 2021

  Abstract

  BACKGROUND: Detection of SARS-CoV-2 nucleocapsid antigen in blood has been described, but the diagnostic and prognostic role of antigenemia is not well understood. This study aimed to determine the frequency, duration, and concentration of nucleocapsid antigen in plasma and its association with COVID-19 severity.METHODS: We utilized an ultrasensitive electrochemiluminescence immunoassay targeting SARS-CoV-2 nucleocapsid antigen to evaluate 777 plasma samples from 104 individuals with COVID-19. We compared plasma antigen to respiratory nucleic acid amplification testing (NAAT) in 74 individuals with COVID-19 from samples collected ± 1day of diagnostic respiratory NAAT, and in 52 SARS-CoV-2-negative individuals. We used Kruskal-Wallis tests, multivariable logistic regression, and mixed-effects modeling to evaluate whether plasma antigen concentration was associated with disease severity.RESULTS: Plasma antigen had 91.9% (95% CI 83.2-97.0%) clinical sensitivity and 94.2% (84.1-98.8%) clinical specificity. Antigen-negative plasma samples belonged to patients with later respiratory cycle thresholds (Ct) when compared with antigen-positive plasma samples. Median plasma antigen concentration (log10 fg/mL) was 5.4 (IQR 3.9-6.0) in outpatients, 6.0 (5.4-6.5) in inpatients, and 6.6 (6.1-7.2) in intensive care unit (ICU) patients. In models adjusted for age, sex, diabetes, and hypertension, plasma antigen concentration at diagnosis was associated with ICU admission (OR 2.8 [95% CI 1.2-6.2], P=.01), but not with non-ICU hospitalization. Rate of antigen decrease was not associated with disease severity.CONCLUSIONS: SARS-CoV-2 plasma nucleocapsid antigen exhibited comparable diagnostic performance to upper respiratory NAAT, especially among those with late respiratory Ct. In addition to currently available tools, antigenemia may facilitate patient triage to optimize intensive care utilization.

  View details for DOI 10.1093/clinchem/hvab216

  View details for PubMedID 34605900

• Case-control study evaluating risk factors for SARS-CoV-2 outbreak amongst healthcare personnel at a tertiary care center. American journal of infection control Rosser, J. I., Tayyar, R., Giardina, R., Kolonoski, P., Kenski, D., Shen, P., Steinmetz, L. M., Hung, L., Xiao, W., Bains, K., Morrison, T., Madison, A., Chang, S., Tompkins, L., Pinsky, B. A., Holubar, M. 2021

  Abstract

  BACKGROUND: Despite several outbreaks of SARS-CoV-2 amongst healthcare personnel (HCP) exposed to COVID-19 patients globally, risk factors for transmission remain poorly understood.METHODS: We conducted an outbreak investigation and case-control study to evaluate SARS-CoV-2 transmission risk in an outbreak among HCP at an academic medical center in California that was confirmed by whole genome sequencing.RESULTS: A total of 7/9 cases and 93/182 controls completed a voluntary survey about risk factors. Compared to controls, cases reported significantly more patient contact time. Cases were also significantly more likely to have performed airway procedures on the index patient, particularly placing the patient on high flow nasal cannula, continuous positive airway pressure (CPAP), or bilevel positive airway pressure (BiPAP) (OR=11.6; 95% CI=1.7-132.1).DISCUSSION: This study highlights the risk of nosocomial infection of SARS-CoV-2 from patients who become infectious midway into their hospitalization. Our findings also reinforce the importance of patient contact and aerosol-generating procedures as key risk factors for HCP infection with SARS-CoV-2.CONCLUSIONS: Re-testing patients for SARS-CoV-2 after admission in suspicious cases and using N95 masks for all aerosol-generating procedures regardless of initial patient SARS-CoV-2 test results can help reduce the risk of SARS-COV-2 transmission to HCP.

  View details for DOI 10.1016/j.ajic.2021.09.004

  View details for PubMedID 34536502

• Nasopharyngeal metabolomics and machine learning approach for the diagnosis of influenza. EBioMedicine Hogan, C. A., Rajpurkar, P., Sowrirajan, H., Phillips, N. A., Le, A. T., Wu, M., Garamani, N., Sahoo, M. K., Wood, M. L., Huang, C., Ng, A. Y., Mak, J., Cowan, T. M., Pinsky, B. A. 2021; 71: 103546

  Abstract

  BACKGROUND: Respiratory virus infections are significant causes of morbidity and mortality, and may induce host metabolite alterations by infecting respiratory epithelial cells. We investigated the use of liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF) combined with machine learning for the diagnosis of influenza infection.METHODS: We analyzed nasopharyngeal swab samples by LC/Q-TOF to identify distinct metabolic signatures for diagnosis of acute illness. Machine learning models were performed for classification, followed by Shapley additive explanation (SHAP) analysis to analyze feature importance and for biomarker discovery.FINDINGS: A total of 236 samples were tested in the discovery phase by LC/Q-TOF, including 118 positive samples (40 influenza A 2009 H1N1, 39 influenza H3 and 39 influenza B) as well as 118 age and sex-matched negative controls with acute respiratory illness. Analysis showed an area under the receiver operating characteristic curve (AUC) of 1.00 (95% confidence interval [95% CI] 0.99, 1.00), sensitivity of 1.00 (95% CI 0.86, 1.00) and specificity of 0.96 (95% CI 0.81, 0.99). The metabolite most strongly associated with differential classification was pyroglutamic acid. Independent validation of a biomarker signature based on the top 20 differentiating ion features was performed in a prospective cohort of 96 symptomatic individuals including 48 positive samples (24 influenza A 2009 H1N1, 5 influenza H3 and 19 influenza B) and 48 negative samples. Testing performed using a clinically-applicable targeted approach, liquid chromatography triple quadrupole mass spectrometry, showed an AUC of 1.00 (95% CI 0.998, 1.00), sensitivity of 0.94 (95% CI 0.83, 0.98), and specificity of 1.00 (95% CI 0.93, 1.00). Limitations include lack of sample suitability assessment, and need to validate these findings in additional patient populations.INTERPRETATION: This metabolomic approach has potential for diagnostic applications in infectious diseases testing, including other respiratory viruses, and may eventually be adapted for point-of-care testing.

  View details for DOI 10.1016/j.ebiom.2021.103546

  View details for PubMedID 34419924

• Diagnosis of Dengue in a returning traveler from Pakistan suspected of COVID-19, California, USA. Diagnostic microbiology and infectious disease Bulterys, P. L., Solis, D., Verghese, M., Huang, C., Sibai, M., Costales, C., Sahoo, M. K., Pinsky, B. A. 2021; 101 (4): 115517

  Abstract

  Dengue and COVID-19 cocirculation presents a diagnostic conundrum for physicians evaluating patients with acute febrile illnesses, both in endemic regions and among returning travelers. We present a case of a returning traveler from Pakistan who, following repeated negative SARS-CoV-2 tests, was found to have a Dengue virus serotype 2 infection.

  View details for DOI 10.1016/j.diagmicrobio.2021.115517

  View details for PubMedID 34537475

• The truth about SARS-CoV-2 cycle threshold values is rarely pure and never simple. Clinical chemistry Rhoads, D. D., Pinsky, B. A. 2021

  Abstract

  Nucleic acid amplification tests (NAATs) are the reference standard methods for SARS-CoV-2 detection because of their high analytical sensitivity and specificity. Many NAATs, which use reverse-transcription real-time PCR (RT-PCR), are clinically validated, technically validated, and authorized by the U.S. Food & Drug Administration (FDA) to be interpreted qualitatively as "detected" (positive for SARS-CoV-2 RNA) or "not detected" (negative for SARS-CoV-2 RNA). As of this writing there are over 250 SARS-CoV-2 molecular diagnostic tests that have obtained emergency use authorization from the FDA. The primary results generated by RT-PCR are fluorescent light emissions; serial detection of this fluorescence is plotted and the amplification curves visualized. Positive or negative interpretation depends on whether or not the curve exceeds a specified signal threshold. Part of the resulting process includes determination of the number of cycles needed before the fluorescent signal crosses this threshold (Ct value). In general, the more viral RNA in the initial specimen, the fewer the number of amplification cycles required to generate a positive signal; thus, the lower the Ct value, the higher the viral burden in the primary sample. Though all current SARS-CoV-2 NAATs are authorized only for qualitative interpretation, as of 10 December 2020, the FDA explicitly states that the Ct value results may be reported by the clinical laboratory in addition to the qualitative interpretation. Throughout the pandemic, many scientists, physicians, politicians, and public citizens have attempted to emphasize the importance (or unimportance) of certain pandemic-related interventions, mitigation strategies, the disease itself, and testing approaches. Some have advocated that a specific variable is most important in a testing approach and should be maximized to the potential detriment of the others: analytical sensitivity and specificity, cost, turnaround time, sample type, or accessibility of collection. If the truth was obvious, then there would be little debate, but the debate has been important and earnest. As Oscar Wilde wrote, "The truth is rarely pure and never simple." We suggest that this quote describes the current situation on the debate over the relevance of Ct values, and we will explore the clinical utility of quantitative SARS-CoV-2 testing here.

  View details for DOI 10.1093/clinchem/hvab146

  View details for PubMedID 34314495

• Effect of Oral Azithromycin vs Placebo on COVID-19 Symptoms in Outpatients With SARS-CoV-2 Infection: A Randomized Clinical Trial. JAMA Oldenburg, C. E., Pinsky, B. A., Brogdon, J., Chen, C., Ruder, K., Zhong, L., Nyatigo, F., Cook, C. A., Hinterwirth, A., Lebas, E., Redd, T., Porco, T. C., Lietman, T. M., Arnold, B. F., Doan, T. 2021

  Abstract

  Importance: Azithromycin has been hypothesized to have activity against SARS-CoV-2.Objective: To determine whether oral azithromycin in outpatients with SARS-CoV-2 infection leads to absence of self-reported COVID-19 symptoms at day 14.Design, Setting, and Participants: Randomized clinical trial of azithromycin vs matching placebo conducted from May 2020 through March 2021. Outpatients from the US were enrolled remotely via internet-based surveys and followed up for 21 days. Eligible participants had a positive SARS-CoV-2 diagnostic test result (nucleic acid amplification or antigen) within 7 days prior to enrollment, were aged 18 years or older, and were not hospitalized at the time of enrollment. Among 604 individuals screened, 297 were ineligible, 44 refused participation, and 263 were enrolled. Participants, investigators, and study staff were masked to treatment randomization.Interventions: Participants were randomized in a 2:1 fashion to a single oral 1.2-g dose of azithromycin (n=171) or matching placebo (n=92).Main Outcomes and Measures: The primary outcome was absence of self-reported COVID-19 symptoms at day 14. There were 23 secondary clinical end points, including all-cause hospitalization at day 21.Results: Among 263 participants who were randomized (median age, 43 years; 174 [66%] women; 57% non-Hispanic White and 29% Latinx/Hispanic), 76% completed the trial. The trial was terminated by the data and safety monitoring committee for futility after the interim analysis. At day 14, there was no significant difference in proportion of participants who were symptom free (azithromycin: 50%; placebo: 50%; prevalence difference, 0%; 95% CI, -14% to 15%; P>.99). Of 23 prespecified secondary clinical end points, 18 showed no significant difference. By day 21, 5 participants in the azithromycin group had been hospitalized compared with 0 in the placebo group (prevalence difference, 4%; 95% CI, -1% to 9%; P=.16).Conclusions and Relevance: Among outpatients with SARS-CoV-2 infection, treatment with a single dose of azithromycin compared with placebo did not result in greater likelihood of being symptom free at day 14. These findings do not support the routine use of azithromycin for outpatient SARS-CoV-2 infection.Trial Registration: ClinicalTrials.gov Identifier: NCT04332107.

  View details for DOI 10.1001/jama.2021.11517

  View details for PubMedID 34269813

• Post-vaccination SARS-CoV-2 infections and incidence of presumptive B.1.427/B.1.429 variant among healthcare personnel at a northern California academic medical center. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Jacobson, K. B., Pinsky, B. A., Montez Rath, M. E., Wang, H., Miller, J. A., Skhiri, M., Shepard, J., Mathew, R., Lee, G., Bohman, B., Parsonnet, J., Holubar, M. 2021

  Abstract

  BACKGROUND: Although mRNA-based SARS-CoV-2 vaccines report ≥90% efficacy, breakthrough infections occur. Little is known about the effectiveness of these vaccines against SARS-CoV-2 variants, including the highly-prevalent B.1.427/B.1.429 variant in California..METHODS: In this quality improvement project, we collected demographic and clinical information from post-vaccine SARS-CoV-2 cases (PVSCs), defined as health care personnel (HCP) with positive SARS-CoV-2 NAAT after receiving ≥1 vaccine dose. Available specimens were tested for L452R, N501Y and E484K mutations by RT-PCR. Mutation prevalence was compared among unvaccinated, early post-vaccinated (<=14 days after dose 1), partially vaccinated (positive test >14 days after dose 1 and ≤14 days after dose 2) and fully vaccinated (>14 days after dose 2) PVSCs.RESULTS: From December 2020-April 2021, >=23,090 HCPS received at least1 dose of an mRNA-based SARS-CoV-2 vaccine, and 660 HCP cases of SARS-CoV-2 occurred of which 189 were PVSCs. Among the PVSCs, 114 (60.3%), 49 (25.9%) and 26 (13.8%) were early post-vaccination, partially vaccinated, and fully vaccinated, respectively. Of 261 available samples from vaccinated and unvaccinated HCP, 103 (39.5%), including 42 PVSCs (36.5%), had L452R mutation presumed to be B.1.427/B.1.429,. When adjusted for community prevalence of B.1.427/B.1.429, PVSCs did not have significantly elevated risk for infection with B.1.427/B.1.429 compared with unvaccinated HCP.CONCLUSIONS: Most PVSCs occurred prior to expected onset of full, vaccine-derived immunity. Presumptive B.1.427/B.1.429 was not more prevalent in post-vaccine cases than in unvaccinated SARS-CoV-2 HCP. Continued infection control measures, particularly ≤14 days post-vaccination, and continued variant surveillance in PVSCs is imperative to control future SARS-CoV-2 surges.

  View details for DOI 10.1093/cid/ciab554

  View details for PubMedID 34137815

• Asthma phenotypes, associated comorbidities, and long-term symptoms in COVID-19. Allergy Eggert, L. E., He, Z., Collins, W., Lee, A. S., Dhondalay, G., Jiang, S. Y., Fitzpatrick, J., Snow, T. T., Pinsky, B. A., Artandi, M., Barman, L., Puri, R., Wittman, R., Ahuja, N., Blomkalns, A., O'Hara, R., Cao, S., Desai, M., Sindher, S. B., Nadeau, K., Chinthrajah, R. S. 2021

  Abstract

  BACKGROUND: It is unclear if asthma and its allergic phenotype are risk factors for hospitalization or severe disease from SARS-CoV-2.METHODS: All patients over 28 days oldtesting positive for SARS-CoV-2 between March 1 and September 30, 2020, were retrospectively identified and characterized through electronic analysis at Stanford. A sub-cohort was followed prospectively to evaluate long-term COVID-19 symptoms.RESULTS: 168,190 patients underwent SARS-CoV-2 testing, and 6,976 (4.15%) tested positive. In a multivariate analysis, asthma was not an independent risk factor for hospitalization (OR 1.12 [95% CI 0.86, 1.45], p=0.40). Among SARS-CoV-2 positive asthmatics, allergic asthma lowered the risk of hospitalization and had a protective effect compared to non-allergic asthma (OR 0.52 (0.28, 0.91), p=0.026); there was no association between baseline medication use as characterized by GINA and hospitalization risk. Patients with severe COVID-19 disease had lower eosinophil levels during hospitalization compared to patients with mild or asymptomatic disease, independent of asthma status (p=0.0014). In a patient sub-cohort followed longitudinally, asthmatics and non-asthmatics had similar time to resolution of COVID-19 symptoms, particularly lower respiratory symptoms.CONCLUSIONS: Asthma is not a risk factor for more severe COVID-19 disease. Allergic asthmatics were half as likely to be hospitalized with COVID-19 compared to non-allergic asthmatics. Lower levels of eosinophil counts (allergic biomarkers) were associated with a more severe COVID-19 disease trajectory. Recovery was similar among asthmatics and non-asthmatics with over 50% of patients reporting ongoing lower respiratory symptoms three months post-infection.

  View details for DOI 10.1111/all.14972

  View details for PubMedID 34080210

• Mapping of SARS-CoV-2 Brain Invasion in COVID-19 Disease Serrano, G., Walkera, J., Arce, R., Glass, M., Vargas, D., Sue, L., Intorcia, A., Nelson, C., Oliver, J., Papa, J., Russell, A., Suszczewicz, K., Borja, C., Sahoo, M., Zhang, H., Solis, D., Montine, T., Zehnder, J., Pinsky, B., Dickson, D., Deture, M., Beach, T. OXFORD UNIV PRESS INC. 2021: 585

  View details for Web of Science ID 000671021700115

• Brain Histopathology in Subjects with COVID-19 Disease Beach, T., Dickson, D., Deture, M., Walker, J., Arce, R., Glass, M., Vargas, D., Sue, L., Intorcia, A., Nelson, C., Oliver, J., Russell, A., Suszczewicz, K., Borja, C., Papa, J., Sahoo, M., Zhang, H., Solis, D., Montine, T., Zehnder, J., Pinsky, B., Serrano, G. OXFORD UNIV PRESS INC. 2021: 585

  View details for Web of Science ID 000671021700114

• Association of Premature Immune Aging and Cytomegalovirus After Solid Organ Transplant FRONTIERS IN IMMUNOLOGY Higdon, L. E., Gustafson, C. E., Ji, X., Sahoo, M. K., Pinsky, B. A., Margulies, K. B., Maecker, H. T., Goronzy, J., Maltzman, J. S. 2021; 12

  View details for DOI 10.3389/fimmu.2021.661551

  View details for Web of Science ID 000659286800001

• Association of Premature Immune Aging and Cytomegalovirus After Solid Organ Transplant. Frontiers in immunology Higdon, L. E., Gustafson, C. E., Ji, X., Sahoo, M. K., Pinsky, B. A., Margulies, K. B., Maecker, H. T., Goronzy, J., Maltzman, J. S. 2021; 12: 661551

  Abstract

  Immune function is altered with increasing age. Infection with cytomegalovirus (CMV) accelerates age-related immunological changes resulting in expanded oligoclonal memory CD8 T cell populations with impaired proliferation, signaling, and cytokine production. As a consequence, elderly CMV seropositive (CMV+) individuals have increased mortality and impaired responses to other infections in comparison to seronegative (CMV-) individuals of the same age. CMV is also a significant complication after organ transplantation, and recent studies have shown that CMV-associated expansion of memory T cells is accelerated after transplantation. Thus, we investigated whether immune aging is accelerated post-transplant, using a combination of telomere length, flow cytometry phenotyping, and single cell RNA sequencing. Telomere length decreased slightly in the first year after transplantation in a subset of both CMV+ and CMV- recipients with a strong concordance between CD57+ cells and short telomeres. Phenotypically aged cells increased post-transplant specifically in CMV+ recipients, and clonally expanded T cells were enriched for terminally differentiated cells post-transplant. Overall, these findings demonstrate a pattern of accelerated aging of the CD8 T cell compartment in CMV+ transplant recipients.

  View details for DOI 10.3389/fimmu.2021.661551

  View details for PubMedID 34122420

  View details for PubMedCentralID PMC8190404

• Multiplex SARS-CoV-2 Genotyping RT-PCR for Population-Level Variant Screening and Epidemiologic Surveillance. Journal of clinical microbiology Wang, H., Miller, J. A., Verghese, M., Sibai, M., Solis, D., Mfuh, K. O., Jiang, B., Iwai, N., Mar, M., Huang, C., Yamamoto, F., Sahoo, M. K., Zehnder, J., Pinsky, B. A. 2021: JCM0085921

  Abstract

  Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population. Between December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

  View details for DOI 10.1128/JCM.00859-21

  View details for PubMedID 34037430

• Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series. EBioMedicine Truong, T. T., Ryutov, A., Pandey, U., Yee, R., Goldberg, L., Bhojwani, D., Aguayo-Hiraldo, P., Pinsky, B. A., Pekosz, A., Shen, L., Boyd, S. D., Wirz, O. F., Roltgen, K., Bootwalla, M., Maglinte, D. T., Ostrow, D., Ruble, D., Han, J. H., Biegel, J. A., Li, M., Huang, C., Sahoo, M. K., Pannaraj, P. S., O'Gorman, M., Judkins, A. R., Gai, X., Dien Bard, J. 2021; 67: 103355

  Abstract

  BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

  View details for DOI 10.1016/j.ebiom.2021.103355

  View details for PubMedID 33915337

• Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies. Genome medicine Lau, B. T., Pavlichin, D., Hooker, A. C., Almeda, A., Shin, G., Chen, J., Sahoo, M. K., Huang, C. H., Pinsky, B. A., Lee, H. J., Ji, H. P. 2021; 13 (1): 62

  Abstract

  BACKGROUND: The genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contact tracing.METHODS: Leveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies variants, and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints.RESULTS: We built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and a combination of mutations that appear in only a small number of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of 100 SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome.CONCLUSIONS: We conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients and determined their general prevalence when compared to over 70,000 other strains. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

  View details for DOI 10.1186/s13073-021-00882-2

  View details for PubMedID 33875001

• Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. medRxiv : the preprint server for health sciences Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021

  Abstract

  COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

  View details for DOI 10.1101/2021.04.10.21255250

  View details for PubMedID 33880485

• Ultra-sensitive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antigen Detection for the Diagnosis of Coronavirus Disease 2019 (COVID-19) in Upper Respiratory Samples. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Wang, H., Hogan, C. A., Verghese, M., Solis, D., Sibai, M., Huang, C., Zehnder, J., Sahoo, M. K., Pinsky, B. A. 2021

  Abstract

  An ultra-sensitive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen assay (S-PLEX, MesoScale Diagnostics) was evaluated in 250 retrospective and 200 prospective upper respiratory specimens. In samples with cycle threshold <35, there was 95%-98% positive and 93%-96% negative percent agreement with reverse transcription-polymerase chain reaction. S-PLEX may provide a high-throughput alternative to nucleic acid-based testing for coronavirus disease 2019 (COVID-19) diagnosis.

  View details for DOI 10.1093/cid/ciab063

  View details for PubMedID 33830203

• mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition. medRxiv : the preprint server for health sciences Röltgen, K., Nielsen, S. C., Arunachalam, P. S., Yang, F., Hoh, R. A., Wirz, O. F., Lee, A. S., Gao, F., Mallajosyula, V., Li, C., Haraguchi, E., Shoura, M. J., Wilbur, J. L., Wohlstadter, J. N., Davis, M. M., Pinsky, B. A., Sigal, G. B., Pulendran, B., Nadeau, K. C., Boyd, S. D. 2021

  Abstract

  During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, new vaccine strategies including lipid nanoparticle delivery of antigen encoding RNA have been deployed globally. The BioNTech/Pfizer mRNA vaccine BNT162b2 encoding SARS-CoV-2 spike protein shows 95% efficacy in preventing disease, but it is unclear how the antibody responses to vaccination differ from those generated by infection. Here we compare the magnitude and breadth of antibodies targeting SARS-CoV-2, SARS-CoV-2 variants of concern, and endemic coronaviruses, in vaccinees and infected patients. We find that vaccination differs from infection in the dominance of IgG over IgM and IgA responses, with IgG reaching levels similar to those of severely ill COVID-19 patients and shows decreased breadth of the antibody response targeting endemic coronaviruses. Viral variants of concern from B.1.1.7 to P.1 to B.1.351 form a remarkably consistent hierarchy of progressively decreasing antibody recognition by both vaccinees and infected patients exposed to Wuhan-Hu-1 antigens.

  View details for DOI 10.1101/2021.04.05.21254952

  View details for PubMedID 33851181

  View details for PubMedCentralID PMC8043478

• SARS-CoV-2 IgG Seropositivity and Acute Asymptomatic Infection Rate Among Firefighter First Responders in an Early Outbreak County in California. Prehospital emergency care : official journal of the National Association of EMS Physicians and the National Association of State EMS Directors Newberry, J. A., Gautreau, M., Staats, K., Carrillo, E., Mulkerin, W., Yang, S., Kohn, M. A., Matheson, L., Boyd, S. D., Pinsky, B. A., Blomkalns, A. L., Strehlow, M. C., D'Souza, P. A. 2021: 1–10

  Abstract

  Objective: Firefighter first responders and other emergency medical services (EMS) personnel have been among the highest risk healthcare workers for illness during the SARS-CoV-2 pandemic. We sought to determine the rate of seropositivity for SARS-CoV-2 IgG antibodies and of acute asymptomatic infection among firefighter first responders in a single county with early exposure in the pandemic.Methods: We conducted a cross-sectional study of clinically active firefighters cross-trained as paramedics or EMTs in the fire departments of Santa Clara County, California. Firefighters without current symptoms were tested between June and August 2020. Our primary outcomes were rates of SARS-CoV-2 IgG antibody seropositivity and SARS-CoV-2 RT-PCR swab positivity for acute infection. We report cumulative incidence, participant characteristics with frequencies and proportions, and proportion positive and associated relative risk (with 95% confidence intervals).Results: We enrolled 983 out of 1339 eligible participants (response rate: 73.4%). Twenty-five participants (2.54%, 95% CI 1.65-3.73) tested positive for IgG antibodies and 9 (0.92%, 95% CI 0.42-1.73) tested positive for SARS-CoV-2 by RT-PCR. Our cumulative incidence, inclusive of self-reported prior positive PCR tests, was 34 (3.46%, 95% CI 2.41-4.80).Conclusion: In a county with one of the earliest outbreaks in the United States, the seroprevalence among firefighter first responders was lower than that reported by other studies of frontline health care workers, while the cumulative incidence remained higher than that seen in the surrounding community.

  View details for DOI 10.1080/10903127.2021.1912227

  View details for PubMedID 33819128

• Comprehensive investigation of sources of misclassification errors in routine HIV testing in Zimbabwe. Journal of the International AIDS Society Gregson, S., Moorhouse, L., Dadirai, T., Sheppard, H., Mayini, J., Beckmann, N., Skovdal, M., Dzangare, J., Moyo, B., Maswera, R., Pinsky, B. A., Mharakurwa, S., Francis, I., Mugurungi, O., Nyamukapa, C. 2021; 24 (4): e25700

  Abstract

  INTRODUCTION: Misclassification errors have been reported in rapid diagnostic HIV tests (RDTs) in sub-Saharan African countries. These errors can lead to missed opportunities for prevention-of-mother-to-child-transmission (PMTCT), early infant diagnosis and adult HIV-prevention, unnecessary lifelong antiretroviral treatment (ART) and wasted resources. Few national estimates or systematic quantifications of sources of errors have been produced. We conducted a comprehensive assessment of possible sources of misclassification errors in routine HIV testing in Zimbabwe.METHODS: RDT-based HIV test results were extracted from routine PMTCT programme records at 62 sites during national antenatal HIV surveillance in 2017. Positive- (PPA) and negative-percent agreement (NPA) for HIV RDT results and the false-HIV-positivity rate for people with previous HIV-positive results ("known-positives") were calculated using results from external quality assurance testing done for HIV surveillance purposes. Data on indicators of quality management systems, RDT kit performance under local climatic conditions and user/clerical errors were collected using HIV surveillance forms, data-loggers and a Smartphone camera application (7 sites). Proportions of cases with errors were compared for tests done in the presence/absence of potential sources of errors.RESULTS: NPA was 99.9% for both pregnant women (N=17224) and male partners (N=2173). PPA was 90.0% (N=1187) and 93.4% (N=136) for women and men respectively. 3.5% (N=1921) of known-positive individuals on ART were HIV negative. Humidity and temperature exceeding manufacturers' recommendations, particularly in storerooms (88.6% and 97.3% respectively), and premature readings of RDT output (56.0%) were common. False-HIV-negative cases, including interpretation errors, occurred despite staff training and good algorithm compliance, and were not reduced by existing external or internal quality assurance procedures. PPA was lower when testing room humidity exceeded 60% (88.0% vs. 93.3%; p=0.007).CONCLUSIONS: False-HIV-negative results were still common in Zimbabwe in 2017 and could be reduced with HIV testing algorithms that use RDTs with higher sensitivity under real-world conditions and greater practicality under busy clinic conditions, and by strengthening proficiency testing procedures in external quality assurance systems. New false-HIV-positive RDT results were infrequent but earlier errors in testing may have resulted in large numbers of uninfected individuals being on ART.

  View details for DOI 10.1002/jia2.25700

  View details for PubMedID 33882190

• Comparison of Anti-Dengue and Anti-Zika IgG on a Plasmonic Gold Platform with Neutralization Testing. The American journal of tropical medicine and hygiene Rojas, A., Natrajan, M. S., Weber, J., Cardozo, F., Cantero, C., Ananta, J. S., Kost, J., Tang, M., Lopez, S., Bernal, C., Guillen, Y., Mendoza, L., Paez, M., Pinsky, B. A., Waggoner, J. J. 2021

  Abstract

  Antibody cross-reactivity confounds testing for dengue virus (DENV) and Zika virus (ZIKV). We evaluated anti-DENV and anti-ZIKV IgG detection using a multiplex serological platform (the pGOLD assay) in patients from the Asuncion metropolitan area in Paraguay, which experiences annual DENV outbreaks but has reported few autochthonous ZIKV infections. Acute-phase sera were tested from 77 patients who presented with a suspected arboviral illness from January to May 2018. Samples were tested for DENV and ZIKV RNA by real-time reverse transcription-PCR, and for DENV nonstructural protein 1 with a lateral-flow immunochromatographic test. Forty-one patients (51.2%) had acute dengue; no acute ZIKV infections were detected. Sixty-five patients (84.4%) had anti-DENV-neutralizing antibodies by focus reduction neutralization testing (FRNT50). Qualitative detection with the pGOLD assay demonstrated good agreement with FRNT50 (kappa = 0.74), and quantitative results were highly correlated between methods (P < 0.001). Only three patients had anti-ZIKV-neutralizing antibodies at titers of 1:55-1:80, and all three had corresponding DENV-neutralizing titers > 1:4,000. Hospitalized dengue cases had significantly higher anti-DENV IgG levels (P < 0.001). Anti-DENV IgG results from the pGOLD assay correlate well with FRNT, and quantitative results may inform patient risk stratification.

  View details for DOI 10.4269/ajtmh.20-1449

  View details for PubMedID 33782214

• Comprehensive pathogen detection for ocular infections JOURNAL OF CLINICAL VIROLOGY Doan, T., Sahoo, M. K., Ruder, K., Huang, C., Zhong, L., Chen, C., Hinterwirth, A., Lin, C., Gonzales, J. A., Pinsky, B. A., Acharya, N. R. 2021; 136

  View details for Web of Science ID 000633136500007

• Comprehensive pathogen detection for ocular infections JOURNAL OF CLINICAL VIROLOGY Doan, T., Sahoo, M. K., Ruder, K., Huang, C., Zhong, L., Chen, C., Hinterwirth, A., Lin, C., Gonzales, J. A., Pinsky, B. A., Acharya, N. R. 2021; 136

  View details for Web of Science ID 000633136500010

• No Evidence of O'nyong-Nyong Viremia among Children with Febrile Illness in Kenya (2015-2018). The American journal of tropical medicine and hygiene Shah, M. M., Ndenga, B. A., Mutuku, F. M., Okuta, V., Ronga, C. O., Chebii, P. K., Maina, P., Jembe, Z., Sahoo, M. K., Huang, C., Weber, J., Pinsky, B. A., LaBeaud, A. D. 2021

  Abstract

  O'nyong-nyong virus (ONNV) is a little-known arbovirus causing intermittent, yet explosive, outbreaks in Africa. It is closely related to chikungunya virus, an emerging infectious disease. O'nyong-nyong virus causes a self-limited illness characterized by bilateral polyarthritis, rash, low-grade fever, and lymphadenopathy. In 1959, an extensive outbreak of ONNV occurred in East Africa, and decades later, another large outbreak was documented in Uganda in 1996. Limited evidence for interepidemic transmission is available, although serologic studies indicate a high prevalence of exposure; 1,045 febrile child participants in western and coastal Kenya were tested for the presence of ONNV using a multiplexed real-time reverse transcriptase-PCR assay. More than half of the participants had malaria parasitemia, and there was no evidence of active ONNV viremia in these participants. Further work is required to better understand the interepidemic circulation of ONNV and to reconcile evidence of high serologic exposure to ONNV among individuals in East Africa.

  View details for DOI 10.4269/ajtmh.20-0580

  View details for PubMedID 33617476

• Mapping of SARS-CoV-2 Brain Invasion and Histopathology in COVID-19 Disease. medRxiv : the preprint server for health sciences Serrano, G. E., Walker, J. E., Arce, R., Glass, M. J., Vargas, D., Sue, L. I., Intorcia, A. J., Nelson, C. M., Oliver, J., Papa, J., Russell, A., Suszczewicz, K. E., Borja, C. I., Belden, C., Goldfarb, D., Shprecher, D., Atri, A., Adler, C. H., Shill, H. A., Driver-Dunckley, E., Mehta, S. H., Readhead, B., Huentelman, M. J., Peters, J. L., Alevritis, E., Bimi, C., Mizgerd, J. P., Reiman, E. M., Montine, T. J., Desforges, M., Zehnder, J. L., Sahoo, M. K., Zhang, H., Solis, D., Pinsky, B. A., Deture, M., Dickson, D. W., Beach, T. G. 2021

  Abstract

  The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal β-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.

  View details for DOI 10.1101/2021.02.15.21251511

  View details for PubMedID 33619496

  View details for PubMedCentralID PMC7899461

• Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19. bioRxiv : the preprint server for biology Feyaerts, D., Hédou, J., Gillard, J., Chen, H., Tsai, E. S., Peterson, L. S., Ando, K., Manohar, M., Do, E., Dhondalay, G. K., Fitzpatrick, J., Artandi, M., Chang, I., Snow, T. T., Chinthrajah, R. S., Warren, C. M., Wittman, R., Meyerowitz, J. G., Ganio, E. A., Stelzer, I. A., Han, X., Verdonk, F., Gaudillière, D. K., Mukherjee, N., Tsai, A. S., Rumer, K. K., Jiang, S., Valdés Ferrer, S. I., Kelly, J. D., Furman, D., Aghaeepour, N., Angst, M. S., Boyd, S. D., Pinsky, B. A., Nolan, G. P., Nadeau, K. C., Gaudillière, B., McIlwain, D. R. 2021

  Abstract

  The biological determinants of the wide spectrum of COVID-19 clinical manifestations are not fully understood. Here, over 1400 plasma proteins and 2600 single-cell immune features comprising cell phenotype, basal signaling activity, and signaling responses to inflammatory ligands were assessed in peripheral blood from patients with mild, moderate, and severe COVID-19, at the time of diagnosis. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identified and independently validated a multivariate model classifying COVID-19 severity (multi-class AUCtraining = 0.799, p-value = 4.2e-6; multi-class AUCvalidation = 0.773, p-value = 7.7e-6). Features of this high-dimensional model recapitulated recent COVID-19 related observations of immune perturbations, and revealed novel biological signatures of severity, including the mobilization of elements of the renin-angiotensin system and primary hemostasis, as well as dysregulation of JAK/STAT, MAPK/mTOR, and NF-κB immune signaling networks. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for the prevention of COVID-19 progression.

  View details for DOI 10.1101/2021.02.09.430269

  View details for PubMedID 33594362

  View details for PubMedCentralID PMC7885914

• Evaluation of a measles virus multiplex, triple-target real-time RT-PCR in three specimen matrices at a U.S. academic medical center. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Broadhurst, M. J., Garamani, N., Hahn, Z., Jiang, B., Weber, J., Huang, C., Sahoo, M. K., Kurzer, J., Hogan, C. A., Pinsky, B. A. 2021; 136: 104757

  Abstract

  BACKGROUND: Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available.METHODS: We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes.RESULTS: The MeV triple-target rRT-PCR was tested against serial dilutions (7.0-2.0 log10 copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log10 copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus.CONCLUSIONS: The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions.

  View details for DOI 10.1016/j.jcv.2021.104757

  View details for PubMedID 33639409

• Cutaneous cytomegalovirus - A case of disseminated cytomegalovirus presenting with extensive ulcerative skin lesions in a renal transplant recipient. Transplant infectious disease : an official journal of the Transplantation Society Ferguson, J., Mooney, K., Saleem, A., Stevens, B. A., Pinsky, B. A., Centkowski, S., Zaba, L. C., Ho, D. Y. 2021

  Abstract

  Cytomegalovirus (CMV) reactivation is common in organ transplant recipients and can lead to significant morbidity and mortality. Cutaneous CMV findings are rarely reported in the literature and diagnosis can be delayed if not clinically recognized. We describe a case of a female patient 20 years post renal transplant who presented with extensive ulcerative skin lesions and diarrhea. She rapidly deteriorated and died on day 5 of hospitalization. Autopsy noted extensive CMV involvement of skin and gastrointestinal (GI) tract by CMV-specific immunohistochemistry.

  View details for DOI 10.1111/tid.13582

  View details for PubMedID 33533137

• Strand Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2 EMERGING INFECTIOUS DISEASES Hogan, C. A., Huang, C., Sahoo, M. K., Wang, H., Jiang, B., Sibai, M., Holubar, M., Mathew, R., Zehnder, J., Pinsky, B. A. 2021; 27 (2): 632–35

  Abstract

  We developed an assay that detects minus-strand RNA as a surrogate for actively replicating severe acute respiratory syndrome coronavirus 2. We detected minus-strand RNA in 41 persons with coronavirus disease up to 30 days after symptom onset. This assay might inform clinical decision-making about patient infectiousness.

  View details for DOI 10.3201/eid2702.204168

  View details for Web of Science ID 000631538500043

  View details for PubMedID 33496233

  View details for PubMedCentralID PMC7853532

• Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Long, D. R., Gombar, S., Hogan, C. A., Greninger, A. L., O'Reilly-Shah, V., Bryson-Cahn, C., Stevens, B., Rustagi, A., Jerome, K. R., Kong, C. S., Zehnder, J., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. E. 2021; 72 (2): 323-326

  Abstract

  Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

  View details for DOI 10.1093/cid/ciaa722

  View details for PubMedID 33501950

• Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results. Clinical chemistry Wang, H. n., Wiredja, D. n., Yang, L. n., Bulterys, P. L., Costales, C. n., Röltgen, K. n., Manalac, J. n., Yee, J. n., Zehnder, J. n., Shi, R. Z., Boyd, S. D., Pinsky, B. A. 2021

  Abstract

  Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results.Over a two-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis.There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] versus 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] versus 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6.Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

  View details for DOI 10.1093/clinchem/hvab045

  View details for PubMedID 33720347

• Mutations in JAK/STAT and NOTCH1 Genes Are Enriched in Post-Transplant Lymphoproliferative Disorders. Frontiers in oncology Butzmann, A., Sridhar, K., Jangam, D., Song, H., Singh, A., Kumar, J., Chisholm, K. M., Pinsky, B., Huang, F., Ohgami, R. S. 1800; 11: 790481

  Abstract

  Post-transplant lymphoproliferative disorders (PTLD) are diseases occurring in immunocompromised patients after hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). Although PTLD occurs rarely, it may be associated with poor outcomes. In most cases, PTLD is driven by Epstein-Barr virus (EBV) infection. Few studies have investigated the mutational landscape and gene expression profile of PTLD. In our study, we performed targeted deep sequencing and RNA-sequencing (RNA-Seq) on 16 cases of florid follicular hyperplasia (FFH) type PTLD and 15 cases of other PTLD types that include: ten monomorphic (M-PTLD), three polymorphic (P-PTLD), and two classic Hodgkin lymphoma type PTLDs (CHL-PTLD). Our study identified recurrent mutations in JAK3 in five of 15 PTLD cases and one of 16 FFH-PTLD cases, as well as 16 other genes that were mutated in M-PTLD, P-PTLD, CHL-PTLD and FFH-PTLD. Digital image analysis demonstrated significant differences in single cell area, major axis, and diameter when comparing cases of M-PTLD and P-PTLD to FFH-PTLD. No morphometric relationship was identified with regards to a specific genetic mutation. Our findings suggest that immune regulatory pathways play an essential role in PTLD, with the JAK/STAT pathway affected in many PTLDs.

  View details for DOI 10.3389/fonc.2021.790481

  View details for PubMedID 35111674

• Comprehensive pathogen detection for ocular infections. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Doan, T. n., Sahoo, M. K., Ruder, K. n., Huang, C. n., Zhong, L. n., Chen, C. n., Hinterwirth, A. n., Lin, C. n., Gonzales, J. A., Pinsky, B. A., Acharya, N. R. 2021; 136: 104759

  Abstract

  Molecular diagnostics such as pathogen-directed PCRs have transformed testing for ocular infections since the late 1990s. Although these assays remain important diagnostic tools for samples with low biomass, the lack of diagnostic range motivates alternative molecular approaches for ocular infections. The aim of this study was to determine the performance of a high-throughput RNA sequencing approach, RNA-seq, to detect infectious agents in ocular samples from patients with presumed ocular infections.We compared the performance of RNA-seq to pathogen-directed PCRs using remnant nucleic acids from 41 aqueous or vitreous samples of patients with presumed ocular infections. Pathogen-directed PCRs were performed at the CLIA-certified Stanford Clinical Virology Laboratory. RNA-seq was performed in a masked manner at the Proctor Foundation at the University of California San Francisco. Percent positive and negative agreement between the two testing approaches were calculated. Discordant results were subjected to orthogonal testing.The positive percent agreement between RNA-seq and pathogen-directed PCRs was 100% (95% confidence interval (CI): 78.5%-100%). The negative percent agreement was 92.6% (95% CI: 76.6%-97.9%). RNA-seq identified pathogens not on the differential diagnosis for 9.7% (4/41) of the samples. Two pathogens solely identified with RNA-seq were confirmed with orthogonal testing.RNA-seq can accurately identify common and rare pathogens in aqueous and vitreous samples of patients with presumed ocular infections. Such an unbiased approach to testing has the potential to improve diagnostics although practical clinical utility warrants additional studies.

  View details for DOI 10.1016/j.jcv.2021.104759

  View details for PubMedID 33609933

• Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion. Frontiers in immunology Wirz, O. F., Roltgen, K., Stevens, B. A., Pandey, S., Sahoo, M. K., Tolentino, L., Verghese, M., Nguyen, K., Hunter, M., Snow, T. T., Singh, A. R., Blish, C. A., Cochran, J. R., Zehnder, J. L., Nadeau, K. C., Pinsky, B. A., Pham, T. D., Boyd, S. D. 2021; 12: 739037

  Abstract

  Background: Transfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings. We sought to identify factors correlating to high antibody titers in convalescent plasma donors and understand the magnitude and pharmacokinetic time course of both transfused antibody titers and the endogenous antibody titers in transfused recipients.Methods: Plasma samples were collected up to 174 days after convalescence from 93 CCP donors with mild disease, and from 16 COVID-19 patients before and after transfusion. Using ELISA, anti-SARS-CoV-2 Spike RBD, S1, and N-protein antibodies, as well as capacity of antibodies to block ACE2 from binding to RBD was measured in an in vitro assay. As an estimate for viral load, viral RNA and N-protein plasma levels were assessed in COVID-19 patients.Results: Anti-SARS-CoV-2 antibody levels and RBD-ACE2 blocking capacity were highest within the first 60 days after symptom resolution and markedly decreased after 120 days. Highest antibody titers were found in CCP donors that experienced fever. Effect of transfused CCP was detectable in COVID-19 patients who received high-titer CCP and had not seroconverted at the time of transfusion. Decrease in viral RNA was seen in two of these patients.Conclusion: Our results suggest that high titer CCP should be collected within 60 days after recovery from donors with past fever. The much lower titers conferred by transfused antibodies compared to endogenous production in the patient underscore the importance of providing CCP prior to endogenous seroconversion.

  View details for DOI 10.3389/fimmu.2021.739037

  View details for PubMedID 34594341

• Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. Nature communications Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021; 12 (1): 5753

  Abstract

  Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

  View details for DOI 10.1038/s41467-021-25576-6

  View details for PubMedID 34599164

• SARS-CoV-2 Neutralization Resistance Mutations in Patient with HIV/AIDS, California, USA. Emerging infectious diseases Hoffman, S. A., Costales, C., Sahoo, M. K., Palanisamy, S., Yamamoto, F., Huang, C., Verghese, M., Solis, D. C., Sibai, M., Subramanian, A., Tompkins, L. S., Grant, P., Shafer, R. W., Pinsky, B. A. 2021; 27 (10)

  Abstract

  We report persistent severe acute respiratory syndrome coronavirus 2 infection in a patient with HIV/AIDS; the virus developed spike N terminal domain and receptor binding domain neutralization resistance mutations. Our findings suggest that immunocompromised patients can harbor emerging variants of severe acute respiratory syndrome coronavirus 2.

  View details for DOI 10.3201/eid2710.211461

  View details for PubMedID 34296992

• Publisher Correction: Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA. Nature communications Natarajan, A., Han, A., Zlitni, S., Brooks, E. F., Vance, S. E., Wolfe, M., Singh, U., Jagannathan, P., Pinsky, B. A., Boehm, A., Bhatt, A. S. 2021; 12 (1): 7100

  View details for DOI 10.1038/s41467-021-27392-4

  View details for PubMedID 34853336

• Performance evaluation and optimized reporting workflow for HIV diagnostic screening and confirmatory tests in a low prevalence setting. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Wiredja, D., Ritchie, T. A., Tam, G., Hogan, C. A., Pinsky, B., Shi, R. Z. 2021; 145: 105020

  Abstract

  Our institution utilizes an antigen/antibody screening test followed by a confirmatory antibody assay for preliminary positive results. Given the low prevalence for HIV infections in our institution's county, we suspect that a substantial portion of the reactive screens are false positives.We aimed to characterize the false positivity rate of the HIV screening test performed at Stanford Health Care. In parallel, we modified our reporting workflow to release both the screening and confirmatory results simultaneously to mitigate the stress of a presumptive positive test.We reviewed 45,296 eligible HIV screen specimens that underwent the Abbott ARCHITECT™ 4th generation HIV antigen/antibody combination assay between August 5, 2016 and March 16, 2021. Final sample signal/cutoff (S/CO) ratios ≥ 1 were deemed positive, which triggers a reflex order for the confirmatory Bio-Rad Geenius™ HIV 1/2 Supplemental Assay. Additional chart review was performed for positive screen cases with negative or indeterminate confirmatory results.Our institution demonstrated a 0.28% (128/45,296) positive screen rate, with 12.5% (16/128) of these samples confirmed as false positives based on a negative HIV-1 RNA test. Median S/CO ratios of true positive screens were significantly higher than those with negative or indeterminate confirmatory tests (602.27vs 2.98; p = 0.0000323). We implemented a new synchronized reporting system for positive screens, which co-releases screen and confirmatory reports without compromise in the overall turnaround time.Our study demonstrates a relatively high percentage of false positive screens. Subsequently, by providing a more complete picture up front, our new reporting pipeline may reduce anxiety of a stand-alone positive screen and optimize downstream clinical decision-making.

  View details for DOI 10.1016/j.jcv.2021.105020

  View details for PubMedID 34736075

• Asthma phenotypes, associated comorbidities, and long-term symptoms in COVID-19 European Journal of Allergy and Clinical Immunology Eggert, L. E., He, Z., Collins, W., Lee, A. S., Nadeau, K., Chinthrajah, R. 2021

  View details for DOI 10.1111/all.14972

• Post-vaccination SARS-CoV-2 infections and incidence of the B.1.427/B.1.429 variant among healthcare personnel at a northern California academic medical center. medRxiv : the preprint server for health sciences Jacobson, K. B., Pinsky, B. A., Rath, M. E., Wang, H., Miller, J. A., Skhiri, M., Shepard, J., Mathew, R., Lee, G., Bohman, B., Parsonnet, J., Holubar, M. 2021

  Abstract

  Distribution of mRNA-based SARS-CoV-2 vaccines to healthcare personnel (HCP) in the United States began in December 2020, with efficacy > 90%. However, breakthrough infections in fully vaccinated individuals have been reported. Meanwhile, multiple SARS-CoV-2 variants of concern have emerged worldwide, including the B.1.427/B.1.429 variant first described in California. Little is known about the real-world effectiveness of the mRNA-based SARS-CoV-2 vaccines against novel variants including B.1.427/B.1.429.In this quality improvement project, post-vaccine SARS-CoV-2 cases (PVSCs) were defined as individuals with positive SARS-CoV-2 nucleic acid amplification test (NAAT) after receiving at least one dose of a SARS-CoV-2 vaccine. Chart extraction of demographic and clinical information was performed, and available specimens meeting cycle threshold value criteria were tested for L452R, N501Y and E484K mutations by RT-PCR.From December 2020 to March 2021, 189 PVSCs were identified out of 22,729 healthcare personnel who received at least one dose of an mRNA-based SARS-CoV-2 vaccine. Of these, 114 (60.3%) occurred within 14 days of first vaccine dose (early post-vaccination), 49 (25.9%) within 14 days of the second vaccine dose (partially vaccinated), and 26 (13.8%) > 14 days after the second dose (fully vaccinated). Of 115 samples available for mutation testing, 42 were positive for L452R alone, presumptive of B.1.427/B.1.429; three had N501Y mutation alone and none were found with E484K mutation. Though on univariate analysis partially- and fully-vaccinated PVSCs were more likely than early post-vaccination PVSCs to be infected with presumptive B.1.427/B.1.429, when adjusted for community prevalence of B.1.427/B.1.429 at the time of infection, partially- and fully-vaccinated PVSC did not have statistically significantly elevated risk ratios for infection with this variant (RR 1.40, 95% CI 0.81-2.43 and RR 1.13, 95% CI 0.59-2.16, respectively).The great majority of PVSCs occurred prior to the expected onset of full, vaccine-derived immunity. Although the B.1.427/B.1.429 variant did not represent a significantly higher proportion of PVSCs than expected, numbers were small and there was a trend towards higher representation in the partially- and fully-vaccinated subset. Continued infection control measures in the workplace and in the community including social distancing and masking, particularly in the early days post-vaccination, as well as continued variant surveillance in PVSCs, is imperative in order to anticipate and control future surges of infection.

  View details for DOI 10.1101/2021.04.14.21255431

  View details for PubMedID 33907767

  View details for PubMedCentralID PMC8077590

• Combined SARS-CoV-2 nucleic acid amplification testing and respiratory virus panel RT-PCR on the Hologic Panther Fusion system. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Stevens, B. A., Hogan, C. A., Mfuh, K. O., Khan, G. n., Sahoo, M. K., Huang, C. n., Garamani, N. n., Zehnder, J. n., Kurzer, J. n., Pinsky, B. A. 2021; 138: 104792

  Abstract

  Significant overlap exists between the symptoms of SARS-CoV-2 and other respiratory viruses. This poses a serious challenge to clinical diagnosis, laboratory testing, and infection control programs.To evaluate the performance of the Hologic Panther Fusion Respiratory Assays (RA) compared to the GenMark ePlex Respiratory Pathogen Panel (RPP) and to assess the ability of the Panther Fusion to perform parallel testing of SARS-CoV-2 and other respiratory viruses from a single sample.A diagnostic comparison study was carried out using 375 clinical nasopharyngeal specimens. Assay performance was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.Overall agreement between the Fusion RA and ePlex RPP was 97.3 % (95 % CI 96.3-98.0), positive percent agreement was 97.2 % (95 % CI 93.0-99.2), negative percent agreement was 97.3 % (95 % CI 96.3-98.0), and the kappa coefficient was 0.85 (95 % CI 0.81-0.89). Forty additional viruses in 30 specimens were detected by Fusion that were not detected by ePlex. The maximum specimen throughput for parallel testing of the Fusion Respiratory Assays with SARS-CoV-2 was 275 samples in 20.7 h for Fusion SARS-CoV-2 and 350 samples in 20.0 h for Aptima Transcription Mediated Amplification SARS-CoV-2.Fusion RA demonstrated substantial agreement compared to the ePlex RPP. However, the Fusion detected respiratory viruses not identified by ePlex, consistent with higher clinical sensitivity. Workflows for parallel testing of respiratory pathogens and SARS-CoV-2 demonstrate that the Panther Fusion instrument provides a flexible, moderate to high throughput testing option for pandemic and seasonal respiratory viruses.

  View details for DOI 10.1016/j.jcv.2021.104792

  View details for PubMedID 33770659

• Peginterferon Lambda-1a for treatment of outpatients with uncomplicated COVID-19: a randomized placebo-controlled trial. Nature communications Jagannathan, P. n., Andrews, J. R., Bonilla, H. n., Hedlin, H. n., Jacobson, K. B., Balasubramanian, V. n., Purington, N. n., Kamble, S. n., de Vries, C. R., Quintero, O. n., Feng, K. n., Ley, C. n., Winslow, D. n., Newberry, J. n., Edwards, K. n., Hislop, C. n., Choong, I. n., Maldonado, Y. n., Glenn, J. n., Bhatt, A. n., Blish, C. n., Wang, T. n., Khosla, C. n., Pinsky, B. A., Desai, M. n., Parsonnet, J. n., Singh, U. n. 2021; 12 (1): 1967

  Abstract

  Type III interferons have been touted as promising therapeutics in outpatients with coronavirus disease 2019 (COVID-19). We conducted a randomized, single-blind, placebo-controlled trial (NCT04331899) in 120 outpatients with mild to moderate COVID-19 to determine whether a single, 180 mcg subcutaneous dose of Peginterferon Lambda-1a (Lambda) within 72 hours of diagnosis could shorten the duration of viral shedding (primary endpoint) or symptoms (secondary endpoint). In both the 60 patients receiving Lambda and 60 receiving placebo, the median time to cessation of viral shedding was 7 days (hazard ratio [HR] = 0.81; 95% confidence interval [CI] 0.56 to 1.19). Symptoms resolved in 8 and 9 days in Lambda and placebo, respectively, and symptom duration did not differ significantly between groups (HR 0.94; 95% CI 0.64 to 1.39). Both Lambda and placebo were well-tolerated, though liver transaminase elevations were more common in the Lambda vs. placebo arm (15/60 vs 5/60; p = 0.027). In this study, a single dose of subcutaneous Peginterferon Lambda-1a neither shortened the duration of SARS-CoV-2 viral shedding nor improved symptoms in outpatients with uncomplicated COVID-19.

  View details for DOI 10.1038/s41467-021-22177-1

  View details for PubMedID 33785743

• Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity. medRxiv : the preprint server for health sciences Truong, T. T., Ryutov, A. n., Pandey, U. n., Yee, R. n., Goldberg, L. n., Bhojwani, D. n., Aguayo-Hiraldo, P. n., Pinsky, B. A., Pekosz, A. n., Shen, L. n., Boyd, S. D., Wirz, O. F., Röltgen, K. n., Bootwalla, M. n., Maglinte, D. T., Ostrow, D. n., Ruble, D. n., Han, J. H., Biegel, J. A., Li, M. n., Huang, C. n., Sahoo, M. K., Pannaraj, P. S., O'Gorman, M. n., Judkins, A. R., Gai, X. n., Bard, J. D. 2021

  Abstract

  There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.The work was partially funded by The Saban Research Institute at Children's Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

  View details for DOI 10.1101/2021.02.27.21252099

  View details for PubMedID 33688673

  View details for PubMedCentralID PMC7941650

• Plasma as an alternative COVID-19 diagnostic specimen in a hospitalized patient negative for SARS-CoV-2 by nasopharyngeal swab. Diagnostic microbiology and infectious disease Lawrence, L. n., Stevens, B. A., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Röltgen, K. n., Wirz, O. n., Zehnder, J. n., Shi, R. Z., Boyd, S. D., Schoolnik, G. n., Pinsky, B. A., Hogan, C. A. 2021; 100 (3): 115365

  Abstract

  We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

  View details for DOI 10.1016/j.diagmicrobio.2021.115365

  View details for PubMedID 33865070

• Evaluation of SARS-CoV-2 total antibody detection via a lateral flow nanoparticle fluorescence immunoassay. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Sibai, M. n., Solis, D. n., Röltgen, K. n., Stevens, B. A., Mfuh, K. O., Sahoo, M. K., Shi, R. Z., Zehnder, J. n., Boyd, S. D., Pinsky, B. A. 2021; 139: 104818

  Abstract

  The coronavirus disease 2019 (COVID-19) endgame may benefit from simple, accurate antibody testing to characterize seroprevalence and immunization coverage.To evaluate the performance of the lateral flow QIAreach anti-SARS-CoV-2 Total rapid nanoparticle fluorescence immunoassay compared to reference isotype-specific IgG, IgM, and IgA SARS-CoV-2 ELISA using S1 or receptor binding domain (RBD) as antigens.A diagnostic comparison study was carried out using 154 well-characterized heparin plasma samples. Agreement between assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.Overall agreement between the QIAreach anti-SARS-CoV-2 Total and any anti-spike domain (S1 or RBD) antibody isotype was 96.0 % (95 % CI 89.8-98.8), the positive percent agreement was 97.6 % (95 % CI 91.0-99.9), the negative percent agreement was 88.2 % (95 % CI 64.4-98.0). The kappa coefficient was 0.86 (95 % CI 0.72 to 0.99).The QIAreach anti-SARS-CoV-2 Total rapid antibody test provides comparable performance to high-complexity, laboratory-based ELISA.

  View details for DOI 10.1016/j.jcv.2021.104818

  View details for PubMedID 33932848

• Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Long, D. R., Gombar, S. n., Hogan, C. A., Greninger, A. L., O'Reilly-Shah, V. n., Bryson-Cahn, C. n., Stevens, B. n., Rustagi, A. n., Jerome, K. R., Kong, C. S., Zehnder, J. n., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. E. 2021; 72 (2): 323–26

  Abstract

  Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

  View details for DOI 10.1093/cid/ciaa722

  View details for PubMedID 33543250

• Performance of Nucleic Acid Amplification Tests for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Prospectively Pooled Specimens EMERGING INFECTIOUS DISEASES Wang, H., Hogan, C. A., Miller, J. A., Sahoo, M. K., Huang, C., Mfuh, K. O., Sibai, M., Zehnder, J., Hickey, B., Sinnott-Armstrong, N., Pinsky, B. A. 2021; 27 (1): 92–103

  Abstract

  Pooled nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2 could increase availability of testing at decreased cost. However, the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. We tested 1,648 prospectively pooled specimens by using 3 nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2: a laboratory-developed real-time reverse transcription PCR targeting the envelope gene, and 2 commercially available Panther System assays targeting open reading frame 1ab. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71.7% to 82.6% for pools of 8 and from 82.9% to 100.0% for pools of 4. We developed and validated an independent stochastic simulation model to estimate effects of dilution on PPA and efficiency of a 2-stage pooled real-time reverse transcription PCR testing algorithm. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection.

  View details for DOI 10.3201/eid2701.203379

  View details for Web of Science ID 000609135300011

  View details for PubMedID 33183494

  View details for PubMedCentralID PMC7774575

• SARS-CoV-2 infection and COVID-19 severity in individuals with prior seasonal coronavirus infection. Diagnostic microbiology and infectious disease Gombar, S. n., Bergquist, T. n., Pejaver, V. n., Hammarlund, N. E., Murugesan, K. n., Mooney, S. n., Shah, N. n., Pinsky, B. A., Banaei, N. n. 2021; 100 (2): 115338

  Abstract

  We show that individuals with documented history of seasonal coronavirus have a similar SARS-CoV-2 infection rate and COVID-19 severity as those with no prior history of seasonal coronavirus. Our findings suggest prior infection with seasonal coronavirus does not provide immunity to subsequent infection with SARS-CoV-2.

  View details for DOI 10.1016/j.diagmicrobio.2021.115338

  View details for PubMedID 33610036

• Cost-Effectiveness of Nasopharyngeal Carcinoma Screening with Epstein-Barr Virus Polymerase Chain Reaction or Serology in High-Incidence Populations Worldwide. Journal of the National Cancer Institute Miller, J. A., Le, Q., Pinsky, B. A., Wang, H. 2020

  Abstract

  BACKGROUND: The incidence of endemic Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) varies considerably worldwide. In high-incidence regions, screening trials have been conducted. We estimated the mortality reduction and cost-effectiveness of EBV-based NPC screening in populations worldwide.METHODS: We identified 380 populations in 132 countries with incident NPC and developed a decision-analytic model to compare ten unique onetime screening strategies to no screening for men and women at age 50years. Screening performance and the stage distribution of undiagnosed NPC were derived from a systematic review of prospective screening trials.RESULTS: Screening was cost-effective in up to 14.5% of populations, depending on the screening strategy. These populations were limited to East Asia, Southeast Asia, North Africa, or were Asian, Pacific Islander, or Inuit populations in North America. A combination of serology and nasopharyngeal polymerase chain reaction (PCR) was most cost-effective, but other combinations of serologic and/or plasma PCR screening were also cost-effective. The estimated reduction in NPC mortality was similar across screening strategies. For a hypothetical cohort of patients in China, 10-year survival improved from 71.0% (95%CI = 68.8%-73.0%) without screening to a median of 86.3% (range = 83.5%-88.2%) with screening. This corresponded to a median 10-year reduction in NPC mortality of 52.9% (range= 43.1%-59.3%). Screening interval impacted absolute mortality reduction and cost-effectiveness.CONCLUSIONS: We observed decreased NPC mortality with EBV-based screening. Screening was cost-effective in many high-incidence populations and could be extended to men and women as early as age 40years in select regions. These findings may be useful when choosing among local public health initiatives.

  View details for DOI 10.1093/jnci/djaa198

  View details for PubMedID 33351145

• SARS-CoV-2 Seroprevalence in Healthcare Personnel in Northern California Early in the COVID-19 Pandemic. Infection control and hospital epidemiology Rosser, J. I., Roltgen, K., Dymock, M., Shepard, J., Martin, A., Hogan, C. A., Blomkalns, A., Mathew, R., Parsonnet, J., Pinsky, B. A., Maldonado, Y. A., Boyd, S. D., Chang, S., Holubar, M., Stanford Healthcare COVID-19 Workforce Response Group 2020: 1–27

  Abstract

  OBJECTIVE: We aimed to assess the magnitude of unidentified SARS-CoV-2 infections in our healthcare personnel (HCP) early in the COVID-19 pandemic and evaluate risk factors for infection in order to identify areas for infection control practice improvement in a northern California academic medical center.METHODS: We reviewed the anti-SARS-CoV-2 receptor binding domain (RBD) IgG serologic test results and self-reported risk factors for seropositivity among 10,449 asymptomatic HCP who underwent voluntary serology testing between April 20 and May 20, 2020.RESULTS: In total, 136 employees (1.3%) tested positive for SARS-CoV-2 IgG. This included 41 (30.1%) individuals who had previously tested positive for SARS-CoV-2 by nasopharyngeal reverse transcription polymerase chain reaction (RT-PCR) between March 13 and April 16, 2020. In multivariable analysis, employees of Hispanic ethnicity (OR = 2.01; 95% CI = 1.22-3.46) and those working in environmental services/food services/patient transport (OR = 4.81; 95% CI = 2.08-10.30) were at increased risk for seropositivity compared to other groups. Employees reporting a household contact with COVID-19 were also at higher risk for seropositivity (OR = 3.25; 95% CI = 1.47-6.44), but those with a work exposure were not (OR = 1.27; 95% CI = 0.58-2.47). Importantly, one-third of seropositive individuals reported no prior symptoms, no suspected exposures, and no prior positive RT-PCR test.CONCLUSION: In this study, SARS-CoV-2 seropositivity among HCP early in the northern California epidemic appeared to be quite low and was more likely attributable to community rather than occupational exposure.

  View details for DOI 10.1017/ice.2020.1358

  View details for PubMedID 33292895

• Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2. Proceedings of the National Academy of Sciences of the United States of America Ramachandran, A., Huyke, D. A., Sharma, E., Sahoo, M. K., Huang, C., Banaei, N., Pinsky, B. A., Santiago, J. G. 2020

  Abstract

  The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

  View details for DOI 10.1073/pnas.2010254117

  View details for PubMedID 33148808

• High Dengue Burden and Circulation of 4 Virus Serotypes among Children with Undifferentiated Fever Kenya, 2014-2017 EMERGING INFECTIOUS DISEASES Shah, M. M., Ndenga, B. A., Mutuku, F. M., Vu, D. M., Grossi-Soyster, E. N., Okuta, V., Ronga, C. O., Chebii, P. K., Maina, P., Jembe, Z., Bosire, C. M., Amugongo, J. S., Sahoo, M. K., Huang, C., Weber, J., Edgerton, S., Hortion, J., Bennett, S. N., Pinsky, B. A., LaBeaud, A. 2020; 26 (11): 2638–50

  Abstract

  Little is known about the extent and serotypes of dengue viruses circulating in Africa. We evaluated the presence of dengue viremia during 4 years of surveillance (2014-2017) among children with febrile illness in Kenya. Acutely ill febrile children were recruited from 4 clinical sites in western and coastal Kenya, and 1,022 participant samples were tested by using a highly sensitive real-time reverse transcription PCR. A complete case analysis with genomic sequencing and phylogenetic analyses was conducted to characterize the presence of dengue viremia among participants during 2014-2017. Dengue viremia was detected in 41.9% (361/862) of outpatient children who had undifferentiated febrile illness in Kenya. Of children with confirmed dengue viremia, 51.5% (150/291) had malaria parasitemia. All 4 dengue virus serotypes were detected, and phylogenetic analyses showed several viruses from novel lineages. Our results suggests high levels of dengue virus infection among children with undifferentiated febrile illness in Kenya.

  View details for DOI 10.3201/eid2611.200960

  View details for Web of Science ID 000596803200012

  View details for PubMedID 33079035

  View details for PubMedCentralID PMC7588514

• Retrospective Screening for SARS-CoV-2 RNA in California, USA, Late 2019 EMERGING INFECTIOUS DISEASES Hogan, C. A., Garamani, N., Sahoo, M. K., Huang, C., Zehnder, J., Pinsky, B. A. 2020; 26 (10): 2487–88

  Abstract

  To investigate the possibility of earlier cases of severe acute respiratory syndrome coronavirus 2 infection than previously recognized, we retrospectively tested pooled samples from 1,700 persons with respiratory signs/symptoms seen at Stanford Health Care, Palo Alto, California, USA, during the last 2 months of 2019. We found no evidence of earlier infection.

  View details for DOI 10.3201/eid2610.202296

  View details for Web of Science ID 000572522100035

  View details for PubMedID 32620178

  View details for PubMedCentralID PMC7510744

• High Frequency of SARS-CoV-2 RNAemia and Association With Severe Disease. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Stevens, B. A., Sahoo, M. K., Huang, C., Garamani, N., Gombar, S., Yamamoto, F., Murugesan, K., Kurzer, J., Zehnder, J., Pinsky, B. A. 2020

  Abstract

  BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in blood, also known as RNAemia, has been reported, but its prognostic implications are poorly understood. This study aimed to determine the frequency of SARS-CoV-2 RNA in plasma and its association with coronavirus disease 2019 (COVID-19) clinical severity.METHODS: An analytical cross-sectional study was performed in a single-center tertiary care institution and included consecutive inpatients and outpatients with confirmed COVID-19. The prevalence of SARS CoV-2 RNAemia and the strength of its association with clinical severity variables were examined and included intensive care unit (ICU) admission, invasive mechanical ventilation, and 30-day all-cause mortality.RESULTS: Paired nasopharyngeal and plasma samples were included from 85 patients. The median age was 55 years, and individuals with RNAemia were older than those with undetectable SARS-CoV-2 RNA in plasma (63 vs 50 years; P = .04). Comorbidities were frequent including obesity (37.6%), hypertension (30.6%), and diabetes mellitus (22.4%). RNAemia was detected in 28/85 (32.9%) of patients, including 22/28 (78.6%) who required hospitalization. In models adjusted for age, RNAemia was detected more frequently in individuals who developed severe disease including ICU admission (32.1 vs 14.0%; P = .04) and invasive mechanical ventilation (21.4% vs 3.5%; P = .02). All 4 deaths occurred in individuals with detectable RNAemia. An additional 121 plasma samples from 28 individuals with RNAemia were assessed longitudinally, and RNA was detected for a maximum duration of 10 days.CONCLUSIONS: This study demonstrated a high proportion of SARS-CoV-2 RNAemia, and an association between RNAemia and clinical severity suggesting the potential utility of plasma viral testing as a prognostic indicator for COVID-19.

  View details for DOI 10.1093/cid/ciaa1054

  View details for PubMedID 32965474

• SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness. medRxiv : the preprint server for health sciences Röltgen, K., Wirz, O. F., Stevens, B. A., Powell, A. E., Hogan, C. A., Najeeb, J., Hunter, M., Sahoo, M. K., Huang, C., Yamamoto, F., Manalac, J., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Nadeau, K. C., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Kim, P. S., Gombar, S., Tibshirani, R., Pinsky, B. A., Boyd, S. D. 2020

  Abstract

  SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

  View details for DOI 10.1101/2020.08.15.20175794

  View details for PubMedID 32839786

  View details for PubMedCentralID PMC7444305

• Mechanisms of Fano-resonant biosensing: Mechanical loading of plasmonic oscillators OPTICS COMMUNICATIONS Zhu, X., Cao, N., Thibeault, B. J., Pinsky, B., Yanik, A. 2020; 469

  View details for DOI 10.1016/j.optcom.2020.125780

  View details for Web of Science ID 000532673400010

• Knowledge, attitudes, and practices of cervical Cancer screening among HIV-positive and HIV-negative women participating in human papillomavirus screening in rural Zimbabwe. BMC women's health Fitzpatrick, M., Pathipati, M. P., McCarty, K., Rosenthal, A., Katzenstein, D., Chirenje, Z. M., Pinsky, B. 2020; 20 (1): 153

  Abstract

  BACKGROUND: Women in low- and middle-income countries are at the highest risk of cervical cancer yet have limited access to and participation in cervical cancer screening programs. Integrating self-collected, community-based screening offers a potential primary screening method in areas of limited resources. In this paper, we present a study evaluating knowledge, attitudes, and practices of cervical cancer and Human Papilloma Virus (HPV) in rural Zimbabwe.METHODS: We performed a community-based cross-sectional knowledge, attitudes and practices of HPV and cervical cancer study in rural Zimbabwe from January 2017-May 2017. Women were selected for the study via random number generation from complete lists of inhabitants in the study area if they satisfied the inclusion criteria (≥30-years-old, ≤65-years-old, not pregnant, intact uterus). If selected, they participated in a 19-question structured knowledge, attitudes and practices survey. The questionnaire included questions on demographics, education, knowledge of HPV, cervical cancer, and risk factors. Chi-squared tests were evaluated comparing knowledge, attitudes and practices relating to HPV and cervical cancer screening with actual infection with HPV. Women were also offered a voluntary HIV and self-collected HPV screening.RESULTS: Six hundred seventy-nine women were included in the knowledge, attitudes and practices survey. Most women (81%) had heard of cervical cancer while the majority had not heard of HPV (12%). The number of women that had been screened previously for cervical cancer was low (5%). There were no significant differences between and within groups regarding knowledge of cervical cancer and actual overall infection with HR-HPV, HPV 16, and HPV 18/45 test results.CONCLUSIONS: Most women in rural Zimbabwe have heard of cervical cancer, but the number that had been screened was low. Extending existing outreach services to include cervical cancer screening, potentially including HPV screening, should include cervical cancer/HPV education and screening triage. This approach would serve to bridge the gap between knowledge and screening availability to address some of the barriers to cervical cancer care still affecting women in many regions of the world.

  View details for DOI 10.1186/s12905-020-01017-2

  View details for PubMedID 32711530

• Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2 EMERGING INFECTIOUS DISEASES Waggoner, J. J., Stittleburg, V., Pond, R., Saklawi, Y., Sahoo, M. K., Babiker, A., Hussaini, L., Kraft, C. S., Pinsky, B. A., Anderson, E. J., Rouphael, N. 2020; 26 (7): 1633–35

  Abstract

  Most reverse transcription PCR protocols for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 2-3 targets for detection. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with singleplex assays. This protocol could streamline detection and decrease reagent use during current high SARS-CoV-2 testing demands.

  View details for DOI 10.3201/eid2607.201285

  View details for Web of Science ID 000551231700052

  View details for PubMedID 32294051

  View details for PubMedCentralID PMC7323516

• Measure what matters: Counts of hospitalized patients are a better metric for health system capacity planning for a reopening. Journal of the American Medical Informatics Association : JAMIA Kashyap, S., Gombar, S., Yadlowsky, S., Callahan, A., Fries, J., Pinsky, B. A., Shah, N. H. 2020

  Abstract

  OBJECTIVE: Responding to the COVID-19 pandemic requires accurate forecasting of health system capacity requirements using readily available inputs. We examined whether testing and hospitalization data could help quantify the anticipated burden on the health system given shelter-in-place (SIP) order.MATERIALS AND METHODS: 16,103 SARS-CoV-2 RT-PCR tests were performed on 15,807 patients at Stanford facilities between March 2 and April 11, 2020. We analyzed the fraction of tested patients that were confirmed positive for COVID-19, the fraction of those needing hospitalization, and the fraction requiring ICU admission over the 40 days between March 2nd and April 11th 2020.RESULTS: We find a marked slowdown in the hospitalization rate within ten days of SIP even as cases continued to rise. We also find a shift towards younger patients in the age distribution of those testing positive for COVID-19 over the four weeks of SIP. The impact of this shift is a divergence between increasing positive case confirmations and slowing new hospitalizations, both of which affects the demand on health systems.CONCLUSION: Without using local hospitalization rates and the age distribution of positive patients, current models are likely to overestimate the resource burden of COVID-19. It is imperative that health systems start using these data to quantify effects of SIP and aid reopening planning.

  View details for DOI 10.1093/jamia/ocaa076

  View details for PubMedID 32548636

• Persistent detection of SARS-CoV-2 RNA in patients and healthcare workers with COVID-19. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Gombar, S., Chang, M., Hogan, C. A., Zehnder, J., Boyd, S., Pinsky, B. A., Shah, N. H. 2020; 129: 104477

  Abstract

  BACKGROUND: Current guidelines for returning health care workers (HCW) to service after a positive SARS-CoV-2 RT-PCR test and ceasing of transmission precautions for patients is based on two general strategies. A test-based strategy that requires negative respiratory RT-PCR tests obtained after the resolution of symptoms. Alternatively, due to the limited availability of testing, many sites employ a symptom-based strategy that recommends excluding HCW from the workforce and keeping patients on contact precautions until a fixed period of time has elapsed from symptom recovery. The underlying assumption of the symptom-based strategy is that waiting for a fixed period of time is a surrogate for negative RT-PCR testing, which itself is a surrogate for the absence of shedding infectious virus.OBJECTIVES: To better understand the appropriate length of symptom based return to work and contact precaution strategies.STUDY DESIGN: We performed an observational analysis of 150 patients and HCW that transitioned from RT-PCR SARS-CoV-2 positive to negative over the course of 2 months at a US academic medical center.RESULTS: We found that the average time to transition from RT-PCR positive to negative was 24 days after symptom onset and 10 % remained positive even 33 days after symptom onset. No difference was seen in HCW and patients.CONCLUSIONS: These findings suggest until definitive evidence of the length of infective viral shedding is obtained that the fixed length of time before returning to work or ceasing contract precautions be revised to over one-month.

  View details for DOI 10.1016/j.jcv.2020.104477

  View details for PubMedID 32505778

• Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients. medRxiv : the preprint server for health sciences Long, D. R., Gombar, S., Hogan, C. A., Greninger, A. L., OReilly Shah, V., Bryson-Cahn, C., Stevens, B., Rustagi, A., Jerome, K. R., Kong, C. S., Zehnder, J., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. 2020

  Abstract

  BACKGROUND: SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) testing remains the cornerstone of laboratory-based identification of patients with COVID-19. As the availability and speed of SARS-CoV-2 testing platforms improve, results are increasingly relied upon to inform critical decisions related to therapy, use of personal protective equipment, and workforce readiness. However, early reports of RT-PCR test performance have left clinicians and the public with concerns regarding the reliability of this predominant testing modality and the interpretation of negative results. In this work, two independent research teams report the frequency of discordant SARS-CoV-2 test results among initially negative, repeatedly tested patients in regions of the United States with early community transmission and access to testing.METHODS: All patients at the University of Washington (UW) and Stanford Health Care undergoing initial testing by nasopharyngeal (NP) swab between March 2nd and April 7th, 2020 were included. SARS-CoV-2 RT-PCR was performed targeting the N, RdRp, S, and E genes and ORF1ab, using a combination of Emergency Use Authorization laboratory-developed tests and commercial assays. Results through April 14th were extracted to allow for a complete 7-day observation period and an additional day for reporting.RESULTS: A total of 23,126 SARS-CoV-2 RT-PCR tests (10,583 UW, 12,543 Stanford) were performed in 20,912 eligible patients (8,977 UW, 11,935 Stanford) undergoing initial testing by NP swab; 626 initially test-negative patients were re-tested within 7 days. Among this group, repeat testing within 7 days yielded a positive result in 3.5% (4.3% UW, 2.8% Stanford) of cases, suggesting an initial false negative RT-PCR result; the majority (96.5%) of patients with an initial negative result who warranted reevaluation for any reason remained negative on all subsequent tests performed within this window.CONCLUSIONS: Two independent research teams report the similar finding that, among initially negative patients subjected to repeat SARS-CoV-2 RT-PCR testing, the occurrence of a newly positive result within 7 days is uncommon. These observations suggest that false negative results at the time of initial presentation do occur, but potentially at a lower frequency than is currently believed. Although it is not possible to infer the clinical sensitivity of NP SARS-CoV-2 RT-PCR testing using these data, they may be used in combination with other reports to guide the use and interpretation of this common testing modality.

  View details for DOI 10.1101/2020.05.03.20089151

  View details for PubMedID 32511542

• A non-optical multiplexed PCR diagnostic platform for serotype-specific detection of dengue virus SENSORS AND ACTUATORS B-CHEMICAL Yin, Z., Ramshani, Z., Waggoner, J. J., Pinsky, B. A., Senapati, S., Chang, H. 2020; 310

  View details for DOI 10.1016/j.snb.2020.127854

  View details for Web of Science ID 000519306300005

• Five-minute point-of-care testing for SARS-CoV-2: Not there yet. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Hogan, C. A., Sahoo, M. K., Huang, C., Garamani, N., Stevens, B., Zehnder, J., Pinsky, B. A. 2020; 128: 104410

  View details for DOI 10.1016/j.jcv.2020.104410

  View details for PubMedID 32403009

• Whole-Genome Analysis of Cervical Human Papillomavirus Type 35 from rural Zimbabwean Women. Scientific reports Fitzpatrick, M. B., Hahn, Z., Mandishora, R. S., Dao, J., Weber, J., Huang, C., Sahoo, M. K., Katzenstein, D. A., Pinsky, B. A. 2020; 10 (1): 7001

  Abstract

  Human papillomavirus (HPV) types differ by geographic location and the ethnicity of the human host, which may have implications for carcinogenicity. HPV35 is one of the least frequently identified high-risk types in North America and Europe but was the most common high-risk HPV (hrHPV) infection in a cohort in rural Zimbabwe. Whole genome analysis is limited for HPV35; no such studies have been performed in Zimbabwe. Of 648 women in the initial cohort in Zimbabwe, 19 (19/648, 2.9%) tested positive for HPV35, and eight samples were successfully sequenced for HPV35. The maximum number of sequence variants for the whole genome was 58 nucleotides (0.7%) compared to the prototype (58/7879). The maximum number of sequence variants in E6 and E7 was 3 (3/450, 0.7%) 2 (2/300, 0.7%), respectively. These are the first HPV35 whole genome sequences from Zimbabwe, and these data further lend support to the carcinogenicity of HPV35 despite limited sequence heterogeneity. Further studies to determine carcinogenic effects and impact of HPV vaccinations are warranted, especially in sub-Saharan Africa.

  View details for DOI 10.1038/s41598-020-63882-z

  View details for PubMedID 32332798

• Rates of Co-infection Between SARS-CoV-2 and Other Respiratory Pathogens. JAMA Kim, D., Quinn, J., Pinsky, B., Shah, N. H., Brown, I. 2020

  View details for DOI 10.1001/jama.2020.6266

  View details for PubMedID 32293646

• Antibody-Dependent Enhancement of Severe Disease Is Mediated by Serum Viral Load in Pediatric Dengue Virus Infections. The Journal of infectious diseases Waggoner, J. J., Katzelnick, L. C., Burger-Calderon, R., Gallini, J., Moore, R. H., Kuan, G., Balmaseda, A., Pinsky, B. A., Harris, E. 2020

  Abstract

  BACKGROUND: Low preexisting anti-dengue virus (DENV) antibody levels are associated with elevated disease severity. While antibody-dependent enhancement of dengue is thought to be driven by viral load, this has not been conclusively shown. We evaluated the association between preinfection anti-DENV antibody titers, viral load, and disease severity among 133 dengue cases in a Nicaraguan pediatric cohort study.METHODS: Viral load was quantified in acute-phase serum by real-time reverse transcription polymerase chain reaction and analyzed in relation to preinfection antibody titer (measured by inhibition enzyme-linked immunosorbent assay) and dengue severity, categorized using 3 definitions.RESULTS: Higher viral load was significantly associated with dengue severity; for each increase of 1.0 log10 copies/mL, the odds of severe dengue increased approximately 50%, regardless of severity definition. Viral load at presentation and the odds of severe disease were highest among patients with low to intermediate preinfection antibody titers and lowest among those with the highest antibody titers. We showed the effect of preinfection antibody titer on disease severity was mediated by viral load for each of 3 dengue severity outcomes.CONCLUSIONS: This study demonstrates the association between preinfection anti-DENV antibody titer, serum viral load, and disease severity, and provides evidence for the mechanism of antibody-dependent enhancement in dengue cases.

  View details for DOI 10.1093/infdis/jiz618

  View details for PubMedID 32236481

• Merkel Cell Carcinoma of Lymph Node is Metastatic Cutaneous Merkel Cell Carcinoma Lawrence, L., Kunder, C., Stehr, H., Saleem, A., Natkunam, Y., Zehnder, J., Pinsky, B., Sahoo, M., Tan, S. NATURE PUBLISHING GROUP. 2020: 824–26

  View details for Web of Science ID 000518328801386

• Merkel Cell Carcinoma of Lymph Node is Metastatic Cutaneous Merkel Cell Carcinoma Lawrence, L., Kunder, C., Stehr, H., Saleem, A., Natkunam, Y., Zehnder, J., Pinsky, B., Sahoo, M., Tan, S. NATURE PUBLISHING GROUP. 2020: 824–26

  View details for Web of Science ID 000518328901386

• Human papillomavirus cytopathic effect in the urine of a 76-year-old man. Diagnostic cytopathology Gottlieb, C., Pinsky, B. A., Mills, A. M., Stelow, E. B. 2020

  View details for DOI 10.1002/dc.24384

  View details for PubMedID 32043839

• Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region. The American journal of tropical medicine and hygiene Cantero, C., Cardozo, F., Waggoner, J. J., Pinsky, B. A., Espinola, A., Infanzon, B., Acosta, M. E., Aria, L., Arevalo de Guillen, Y., Cuevas, T., Rojas, V., Segovia, C., Centurion, A., Rojas, A. 2020

  Abstract

  Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clinicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

  View details for DOI 10.4269/ajtmh.19-0707

  View details for PubMedID 31933462

• Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens. Journal of clinical microbiology Hogan, C. A., Garamani, N. n., Lee, A. S., Tung, J. K., Sahoo, M. K., Huang, C. n., Stevens, B. n., Zehnder, J. n., Pinsky, B. A. 2020

  Abstract

  Background: Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use.Objectives: The aim of this study was to assess test performance of the Accula SARS-CoV-2 test.Study design: The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa coefficient.Results: Overall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%).Conclusion: Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

  View details for DOI 10.1128/JCM.01072-20

  View details for PubMedID 32461285

• Comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of SARS-CoV-2. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Bulterys, P. L., Garamani, N. n., Stevens, B. n., Sahoo, M. K., Huang, C. n., Hogan, C. A., Zehnder, J. n., Pinsky, B. A. 2020; 129: 104427

  Abstract

  Numerous nucleic acid amplification tests, including real-time, reverse transcription PCR (rRT-PCR) and isothermal amplification methods, have been developed to detect SARS-CoV-2 RNA, including many that have received emergency use authorization (EUA). There is a need to assess their test performance relative to one another.The aim of this study was to compare the test performance of a high complexity laboratory-developed rRT-PCR EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene with other tests: the Atila isothermal amplification assay targeting the nucleocapsid (N) gene and open reading frame 1ab (ORF1ab), the Altona E and spike (S) multiplex, real-time RT-PCR, and the US Centers for Disease Control and Prevention (CDC) N1 and N2 rRT-PCRs.A diagnostic comparison study was performed by testing nasopharyngeal samples from persons under investigation for coronavirus disease 2019 (COVID-19). Assay performance was assessed by percent agreement and Cohen's kappa coefficient.Positive percent agreement with the SHC EUA reference assay was 82.8 % (95 % confidence interval (CI) 65.0 to 92.9) for Atila, 86.7 % (95 % CI 69.7 to 95.3) for the Altona E and S targets, and 86.7 % (95 % CI 69.7 to 95.3) and 90.0 % (95 % CI 73.6 to 97.3), for the CDC N1 and N2 targets, respectively. All assays demonstrated 100 % negative percent agreement. Kappa coefficients ranged from 0.86 to 0.92, indicating excellent agreement.Performance was comparable among the SARS-CoV-2 nucleic acid amplification methods tested, with a limited number of discrepancies observed in specimens with low viral loads.

  View details for DOI 10.1016/j.jcv.2020.104427

  View details for PubMedID 32535398

  View details for PubMedCentralID PMC7207111

• Comparison of the Panther Fusion and a laboratory-developed test targeting the envelope gene for detection of SARS-CoV-2. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Hogan, C. A., Sahoo, M. K., Huang, C. n., Garamani, N. n., Stevens, B. n., Zehnder, J. n., Pinsky, B. A. 2020; 127: 104383

  Abstract

  Numerous nucleic acid amplification assays have recently received emergency use authorization (EUA) for the diagnosis of SARS-CoV-2 infection, and there is a need to assess their test performance relative to one another.The aim of this study was to compare the test performance of the Hologic Panther Fusion SARS-CoV-2 assay targeting two regions of open reading frame 1ab (ORF1ab) to a high complexity molecular-based, laboratory-developed EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene.We performed a diagnostic comparison study by testing nasopharyngeal samples on the two assays. Assay agreement was assessed by overall percent agreement and Cohen's kappa coefficient.A total of 184 nasopharyngeal samples were tested using the two assays, of which 180 showed valid results and were included for the comparative analysis. Overall percent agreement between the assays was 98.3 % (95 % confidence interval (CI) 95.2-99.7) and kappa coefficient was 0.97 (95 % CI 0.93-1.0). One sample was detected on the SHC laboratory developed test (LDT) and not on the Panther Fusion, and had a Ct of 35.9. Conversely, 2 samples were detected on the Panther Fusion and not on the LDT, and had Ct values of 37.2 and 36.6.The Panther Fusion SARS-CoV-2 assay and the SHC LDT perform similarly on clinical nasopharyngeal swab specimens. Other considerations, including reagent availability, turnaround time, labor requirements, cost and instrument throughput should guide the decision of which assay to perform.

  View details for DOI 10.1016/j.jcv.2020.104383

  View details for PubMedID 32353760

• A comprehensive analysis of RHOA mutation positive and negative angioimmunoblastic T-cell lymphomas by targeted deep sequencing, expression profiling and single cell digital image analysis. International journal of molecular medicine Butzmann, A. n., Sridhar, K. n., Jangam, D. n., Kumar, J. n., Sahoo, M. K., Shahmarvand, N. n., Warnke, R. n., Rangasamy, E. n., Pinsky, B. A., Ohgami, R. S. 2020

  Abstract

  Angioimmunoblastic T‑cell lymphoma (AITL) is a uniquely aggressive mature T‑cell neoplasm. In recent years, recurrent genetic mutations in ras homolog family member A (RHOA), tet methylcytosine dioxygenase 2 (TET2), DNA methyltransferase 3 alpha (DNMT3A) and isocitrate dehydrogenase [NADP(+)] 2 (IDH2) have been identified as associated with AITL. However, a deep molecular study assessing both DNA mutations and RNA expression profile combined with digital image analysis is lacking. The present study aimed to evaluate the significance of molecular and morphologic features by high resolution digital image analysis in several cases of AITL. To do so, a total of 18 separate tissues from 10 patients with AITL were collected and analyzed. The results identified recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrated increased DNA mutations in coding, promoter and CCCTC binding factor (CTCF) binding sites in RHOA mutated AITLs vs. RHOA non‑mutated cases, as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed that RHOA mutated cells have decreased eccentricity (more circular) compared with RHOA non‑mutated AITL cells. In conclusion, the results from the present study expand our understanding of AITL and demonstrate that there are specific cell biological and morphological manifestations of RHOA mutations in cases of AITL.

  View details for DOI 10.3892/ijmm.2020.4686

  View details for PubMedID 32945366

• Carving out a niche for SARS-CoV-2 plasma RNA testing. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Pinsky, B. A., Hogan, C. A. 2020

  View details for DOI 10.1093/cid/ciaa1412

  View details for PubMedID 32941602

• Evidence of transovarial transmission of Chikungunya and Dengue viruses in field-caught mosquitoes in Kenya. PLoS neglected tropical diseases Heath, C. J., Grossi-Soyster, E. N., Ndenga, B. A., Mutuku, F. M., Sahoo, M. K., Ngugi, H. N., Nbakaya, J. O., Siema, P. n., Kitron, U. n., Zahiri, N. n., Hortion, J. n., Waggoner, J. J., King, C. H., Pinsky, B. A., LaBeaud, A. D. 2020; 14 (6): e0008362

  Abstract

  Arboviruses are among the most important emerging pathogens due to their increasing public health impact. In Kenya, continued population growth and associated urbanization are conducive to vector spread in both urban and rural environments, yet mechanisms of viral amplification in vector populations is often overlooked when assessing risks for outbreaks. Thus, the characterization of local arbovirus circulation in mosquito populations is imperative to better inform risk assessments and vector control practices. Aedes species mosquitoes were captured at varying stages of their life cycle during different seasons between January 2014 and May 2016 at four distinct sites in Kenya, and tested for chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV) viruses by RT-PCR. CHIKV was detected in 45 (5.9%) and DENV in 3 (0.4%) mosquito pools. No ZIKV was detected. Significant regional variation in prevalence was observed, with greater frequency of CHIKV on the coast. DENV was detected exclusively on the coast. Both viruses were detected in immature mosquitoes of both sexes, providing evidence of transovarial transmission of these arboviruses in local mosquitoes. This phenomenon may be driving underlying viral maintenance that may largely contribute to periodic re-emergence among humans in Kenya.

  View details for DOI 10.1371/journal.pntd.0008362

  View details for PubMedID 32559197

• Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies. medRxiv : the preprint server for health sciences Lau, B. T., Pavlichin, D. n., Hooker, A. C., Almeda, A. n., Shin, G. n., Chen, J. n., Sahoo, M. K., Huang, C. n., Pinsky, B. A., Lee, H. n., Ji, H. P. 2020

  Abstract

  The genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contract tracing.Leveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints.We built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and rare ones that occur in only small fraction of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome.We conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients, mutations demarcating dominant species and the prevalence of mutation signatures, of which a significant number were relatively unique. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

  View details for DOI 10.1101/2020.11.02.20224816

  View details for PubMedID 33173909

  View details for PubMedCentralID PMC7654905

• Reply to Muller and Chaudhury. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Pinsky, B. A., Banaei, N. n. 2020

  View details for DOI 10.1093/cid/ciaa220

  View details for PubMedID 32140706

• A predictive tool for identification of SARS-CoV-2 PCR-negative emergency department patients using routine test results. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Joshi, R. P., Pejaver, V. n., Hammarlund, N. E., Sung, H. n., Lee, S. K., Furmanchuk, A. n., Lee, H. Y., Scott, G. n., Gombar, S. n., Shah, N. n., Shen, S. n., Nassiri, A. n., Schneider, D. n., Ahmad, F. S., Liebovitz, D. n., Kho, A. n., Mooney, S. n., Pinsky, B. A., Banaei, N. n. 2020; 129: 104502

  Abstract

  Testing for COVID-19 remains limited in the United States and across the world. Poor allocation of limited testing resources leads to misutilization of health system resources, which complementary rapid testing tools could ameliorate.To predict SARS-CoV-2 PCR positivity based on complete blood count components and patient sex.A retrospective case-control design for collection of data and a logistic regression prediction model was used. Participants were emergency department patients > 18 years old who had concurrent complete blood counts and SARS-CoV-2 PCR testing. 33 confirmed SARS-CoV-2 PCR positive and 357 negative patients at Stanford Health Care were used for model training. Validation cohorts consisted of emergency department patients > 18 years old who had concurrent complete blood counts and SARS-CoV-2 PCR testing in Northern California (41 PCR positive, 495 PCR negative), Seattle, Washington (40 PCR positive, 306 PCR negative), Chicago, Illinois (245 PCR positive, 1015 PCR negative), and South Korea (9 PCR positive, 236 PCR negative).A decision support tool that utilizes components of complete blood count and patient sex for prediction of SARS-CoV-2 PCR positivity demonstrated a C-statistic of 78 %, an optimized sensitivity of 93 %, and generalizability to other emergency department populations. By restricting PCR testing to predicted positive patients in a hypothetical scenario of 1000 patients requiring testing but testing resources limited to 60 % of patients, this tool would allow a 33 % increase in properly allocated resources.A prediction tool based on complete blood count results can better allocate SARS-CoV-2 testing and other health care resources such as personal protective equipment during a pandemic surge.

  View details for DOI 10.1016/j.jcv.2020.104502

  View details for PubMedID 32544861

• Is Merkel Cell Carcinoma of Lymph Node Actually Metastatic Cutaneous Merkel Cell Carcinoma? American journal of clinical pathology Lawrence, L. E., Saleem, A. n., Sahoo, M. K., Tan, S. K., Pinsky, B. A., Natkunam, Y. n., Kunder, C. A., Stehr, H. n., Zehnder, J. L. 2020

  Abstract

  The possibility of a so-called primary lymph node neuroendocrine carcinoma has been described in the literature. Here we evaluate cases fitting such a diagnosis and find that the cases demonstrate a convincing and pervasive pattern consistent with metastatic Merkel cell carcinoma.Six cases of primary lymph node Merkel cell carcinoma and one case of metastatic neuroendocrine carcinoma at a bony site, all with unknown primary, were sequenced using a combination of whole-exome and targeted panel methods. Sequencing results were analyzed for the presence of an ultraviolet (UV) mutational signature or off-target detection of Merkel cell polyomavirus (MCPyV).Four of six primary lymph node cases were positive for a UV mutational signature, with the remaining two cases positive for off-target alignment of MCPyV. One case of neuroendocrine carcinoma occurring at a bony site was also positive for a UV mutational signature.We find no evidence to corroborate the existence of so-called primary Merkel cell carcinoma of lymph node.

  View details for DOI 10.1093/ajcp/aqaa051

  View details for PubMedID 32445471

• Comparison of a Point-of-Care Assay and a High-Complexity Assay for Detection of SARS-CoV-2 RNA. The journal of applied laboratory medicine Stevens, B. n., Hogan, C. A., Sahoo, M. K., Huang, C. n., Garamani, N. n., Zehnder, J. n., Kurzer, J. n., Pinsky, B. A. 2020

  Abstract

  Numerous nucleic acid amplification assays utilizing different target genes of the SARS-CoV-2 genome have received emergency use authorization (EUA) by the United States Food and Drug Administration (FDA). Limited data are available comparing the test performance characteristics of these assays.A diagnostic comparison study was performed to evaluate the performance of the Cepheid Xpert Xpress SARS-CoV-2 assay compared to the Hologic Panther Fusion SARS-CoV-2 assay using clinical nasopharyngeal specimens. Agreement between the two assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.A total of 104 (54 positive and 50 negative) clinical nasopharyngeal samples were tested by both assays. Using the Panther Fusion as a reference standard, the Xpert demonstrated an overall agreement of 99.0% (95% confidence interval (CI): 94.8 - 100), positive percent agreement of 98.1% (95% CI: 90.1 - 100), and a negative percent agreement of 100% (95% CI: 94.2 - 100). The kappa coefficient was 0.98 (95% CI: 0.94 - 1.0). One sample positive by the Panther Fusion with a cycle threshold (Ct) of 38.6 was found to be reproducibly negative by the Xpert assay.The Cepheid Xpert Xpress SARS-CoV-2 assay provides test performance comparable to the Hologic Panther Fusion SARS-CoV-2 assay while offering laboratories rapid, on-demand testing capacity.

  View details for DOI 10.1093/jalm/jfaa135

  View details for PubMedID 32761092

• Assessment of Sensitivity and Specificity of Patient-Collected Lower Nasal Specimens for Sudden Acute Respiratory Syndrome Coronavirus 2 Testing. JAMA network open Altamirano, J. n., Govindarajan, P. n., Blomkalns, A. L., Kushner, L. E., Stevens, B. A., Pinsky, B. A., Maldonado, Y. n. 2020; 3 (6): e2012005

  View details for DOI 10.1001/jamanetworkopen.2020.12005

  View details for PubMedID 32530469

• Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2. Emerging infectious diseases Hogan, C. A., Gombar, S. n., Wang, H. n., Röltgen, K. n., Shi, R. Z., Holubar, M. n., Chang, S. I., Lee, G. M., Boyd, S. D., Zehnder, J. n., Pinsky, B. A. 2020; 27 (1)

  Abstract

  Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.

  View details for DOI 10.3201/eid2701.203892

  View details for PubMedID 33256889

• Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2. Cell host & microbe Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Jean, G. H., Stevens, B. A., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Hunter, M. n., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Nadeau, K. C., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

  Abstract

  B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.

  View details for DOI 10.1016/j.chom.2020.09.002

  View details for PubMedID 32941787

• Virological Failure and Acquired Genotypic Resistance Associated With Contemporary Antiretroviral Treatment Regimens. Open forum infectious diseases Rhee, S. Y., Clutter, D. n., Hare, C. B., Tchakoute, C. T., Sainani, K. n., Fessel, W. J., Hurley, L. n., Slome, S. n., Pinsky, B. A., Silverberg, M. J., Shafer, R. W. 2020; 7 (9): ofaa316

  Abstract

  There are few descriptions of virologic failure (VF) and acquired drug resistance (HIVDR) in large cohorts initiating contemporary antiretroviral therapy (ART).We studied all persons with HIV (PWH) in a California clinic population initiating ART between 2010 and 2017. VF was defined as not attaining virologic suppression, discontinuing ART, or virologic rebound prompting change in ART.During the study, 2315 PWH began ART. Six companion drugs were used in 93.3% of regimens: efavirenz, elvitegravir/c, dolutegravir, b-darunavir, rilpivirine, and raltegravir. During a median follow-up of 36 months, 214 (9.2%) PWH experienced VF (2.8 per 100 person-years) and 62 (2.7%) experienced HIVDR (0.8 per 100 person-years). In multivariable analyses, younger age, lower CD4 count, higher virus load, and b-atazanavir were associated with increased VF risk; lower CD4 count, higher virus load, and nevirapine were associated with increased HIVDR risk. Compared with efavirenz, dolutegravir, raltegravir, and b-darunavir were associated with reduced HIVDR risk. Risks of VF and HIVDR were not significantly associated with ART initiation year. Of the 62 PWH with HIVDR, 42 received an non-nucleoside RT inhibitor (NNRTI), 15 an integrase-strand transfer inhibitor (INSTI), and 5 a protease inhibitor (PI). Among those with HIVDR on an NNRTI or first-generation INSTI, 59% acquired dual class resistance and 29% developed tenofovir resistance; those receiving a PI or dolutegravir developed just M184V.Despite the frequent use of contemporary ART regimens, VF and HIVDR continue to occur. Further efforts are required to improve long-term ART virological responses to prevent the consequences of ongoing HIV-1 replication including virus transmission and HIVDR.

  View details for DOI 10.1093/ofid/ofaa316

  View details for PubMedID 32904894

  View details for PubMedCentralID PMC7462367

• Utilization, yield, and accuracy of the FilmArray Meningitis/Encephalitis panel with diagnostic stewardship and testing algorithm. Journal of clinical microbiology Broadhurst, M. J., Dujari, S. n., Budvytiene, I. n., Pinsky, B. A., Gold, C. A., Banaei, N. n. 2020

  Abstract

  Background: The impact of diagnostic stewardship and testing algorithms on utilization and performance of the FilmArray® Meningitis/Encephalitis (ME) Panel has received limited investigation.Methods: We performed a retrospective single-center cohort study assessing all individuals with suspected ME between February 2017 and April 2019 for whom the ME Panel was ordered. Testing was restricted to patients with cerebrospinal fluid (CSF) pleocytosis. Positive ME Panel results were confirmed before reporting through correlation with direct stain (Gram and Calcofluor white) and CSF Cryptococcal antigen or by repeat ME Panel testing. Outcomes included ME Panel test utilization rate, negative predictive value of non-pleocytic CSF samples, test yield and false-positivity rate, and time to appropriate de-escalation of acyclovir.Results: Restricting testing to pleocytic CSF samples reduced ME Panel utilization by 42.7% (263 vs 459 tests performed) and increased test yield by 61.8% (18.6% vs 11.5% positivity rate; P < 0.01) with application of criteria. The negative predictive value of normal CSF WBC for ME Panel targets was 100% (195/195) for non-viral targets and 98.0% (192/196) overall. All pathogens detected in non-pleocytic CSF samples were herpesviruses. Application of a selective testing algorithm based on repeat testing of non-viral targets avoided 75% (3/4) of false-positive results without generating false-negative results. Introduction of the ME panel reduced the duration of acyclovir treatment from an average of 66 hours (SD, 43) to 46 hours (SD, 36) (P = 0.03).Conclusions: Implementation of the ME Panel with restriction criteria and a selective testing algorithm for non-viral targets optimizes its utilization, yield and accuracy.

  View details for DOI 10.1128/JCM.00311-20

  View details for PubMedID 32493787

• Interferon-gamma release assay for accurate detection of SARS-CoV-2 T cell response. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K. n., Jagannathan, P. n., Pham, T. D., Pandey, S. n., Bonilla, H. F., Jacobson, K. n., Parsonnet, J. n., Andrews, J. R., Weiskopf, D. n., Sette, A. n., Pinsky, B. A., Singh, U. n., Banaei, N. n. 2020

  Abstract

  We investigated feasibility and accuracy of an interferon-gamma release assay (IGRA) for detection of T cell responses to SARS-CoV-2. Whole blood IGRA accurately distinguished between convalescents and uninfected healthy blood donors with a predominantly CD4+ T cell response. SARS-CoV-2 IGRA may serve as a useful diagnostic tool in managing the COVID-19 pandemic.

  View details for DOI 10.1093/cid/ciaa1537

  View details for PubMedID 33035306

• Proinflammatory IgG Fc structures in patients with severe COVID-19 Nature Immunology Chakraborty, S., Gonzales, J., Edwards, K., Mallajosyulla, V., Buzzanco, A. S., Sherwood, R., Buffone, C., Kathale, N., Providenza, S., Xie, M. M., Andrews, J. R., Blish, C. A., Singh, U., Dugan, H., Wilson, P. C., Pham, T. D., Boyd, S. D., Nadeau, K. C., Pinsky, B. A., Zhang, S., Memoli, M. J., Taubenberger, J. K., Morales, T., Schapiro, J. M., Tan, G. S., et al 2020

  View details for DOI 10.1038/s41590-020-00828-7

• Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2. bioRxiv : the preprint server for biology Nielsen, S. C., Yang, F. n., Jackson, K. J., Hoh, R. A., Röltgen, K. n., Stevens, B. n., Lee, J. Y., Rustagi, A. n., Rogers, A. J., Powell, A. E., Najeeb, J. n., Otrelo-Cardoso, A. R., Yost, K. E., Daniel, B. n., Chang, H. Y., Satpathy, A. T., Jardetzky, T. S., Kim, P. S., Wang, T. T., Pinsky, B. A., Blish, C. A., Boyd, S. D. 2020

  Abstract

  During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

  View details for DOI 10.1101/2020.07.08.194456

  View details for PubMedID 32676593

  View details for PubMedCentralID PMC7359515

• Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Yang, S. n., Garner, O. B., Green, D. A., Gomez, C. A., Dien Bard, J. n., Pinsky, B. A., Banaei, N. n. 2020

  Abstract

  Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice.We performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 U.S. institutions over 1.5 years. Comprehensive chart review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established.A total of 82 Karius tests were evaluated, from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. Karius positivity rate was 50/82 (61.0%), with 25 (50.0%) showing two or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only one was associated with clinical impact.The real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods and how best to integrate mNGS into current testing algorithms.

  View details for DOI 10.1093/cid/ciaa035

  View details for PubMedID 31942944

• Metagenomic Next-Generation Sequencing for the Identification and Quantitation of Transplant-Related DNA Viruses. Journal of clinical microbiology Carpenter, M. L., Tan, S. K., Watson, T., Bacher, R., Nagesh, V., Watts, A., Bentley, G., Weber, J., Huang, C., Sahoo, M. K., Hinterwirth, A., Doan, T., Carter, T., Dong, Q., Gourguechon, S., Harness, E., Kermes, S., Radhakrishnan, S., Wang, G., Quiroz-Zarate, A., Ching, J., Pinsky, B. A. 2019

  Abstract

  Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related dsDNA viruses (ADV, BKV, CMV, EBV, HHV-6A, HHV-6B, HSV-1, HSV-2, JCV, and VZV). The mNGS 95% limit of detection ranged from 14 international units (IU)/mL (HHV-6) to 191 copies/mL (BKV), and the lower limit of quantitation ranged from 442 IU/mL (EBV) to 661 copies/mL (VZV). Evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and qPCR of 89.2% (306/343), with a kappa statistic of 0.725. The positive percent agreement was 84.9% (73/86) and negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed co-infections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of Y = 0.953*X + 0.075 [95% CI of the slope (0.883 to 1.011) and intercept (-0.100 to 0.299)], and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log10 concentration, 95% limits of agreement of -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates comparable analytical and clinical performance to qPCR for transplant-related DNA viruses.

  View details for DOI 10.1128/JCM.01113-19

  View details for PubMedID 31554674

• Cost-Effective Respiratory Virus Testing. Journal of clinical microbiology Pinsky, B. A., Hayden, R. T. 2019; 57 (9)

  Abstract

  The timely and accurate diagnosis of respiratory virus infections has the potential to optimize downstream (posttesting) use of limited health care resources, including antibiotics, antivirals, ancillary testing, and inpatient and emergency department beds. Cost-effective algorithms for respiratory virus testing must take into consideration numerous factors, including which patients should be tested, what testing should be performed (for example, antigen testing versus reverse transcription-PCR testing or influenza A/B testing versus testing with a comprehensive respiratory virus panel), and the turnaround time necessary to achieve the desired posttesting outcomes. Despite the clinical impact of respiratory virus infections, the cost-effectiveness of respiratory virus testing is incompletely understood. In this article, we review the literature pertaining to the cost-effectiveness of respiratory virus testing in pediatric and adult patient populations, in emergency department, outpatient, and inpatient clinical settings. Furthermore, we consider the cost-effectiveness of a variety of testing methods, including rapid antigen tests, direct fluorescent antibody assays, and nucleic acid amplification tests.

  View details for DOI 10.1128/JCM.00373-19

  View details for PubMedID 31142607

  View details for PubMedCentralID PMC6711893

• Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing. The Journal of molecular diagnostics : JMD Weber, J., Volkova, I., Sahoo, M. K., Tzou, P. L., Shafer, R. W., Pinsky, B. A. 2019

  Abstract

  Genotypic antiretroviral drug resistance testing is a critical component of the global efforts to control the HIV-1 epidemic. This study investigates the semi-automated, next-generation sequencing (NGS)-based Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay in a prospective cohort of HIV-1 infected patients. Two-hundred and sixty-nine samples were successfully sequenced by both NGS and Sanger sequencing. Among the 261 protease/reverse transcriptase (PR/RT) sequences, a mean of 0.37 drug resistance mutations were identified by both Sanger and NGS, 0.08 by NGS alone, and 0.03 by Sanger alone. Among the 50 integrase sequences, a mean of 0.3 drug resistance mutations were detected by both Sanger and NGS, and 0.08 by NGS alone. NGS estimated higher levels of drug resistance to one or more antiretroviral drugs for 6.5% of PR/RT sequences and 4.0% of integrase sequences, whereas Sanger estimated higher levels of drug resistance for 3.8% of PR/RT sequences. Although the samples successfully sequenced by the Sentosa SQ HIV Genotyping Assay demonstrated similar predicted resistance compared with Sanger, 44% of Sentosa runs failed QC requiring 17 additional runs. This semi-automated NGS-based assay may aid in HIV-1 genotypic drug resistance testing, though numerous QC issues were observed when this platform was used in a clinical laboratory setting. With additional refinement, the Sentosa SQ HIV-1 Genotyping Assay may contribute to the global efforts to control HIV-1.

  View details for DOI 10.1016/j.jmoldx.2019.06.003

  View details for PubMedID 31382033

• Unbiased Pathogen Detection and Host Gene Profiling for Conjunctivitis OPHTHALMOLOGY Lalitha, P., Seitzman, G. D., Kotecha, R., Hinterwirth, A., Chen, C., Zhong, L., Cummings, S., Lebas, E., Sahoo, M. K., Pinsky, B. A., Lietman, T. M., Thuy Doan 2019; 126 (8): 1090–94

  View details for DOI 10.1016/j.ophtha.2019.03.039

  View details for Web of Science ID 000475845500014

• Evaluating for Human Herpesvirus 6 in the Liver Explants of Children With Liver Failure of Unknown Etiology JOURNAL OF INFECTIOUS DISEASES Yang, C. H., Sahoo, M. K., Fitzpatrick, M., Lau, A. H., Pinsky, B. A., Martinez, O. M. 2019; 220 (3): 361–69

  View details for DOI 10.1093/infdis/jiy644

  View details for Web of Science ID 000477595700004

• Impact of Pretransplant Donor BK Viruria in Kidney Transplant Recipients JOURNAL OF INFECTIOUS DISEASES Tan, S. K., Huang, C., Sahoo, M. K., Weber, J., Kurzer, J., Stedman, M. R., Concepcion, W., Gallo, A. E., Alonso, D., Srinivas, T., Storch, G. A., Subramanian, A. K., Tan, J. C., Pinsky, B. A. 2019; 220 (3): 370–76

  View details for DOI 10.1093/infdis/jiz114

  View details for Web of Science ID 000477595700005

• Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance JOURNAL OF MOLECULAR DIAGNOSTICS Clutter, D. S., Mazarei, G., Sinha, R., Manasa, J., Nouhin, J., LaPrade, E., Bolouki, S., Tzou, P. L., Hannita-Hui, J., Sahoo, M. K., Kuimelis, P., Kuimelis, R. G., Pinsky, B. A., Schoolnik, G. K., Hassibi, A., Shafer, R. W. 2019; 21 (4): 580–92

  View details for DOI 10.1016/j.jmoldx.2019.02.005

  View details for Web of Science ID 000475412100005

• Community-based self-collected human papillomavirus screening in rural Zimbabwe. BMC public health Fitzpatrick, M. B., El-Khatib, Z., Katzenstein, D., Pinsky, B. A., Chirenje, Z. M., McCarty, K. 2019; 19 (Suppl 1): 603

  Abstract

  BACKGROUND: In low- and middle-income countries (LMIC), women have limited access to and uptake of cervical cancer screening. Delayed diagnosis leads to poorer outcomes and early mortality, and continues to impede cancer control disproportionately in LMIC. Integrating self-collected, community-based screening for High Risk-Human Papilloma Virus (HR-HPV) into existent HIV programs is a potential screening method to identify women at high risk for developing high-risk cervical lesions.METHODS: We implemented community-based cross-sectional study on self-collection HR-HPV screening in conjunction with existing community outreach models for the distribution of antiretroviral therapy (ART) and the World Health Organization Expanded Program on Immunization (EPI) outreach in villages in rural Zimbabwe from January 2017 through May 2017.RESULTS: Overall, there was an 82% response rate: 70% of respondents participated in self-collection and 12% were ineligible for the study (inclusion criteria: age 30-65, not pregnant, with an intact uterus). Women recruited in the first 2-3months of the study had more opportunities to participate and therefore significantly higher participation: 81% participation (additional 11% ineligible), while those with fewer opportunities also had lower participation: 63% (additional 13% ineligible) (p<0.001). Some village outreach centers (N=5/12) had greater than 89% participation.CONCLUSIONS: Integration of HR-HPV screening into existing community outreach models for HIV and immunizations could facilitate population-based screening to scale cancer control and prevention programs in sub-Saharan Africa. Community/village health workers (CHW/VHW) and village outreach programs offer a potential option for cervical cancer screening programs to move towards improving access of sexual and reproductive health resources for women at highest risk.

  View details for DOI 10.1186/s12889-019-6810-5

  View details for PubMedID 31138174

• Evaluation of the Aptima HCV Quant Dx Assay Using Serum and Dried Blood Spots JOURNAL OF CLINICAL MICROBIOLOGY Weber, J., Sahoo, M. K., Taylor, N., Shi, R., Pinsky, B. A. 2019; 57 (4)

  View details for DOI 10.1128/JCM.00030-19

  View details for Web of Science ID 000462714100002

• Dual-target, real-time PCR for the diagnosis of intraocular Toxoplasma gondii infections BRITISH JOURNAL OF OPHTHALMOLOGY Gomez, C. A., Sahoo, M. K., Kahn, G., Zhong, L., Montoya, J. G., Pinsky, B. A., Thuy Doan 2019; 103 (4): 569–72

  View details for DOI 10.1136/bjophthalmol-2018-313064

  View details for Web of Science ID 000471871400024

• A 20-Gene Set Predictive of Progression to Severe Dengue. Cell reports Robinson, M., Sweeney, T. E., Barouch-Bentov, R., Sahoo, M. K., Kalesinskas, L., Vallania, F., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Pinsky, B. A., Khatri, P., Einav, S. 2019; 26 (5): 1104

  Abstract

  There is a need to identify biomarkers predictive of severe dengue. Single-cohort transcriptomics has not yielded generalizable results or parsimonious, predictive gene sets. We analyzed blood samples of dengue patients from seven gene expression datasets (446 samples, five countries) using an integrated multi-cohort analysis framework and identified a 20-gene set that predicts progression to severe dengue. We validated the predictive power of this 20-gene set in three retrospective dengue datasets (84 samples, three countries) and a prospective Colombia cohort (34 patients), with an area under the receiver operating characteristic curve of 0.89, 100% sensitivity, and 76% specificity. The 20-gene dengue severity scores declined during the diseasecourse, suggesting an infection-triggered host response. This 20-gene set is strongly associated with the progression to severe dengue and represents a predictive signature, generalizable across ages, host genetic factors, and virus strains, with potential implications for the development of a host response-based dengue prognostic assay.

  View details for PubMedID 30699342

• Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted Human Immunodeficiency Virus Type 1 Drug Resistance in a Large US Clinic Population CLINICAL INFECTIOUS DISEASES Rhee, S., Clutter, D., Fessel, W., Klein, D., Slome, S., Pinsky, B. A., Marcus, J. L., Hurley, L., Silverberg, M. J., Pond, S., Shafer, R. W. 2019; 68 (2): 213–21

  View details for DOI 10.1093/cid/ciy453

  View details for Web of Science ID 000459636700010

• Molecular profiling of clear cell adenocarcinoma of the urinary tract. Virchows Archiv : an international journal of pathology Lin, C. Y., Saleem, A. n., Stehr, H. n., Zehnder, J. L., Pinsky, B. A., Kunder, C. A. 2019

  Abstract

  Clear cell adenocarcinoma (CCA) of the urinary tract is a rare type of malignancy whose molecular profiles remain undefined. Here we reported an integrated clinicopathologic and molecular profiling analysis of four cases of clear cell adenocarcinoma arising in the urethra or the bladder. Utilizing a clinically validated 130-gene exon-sequencing assay, we identified recurrent pathogenic PIK3CA (p. E545K) and KRAS (p.G12D) variants in three of four (75%) of the cases. In addition, an APC variant (P.S2310X), a TP53 variant (p.R273C), and a MYC amplification event were identified. The only CCA case without either PIK3CA or KRAS variants has a distinct pathogenesis through BK virus, demonstrated by positive BK virus PCR and SV40 immunohistochemistry. The novel finding of recurrent variants in the PI3K/AKT/mTOR pathway provides not only insights into oncogenesis but also potential clinical therapeutic targets for patients with clear cell adenocarcinoma of the urinary tract.

  View details for DOI 10.1007/s00428-019-02634-5

  View details for PubMedID 31372739

• Real-time RT-PCR for the detection and quantitation of Oropouche virus. Diagnostic microbiology and infectious disease Rojas, A. n., Stittleburg, V. n., Cardozo, F. n., Bopp, N. n., Cantero, C. n., López, S. n., Bernal, C. n., Mendoza, L. n., Aguilar, P. n., Pinsky, B. A., Guillén, Y. n., Páez, M. n., Waggoner, J. J. 2019: 114894

  Abstract

  Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/μL. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/μL were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ≤10 copies/μL (8/14 vs 0/13, respectively, P = 0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.

  View details for DOI 10.1016/j.diagmicrobio.2019.114894

  View details for PubMedID 31727377

• Characterization of dengue cases among patients with an acute illness, Central Department, Paraguay. PeerJ Rojas, A. n., Cardozo, F. n., Cantero, C. n., Stittleburg, V. n., López, S. n., Bernal, C. n., Gimenez Acosta, F. E., Mendoza, L. n., Pinsky, B. A., Arévalo de Guillén, I. n., Páez, M. n., Waggoner, J. n. 2019; 7: e7852

  Abstract

  In 2018, Paraguay experienced a large dengue virus (DENV) outbreak. The primary objective of this study was to characterize dengue cases in the Central Department, where the majority of cases occur, and identify factors associated with DENV infection.Patients were enrolled from January-May 2018 if they presented with a suspected arboviral illness. Acute-phase specimens (≤8 days after symptom onset) were tested using rRT-PCR, a rapid diagnostic test for DENV nonstructural protein 1 (NS1) and anti-DENV IgM and IgG, and ELISA for IgG against NS1 from Zika virus (ZIKV).A total of 231 patients were enrolled (95.2% adults) at two sites: emergency care and an outpatient clinical site. Patients included 119 (51.5%) dengue cases confirmed by rRT-PCR (n = 115, 96.6%) and/or the detection of NS1 and anti-DENV IgM (n = 4, 3.4%). DENV-1 was the predominant serotype (109/115, 94.8%). Epidemiologically, dengue cases and non-dengue cases were similar, though dengue cases were less likely to reside in a house/apartment or report a previous dengue case. Clinical and laboratory findings associated with dengue included red eyes, absence of sore throat, leucopenia and thrombocytopenia. At an emergency care site, 26% of dengue cases (26/100) required hospitalization. In univariate analysis, hospitalization was associated with increased viral load, anti-DENV IgG, and thrombocytopenia. Among dengue cases that tested positive for IgG against ZIKV NS1, the odds of DENV NS1 detection in the acute phase were decreased 10-fold (OR 0.1, 0.0-0.3).Findings from a predominantly adult population demonstrate clinical and laboratory factors associated with DENV infections and the potential severity of dengue in this group. The combination of viral load and specific IgG antibodies warrant further study as a prognostic to identify patients at risk for severe disease.

  View details for DOI 10.7717/peerj.7852

  View details for PubMedID 31616598

  View details for PubMedCentralID PMC6790102

• Risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the Colombo dengue study. BMC infectious diseases Sigera, P. C., Amarasekara, R. n., Rodrigo, C. n., Rajapakse, S. n., Weeratunga, P. n., De Silva, N. L., Huang, C. H., Sahoo, M. K., Pinsky, B. A., Pillai, D. R., Tissera, H. A., Jayasinghe, S. n., Handunnetti, S. n., Fernando, S. D. 2019; 19 (1): 680

  Abstract

  A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before.Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers.Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05).Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.

  View details for DOI 10.1186/s12879-019-4304-9

  View details for PubMedID 31370795

• Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. Journal of clinical microbiology Murugesan, K. n., Hogan, C. A., Palmer, Z. n., Reeve, B. n., Theron, G. n., Andama, A. n., Somoskovi, A. n., Steadman, A. n., Madan, D. n., Andrews, J. n., Croda, J. n., Sahoo, M. K., Cattamanchi, A. n., Pinsky, B. A., Banaei, N. n. 2019

  Abstract

  Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood.Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus and EBV, and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. PCR cycle threshold (CT) was used to measure amplifiable cfDNA.In spiked samples, median CT for M. tuberculosis, S. enterica, and EBV cfDNA was significantly lower in blood collected in K2EDTA than Streck and PAXgene blood collection tubes, and significantly lower in EDTA-urine than Streck-urine. Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT compared with Streck blood collection tube and urine preservative. Processing delay increased median pathogen CT for Streck and PAXgene but not K2EDTA blood samples, and for urine preserved with Streck reagent but not EDTA. Double spin compared with single spin plasma separation increased median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant, and between fresh and thawed plasma and urine after 24 weeks at -80 °C. Larger plasma and urine volume in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest large volume single spin K2EDTA-plasma and EDTA-whole urine with up to 24-hour processing delay may optimize pcfDNA detection.

  View details for DOI 10.1128/JCM.00782-19

  View details for PubMedID 31511335

• Comparison of Transcription-Mediated Amplification and Real-Time PCR Assays for Hepatitis B Virus DNA Quantitation in Serum. The journal of applied laboratory medicine Weber, J. n., Sahoo, M. K., Taylor, N. n., Uy, E. n., Shi, R. Z., Pinsky, B. A. 2019; 4 (3): 383–90

  Abstract

  The quantification of hepatitis B (HBV) DNA in serum is critical to identify patients requiring antiviral therapy and to monitor the response to treatment.This study describes the evaluation of the Aptima HBV Quant Dx assay (Aptima HBV) performed on the automated Panther system.Aptima HBV was linear from 1.70 to 7.70 log10 IU/mL with a commercial reference panel, as well as clinical specimens representing genotypes B and C, and total imprecision, as measured by the percentage coefficient of variation (%CV) at 2.0 log10 IU/mL was <10%. The specificity of Aptima HBV was 94.7% (126/133) and 96.6% (84/87) for serum specimens from individuals without HBV exposure and individuals with resolved HBV infection, respectively. The qualitative agreement and quantitative accuracy of Aptima HBV was compared to the COBAS AmpliPrep/COBAS TaqMan HBV Test v2.0 (CAP/CTM). Overall agreement was 90.8% (187/206) with a κ statistic of 0.708 (standard error, 0.063; 95% CI, 0.585-0.831). Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% CI of the slope, 0.883-1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (Aptima - CAP/CTM) showed a slight negative bias (-0.054 log10 IU/mL, and 95% limits of agreement of -1.093 to 0.984).The Aptima HBV test affords a suitable alternative to CAP/CTM for serum virus load testing and provides a key component of the diagnostic algorithm for the global eradication of viral hepatitis.

  View details for DOI 10.1373/jalm.2019.029058

  View details for PubMedID 31659075

• Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe dengue. Proceedings of the National Academy of Sciences of the United States of America Zanini, F., Robinson, M. L., Croote, D., Sahoo, M. K., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Goo, L., Pinsky, B. A., Quake, S. R., Einav, S. 2018

  Abstract

  Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA-containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.

  View details for PubMedID 30530648

• Deep Sequencing Prompts the Modification of a Real-time RT-PCR for the Serotype-Specific Detection of Polioviruses. Journal of virological methods Holubar, M., Sahoo, M. K., Huang, C., Mohamed-Hadley, A., Liu, Y., Waggoner, J. J., Troy, S. B., Garcia-Garcia, L., Ferreyra-Reyes, L., Maldonado, Y., Pinsky, B. A. 2018

  Abstract

  Polioviruses are members of the Enterovirus C species and asymptomatic fecal shedding allows for their transmission and persistence in a community, as well as the emergence of vaccine-derived polioviruses. Using three serotype-specific real-time RT-PCR (rRT-PCR) assays, the shedding and circulation of oral poliovirus vaccine (OPV) strains was previously investigated in a prospective cohort of MFexican children, their contacts, and nearby sewage. Subsequently, a deep sequencing approach targeting the P1 genomic region was applied to characterize OPV strains previously detected by rRT-PCR. Amplifiable RNA was obtained for sequencing from 40.3% (58/144) of stool samples and 71.4% (15/21) of sewage using nucleic acids extracted directly from primary rRT-PCR-positive specimens. Sequencing detected one or more OPV serotypes in 62.1% (36/58) of stool and 53.3% (8/15) of sewage samples. All stool and sewage samples in which poliovirus was not detected by deep sequencing contained at least one non-polio enterovirus C (NPEV-C) strain. To improve screening specificity, a modified, two-step, OPV serotype-specific multiplex rRT-PCR was evaluated. In stool specimens, the overall agreement between the original assays and the multiplex was 70.3%. By serotype, the overall agreement was 95.7% for OPV serotype-1 (S1), 65.6% for S2, and 96.1% for S3. Furthermore, most original rRT-PCR positive/multiplex rRT-PCR negative results were collected in the summer and fall months, consistent with NPEV-C circulation patterns. In conclusion, this deep sequencing approach allowed for the characterization of OPV sequences directly from clinical samples and facilitated the implementation of a more specific multiplex rRT-PCR for OPV detection and serotyping.

  View details for PubMedID 30447245

• A Case Report of Pediatric Clear Cell Carcinoma of the Urinary Bladder Associated With Polyomavirus AJSP-REVIEWS AND REPORTS Saleem, A., Brown, R. A., Higgins, J. T., Troxell, M. L., Kunder, C. A., Pinsky, B. A., Zambrano, E., Kao, C. 2018; 23 (6): 291–95

  View details for DOI 10.1097/PCR.0000000000000284

  View details for Web of Science ID 000457629000012

• Native Kidney Cytomegalovirus Nephritis and Cytomegalovirus Prostatitis in a Kidney Transplant Recipient. Transplant infectious disease : an official journal of the Transplantation Society Tan, S. K., Cheng, X. S., Kao, C., Weber, J., Pinsky, B. A., Gill, H. S., Busque, S., Subramanian, A. K., Tan, J. C. 2018: e12998

  Abstract

  We present a case of cytomegalovirus (CMV) native kidney nephritis and prostatitis in a CMV D+/R- kidney transplant recipient who had completed six months of CMV prophylaxis four weeks prior to the diagnosis of genitourinary CMV disease. The patient had a history of benign prostatic hypertrophy and urinary retention that required self-catheterization to relieve high post-voiding residual volumes. At 7 months post-transplant, he was found to have a urinary tract infection, moderate hydronephrosis of the transplanted kidney, and severe hydroureteronephrosis of the native left kidney and ureter, and underwent native left nephrectomy and transurethral resection of the prostate. Histopathologic examination of kidney and prostate tissue revealed CMV inclusions consistent with invasive CMV disease. This case highlights that CMV may extend beyond the kidney allograft to involve other parts of the genitourinary tract, including the native kidneys and prostate. Furthermore, we highlight the tissue-specific risk factors that preceded CMV tissue invasion. In addition to concurrent diagnoses, health care providers should have a low threshold for considering late-onset CMV disease in high-risk solid organ transplant recipients presenting with signs and symptoms of genitourinary tract pathology. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30203504

• Poor Immunogenicity, Not Vaccine Strain Egg Adaptation, May Explain the Low H3N2 Influenza Vaccine Effectiveness in 2012-2013 CLINICAL INFECTIOUS DISEASES Cobey, S., Gouma, S., Parkhouse, K., Chambers, B. S., Ertl, H. C., Schmader, K. E., Halpin, R. A., Lin, X., Stockwell, T. B., Das, S. R., Landon, E., Tesic, V., Youngster, I., Pinsky, B. A., Wentworth, D. E., Hensley, S. E., Grad, Y. H. 2018; 67 (3): 327–33

  Abstract

  Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012-2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3).We investigated the basis of low VE in 2012-2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination.We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer.In contrast to analyses based on ferret studies, low H3N2 VE in 2012-2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

  View details for PubMedID 29471464

  View details for PubMedCentralID PMC6051447

• FOXP3-positive T-cell lymphomas in non-HTLV1 carriers include ALK-negative anaplastic large cell lymphoma: expanding the Spectrum of T-cell lymphomas with regulatory phenotype. Human pathology Ferreira, C. R., Zhao, S., Sahoo, M. K., Pinsky, B., Weber, J., Lage, L. A., Pereira, J., Zerbini, M. C., Natkunam, Y. 2018

  Abstract

  Forkhead box P3 (FOXP3) is a specific marker for regulatory T-cells (Tregs). We report 6 cases of T-cell lymphomas with Treg phenotype based on diffuse positivity for FOXP3 in tumor cells. The patients showed a median age of 56years with a male predominance. Sites of disease included lymph nodes (4), skin (2), subcutaneous tissue (1) and bone marrow (1). All cases showed monomorphic large cells, some with Hodgkin-like or anaplastic cells. All cases expressed pan T cell markers and lacked cytotoxic markers; one case showed diffuse PD1 staining. Only one case harbored human T-lymphotrophic virus (HTLV)-1 DNA within tumor cells and was classified as adult T-cell leukemia/lymphoma (ATLL). Among 5 HTLV1-negative cases, 3 were classified as peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 2 fulfilled criteria for ALK-negative anaplastic large cell lymphoma (ALCL) with diffuse and strong CD30 positivity. We concluded that Treg phenotype may be rarely seen in HTLV1-negative cases, such as PTCL, NOS and ALK-negative ALCL. Our findings expand the spectrum of T-cell lymphomas with regulatory phenotype and suggest that consideration should be given to HTLV1 DNA testing in the appropriate clinical setting to rule out ATLL.

  View details for PubMedID 29898383

• Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing JOURNAL OF CLINICAL MICROBIOLOGY Tzou, P. L., Ariyaratne, P., Varghese, V., Lee, C., Rakhmanaliev, E., Villy, C., Yee, M., Tan, K., Michel, G., Pinsky, B. A., Shafer, R. W. 2018; 56 (6)

  Abstract

  The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy.

  View details for PubMedID 29618499

  View details for PubMedCentralID PMC5971553

• High human herpesvirus 6 viral load in pediatric allogeneic hematopoietic stem cell transplant patients is associated with detection in end organs and high mortality PEDIATRIC TRANSPLANTATION Winestone, L. E., Punn, R., Tamaresis, J. S., Buckingham, J., Pinsky, B. A., Waggoner, J. J., Kharbanda, S. 2018; 22 (2)

  Abstract

  Human Herpes Virus 6 (HHV-6) reactivation occurs in approximately half of patients following allogeneic hematopoietic stem cell transplant (HSCT). While encephalitis and delayed engraftment are well-documented complications of HHV-6 following HSCT, the extent to which HHV-6 viremia causes disease in children is controversial. We performed a retrospective review of HHV-6 reactivation and possible manifestations in pediatric allogeneic HSCT patients at a single institution. Of 89 children and young adults who underwent allogeneic HSCT over a three-and-a-half-year period, 34 patients reactivated HHV-6 early post-transplant. Unrelated donor stem cell source and lack of antiviral prophylaxis were risk factors for the development of HHV-6 viremia. Viremia correlated with the presence of acute graft-versus-host disease, but not chronic graft-versus-host disease. We identified two subgroups within the viremic patients-a high-risk viremic and tissue-positive group that reactivated HHV-6 and had suspected end-organ disease and a low-risk viremic but asymptomatic group that reactivated HHV-6 but did not exhibit symptoms or signs of end-organ disease. Peak viral load was found to be strongly associated with mortality. Prospective studies in larger numbers of patients are needed to further investigate the role of HHV-6 in causing symptomatic end-organ disease as well as the association of viral load with mortality.

  View details for PubMedID 29181879

  View details for PubMedCentralID PMC5820136

• Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Rojas, A., Diagne, C. T., Stittleburg, V. D., Mohamed-Hadley, A., Arevalo de Guillen, Y., Balmaseda, A., Faye, O., Faye, O., Sall, A. A., Harris, E., Pinsky, B. A., Waggoner, J. J. 2018; 98 (6): 1833–36

  Abstract

  The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/μL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

  View details for PubMedID 29611509

• ARBOVIRUS AND MALARIA CO-INFECTIONS AMONG FEBRILE KENYAN CHILDREN Shah, M., Sahoo, M., Krystosik, A., Mutuku, F., Ndenga, B., Otuka, V., Ronga, C., Chebii, P., Maina, P., Jembe, Z., Pinsky, B., LaBeaud, A. AMER SOC TROP MED & HYGIENE. 2018: 162–63

  View details for Web of Science ID 000461386602521

• Real-time RT-PCR for Mayaro virus detection in plasma and urine JOURNAL OF CLINICAL VIROLOGY Waggoner, J. J., Rojas, A., Mohamed-Hadley, A., Arevalo de Guillen, Y., Pinsky, B. A. 2018; 98: 1–4

  Abstract

  Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature.To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains.A MAYV rRT-PCR targeting a region of the 5'UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes.The linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/μL, and the lower limit of 95% detection was 8.2copies/μL. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine.We report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.

  View details for PubMedID 29172075

  View details for PubMedCentralID PMC5742299

• Zika and Chikungunya virus detection in naturally infected Aedes aegypti in Ecuador ACTA TROPICA Cevallos, V., Ponce, P., Waggoner, J. J., Pinsky, B. A., Coloma, J., Quiroga, C., Morales, D., Cardenas, M. 2018; 177: 74–80

  Abstract

  The wide and rapid spread of Chikungunya (CHIKV) and Zika (ZIKV) viruses represent a global public health problem, especially for tropical and subtropical environments. The early detection of CHIKV and ZIKV in mosquitoes may help to understand the dynamics of the diseases in high-risk areas, and to design data based epidemiological surveillance to activate the preparedness and response of the public health system and vector control programs. This study was done to detect ZIKV and CHIKV viruses in naturally infected fed female Aedes aegypti (L.) mosquitoes from active epidemic urban areas in Ecuador. Pools (n=193; 22 pools) and individuals (n=22) of field collected Ae. aegypti mosquitoes from high-risk arboviruses infection sites in Ecuador were analyzed for the presence of CHIKV and ZIKV using RT-PCR. Phylogenetic analysis demonstrated that both ZIKV and CHIKV viruses circulating in Ecuador correspond to the Asian lineages. Minimum infection rate (MIR) of CHIKV for Esmeraldas city was 2.3% and the maximum likelihood estimation (MLE) was 3.3%. The minimum infection rate (MIR) of ZIKV for Portoviejo city was 5.3% and for Manta city was 2.1%. Maximum likelihood estimation (MLE) for Portoviejo city was 6.9% and 2.6% for Manta city. Detection of arboviruses and infection rates in the arthropod vectors may help to predict an outbreak and serve as a warning tool in surveillance programs.

  View details for PubMedID 28982578

• Transplant Virus Detection Using Multiplex Targeted Sequencing. The journal of applied laboratory medicine Tan, S. K., Shen, P. n., Lefterova, M. I., Sahoo, M. K., Fung, E. n., Odegaard, J. I., Davis, R. W., Pinsky, B. A., Scharfe, C. n. 2018; 2 (5): 757–69

  Abstract

  Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation.We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients.For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR.Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.

  View details for DOI 10.1373/jalm.2017.024521

  View details for PubMedID 31245786

  View details for PubMedCentralID PMC6594177

• Molecular diagnosis of Zika virus infections REVIEWS IN MEDICAL MICROBIOLOGY St George, K., Pinsky, B. A. 2018; 29 (1): 8–16

  View details for DOI 10.1097/MRM.0000000000000125

  View details for Web of Science ID 000419432200002

• Global epidemiology of non-influenza RNA respiratory viruses: data gaps and a growing need for surveillance. The Lancet. Infectious diseases Tang, J. W., Lam, T. T., Zaraket, H., Lipkin, W. I., Drews, S. J., Hatchette, T. F., Heraud, J. M., Koopmans, M. P. 2017; 17 (10): e320-e326

  Abstract

  Together with influenza, the non-influenza RNA respiratory viruses (NIRVs), which include respiratory syncytial virus, parainfluenza viruses, coronavirus, rhinovirus, and human metapneumovirus, represent a considerable global health burden, as recognised by WHO's Battle against Respiratory Viruses initiative. By contrast with influenza viruses, little is known about the contemporaneous global diversity of these viruses, and the relevance of such for development of pharmaceutical interventions. Although far less advanced than for influenza, antiviral drugs and vaccines are in different stages of development for several of these viruses, but no interventions have been licensed. This scarcity of global genetic data represents a substantial knowledge gap and impediment to the eventual licensing of new antiviral drugs and vaccines for NIRVs. Enhanced genetic surveillance will assist and boost research and development into new antiviral drugs and vaccines for these viruses. Additionally, understanding the global diversity of respiratory viruses is also part of emerging disease preparedness, because non-human coronaviruses and paramyxoviruses have been listed as priority concerns in a recent WHO research and development blueprint initiative for emerging infectious diseases. In this Personal View, we explain further the rationale for expanding the genetic database of NIRVs and emphasise the need for greater investment in this area of research.

  View details for DOI 10.1016/S1473-3099(17)30238-4

  View details for PubMedID 28457597

• Malaria and Chikungunya Detected Using Molecular Diagnostics Among Febrile Kenyan Children OPEN FORUM INFECTIOUS DISEASES Waggoner, J., Brichard, J., Mutuku, F., Ndenga, B., Heath, C., Mohamed-Hadley, A., Sahoo, M. K., Vulule, J., Lefterova, M., BanaeiA, N., Mukoko, D., Pinsky, B. A., LaBeaud, A. 2017; 4 (3): ofx110

  Abstract

  In sub-Saharan Africa, malaria is frequently overdiagnosed as the cause of an undifferentiated febrile illness, whereas arboviral illnesses are presumed to be underdiagnosed.Sera from 385 febrile Kenyan children, who presented to 1 of 4 clinical sites, were tested using microscopy and real-time molecular assays for dengue virus (DENV), chikungunya virus (CHIKV), malaria, and Leptospira.Malaria was the primary clinical diagnosis for 254 patients, and an arboviral infection (DENV or CHIKV) was the primary diagnosis for 93 patients. In total, 158 patients (41.0%) had malaria and 32 patients (8.3%) had CHIKV infections. Compared with real-time polymerase chain reaction, microscopy demonstrated a percent positive agreement of 49.7%. The percentage of malaria cases detected by microscopy varied significantly between clinical sites. Arboviral infections were the clinical diagnosis for patients on the Indian Ocean coast (91 of 238, 38.2%) significantly more often than patients in the Lake Victoria region (2 of 145, 1.4%; P < .001). However, detection of CHIKV infections was significantly higher in the Lake Victoria region (19 of 145 [13.1%] vs 13 of 239 [5.4%]; P = .012).The clinical diagnosis of patients with an acute febrile illness, even when aided by microscopy, remains inaccurate in malaria-endemic areas, contributing to inappropriate management decisions.

  View details for PubMedID 28702473

• Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections. Ophthalmology Doan, T., Acharya, N. R., Pinsky, B. A., Sahoo, M. K., Chow, E. D., Banaei, N., Budvytiene, I., Cevallos, V., Zhong, L., Zhou, Z., Lietman, T. M., DeRisi, J. L. 2017

  View details for DOI 10.1016/j.ophtha.2017.03.045

  View details for PubMedID 28526549

• Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing. Journal of clinical microbiology Sahoo, M. K., Holubar, M., Huang, C., Mohamed-Hadley, A., Liu, Y., Waggoner, J. J., Troy, S. B., Garcia-Garcia, L., Ferreyra-Reyes, L., Maldonado, Y., Pinsky, B. A. 2017

  Abstract

  Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication.

  View details for DOI 10.1128/JCM.00144-17

  View details for PubMedID 28468861

• Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine. Journal of virological methods Tan, S. K., Sahoo, M. K., Milligan, S., Taylor, N., Pinsky, B. A. 2017

  Abstract

  Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine.To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics(®) ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4°C, or -80°C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA).ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48hours (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4°C, suggesting that storage at 4°C can slow the progression of RNA degradation. Freezing urine samples at -80°C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3 days, 1/6 replicates at 10 days, and 1/3 replicates at 30 days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA.ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80°C for 10 days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing.

  View details for DOI 10.1016/j.jviromet.2017.04.018

  View details for PubMedID 28472623

• Pericardial Effusion Following Hematopoietic Cell Transplantation in Children and Young Adults Is Associated with Increased Risk of Mortality. Biology of blood and marrow transplantation Cox, K., Punn, R., Weiskopf, E., Pinsky, B. A., Kharbanda, S. 2017

  Abstract

  Hematopoietic cell transplantation (HCT) is curative for many pediatric malignant and nonmalignant disorders but is associated with significant morbidity and mortality, including the development of pericardial effusion (PEF). We report the results of a retrospective chart review performed to assess the incidence, risk factors, and prognostic significance of PEF in pediatric HCT recipient at Lucile Packard Children's Hospital of Stanford University. A total of 119 patients undergoing HCT between January 2010 and December 2013 were selected through the hospital's Pediatric Stem Cell Transplant Program database. A retrospective chart review, including review of documentation, correspondence, imaging reports, laboratory values, and death records, was performed to collect data. The overall incidence of PEF in our population was 21%. Risk factors for the development of PEF included unrelated donor transplants and cord blood as the stem cell source (P = .005), whereas HLA mismatch approached significance (P = .05). The risk for development of PEF was found to not be significantly associated with acute or chronic graft-versus-host disease (GVHD), age at transplantation, sex, conditioning regimen, or viral reactivation status. Of interest, 6 of the 119 patients were found to have transplant-associated thrombotic microangiopathy (TA-TMA). Four of those 6 patients developed PEF, suggesting TA-TMA as a risk factor for PEF. Eight of the 25 patients who developed PEF (32%) required pericardiocentesis. Five out of the 8 patients requiring pericardiocentesis died owing to causes unrelated to the procedure or to PEF itself. Pericardial fluid testing in 4 of these patients (50%) was positive for human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, and/or adenovirus. Finally, of significant interest, patients with PEF had a statistically significant higher likelihood of mortality compared with those without PEF (44% versus 17%; P = .007).

  View details for DOI 10.1016/j.bbmt.2017.03.028

  View details for PubMedID 28390986

• Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE Kim, K. Y., Le, Q., Yom, S. S., Pinsky, B. A., Bratman, S. V., Ng, R. W., El Mubarak, H. S., Chan, K., Sander, M., Conley, B. A. 2017; 109 (4)

  Abstract

  Clinical studies have shown plasma Epstein-Barr virus (EBV) DNA level to be an independent prognostic biomarker for nasopharyngeal carcinoma (NPC). However, the proportion of NPC patients whose tumors are associated with EBV vary with geographic location, and there are a variety of assays for plasma EBV. To develop the level of evidence needed to demonstrate the clinical utility of plasma EBV DNA detection for NPC patients and encourage widespread adoption of this biomarker test in clinical laboratories, validated harmonized assays are needed. In 2015, the National Cancer Institute (NCI) convened a Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer, where experts in head and neck oncology and laboratory medicine addressed the limitations of currently available polymerase chain reaction-based EBV DNA quantitation assays and discussed strategies for advancing the development of harmonized EBV DNA assays and their appropriate clinical use. This article presents the key recommendations to direct future efforts in assay harmonization and validation.

  View details for PubMedID 28376165

• Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus. Journal of clinical microbiology Tan, S. K., Milligan, S., Sahoo, M. K., Taylor, N., Pinsky, B. A. 2017; 55 (3): 923-930

  Abstract

  Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R(2) = 0.99; secondary standard, Y = 1.04X - 0.26, R(2) = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

  View details for DOI 10.1128/JCM.02315-16

  View details for PubMedID 28053213

  View details for PubMedCentralID PMC5328461

• T cells expand after solid organ transplantation in the absence of CMV disease. American journal of transplantation Higdon, L. E., Trofe-Clark, J., Liu, S., Margulies, K. B., Sahoo, M. K., Blumberg, E., Pinsky, B. A., Maltzman, J. S. 2017

  Abstract

  Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid-organ transplant recipients. Approximately 60% of adults are CMV seropositive indicating previous exposure. Following resolution of primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8(+) T cell responses to CMV polypeptides IE-1 and pp65 were analyzed in sixteen CMV seropositive renal and cardiac transplant recipients longitudinally pre- and post-transplant. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis and CMV viral load monitoring, with approximately half receiving T cell depleting induction therapy. The frequency of CMV-responsive CD8(+) T cells, defined by production of effector molecules in response to CMV peptides, increased during the course of a year post-transplant. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV-responsive T cell population post-transplant. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1111/ajt.14227

  View details for PubMedID 28199780

• High incidence of Zika virus infection detected in plasma and cervical cytology specimens from pregnant women in Guayaquil, Ecuador. American journal of reproductive immunology Zambrano, H., Waggoner, J., León, K., Pinsky, B., Vera, K., Schettino, M., Rivera, L., Landivar, J., Granda, M., Lee, A., Mor, G. 2017; 77 (2)

  Abstract

  Zika virus (ZIKV) infection during pregnancy has been linked to severe birth defects, and the epidemiologic situation of the ZIKV epidemic in Ecuador is poorly understood. Guayaquil, Ecuador, has a tropical climate and experiences frequent outbreaks of dengue and chikungunya virus, and in December 2015, ZIKV was identified. Given the well-documented effects of ZIKV in pregnancy, including microcephaly, we tested for the presence of ZIKV in both plasma and cervical cytology of pregnant women. We report the identification of a population of pregnant women with a high incidence of ZIKV infection detected in the plasma and lower reproductive tract. A case-control study was performed to determine the incidence of ZIKV infection among low-income, pregnant women at risk for preterm delivery compared to matched controls. Plasma and cervical cytology specimens were tested for ZIKV by rRT-PCR. Fifty-nine pregnant women were enrolled. The incidence of ZIKV was 54% (32/59) overall: 18/31 (58.1%) in cases and 14/28 (50.0%) in controls. ZIKV detection in plasma and cervical cytology specimens demonstrated good agreement. Overall, outcomes for neonates born to ZIKV-positive and ZIKV-negative mothers were similar. However, two neonates were born with microcephaly to case mothers who were ZIKV positive. We report a high incidence of ZIKV infection (54%) in a distinctive population in Guayaquil, Ecuador. We identify ZIKV in cervical samples that correlates with ZIKV in the plasma. These data raise concerns regarding the breadth of the ZIKV epidemic in Ecuador and demonstrate the utility of cervical cytology specimens for ZIKV testing.

  View details for DOI 10.1111/aji.12630

  View details for PubMedID 28177195

• Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards. Journal of clinical microbiology Hayden, R. T., Sun, Y., Tang, L., Procop, G. W., Hillyard, D. R., Pinsky, B. A., Young, S. A., Caliendo, A. M. 2017; 55 (2): 423-430

  Abstract

  It has been hoped that the recent availability of WHO quantitative standards would improve interlaboratory agreement for viral load testing; however, insufficient data are available to evaluate whether this has been the case. Results from 554 laboratories participating in proficiency testing surveys for quantitative PCR assays of cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), adenovirus (ADV), and human herpesvirus 6 (HHV6) were evaluated to determine overall result variability and then were stratified by assay manufacturer. The impact of calibration to international units/ml (CMV and EBV) on variability was also determined. Viral loads showed a high degree of interlaboratory variability for all tested viruses, with interquartile ranges as high as 1.46 log10 copies/ml and the overall range for a given sample up to 5.66 log10 copies/ml. Some improvement in result variability was seen when international units were adopted. This was particularly the case for EBV viral load results. Variability in viral load results remains a challenge across all viruses tested here; introduction of international quantitative standards may help reduce variability and does so more or less markedly for certain viruses.

  View details for DOI 10.1128/JCM.02044-16

  View details for PubMedID 27852673

  View details for PubMedCentralID PMC5277511

• IgG antibodies to dengue enhanced for FcγRIIIA binding determine disease severity. Science (New York, N.Y.) Wang, T. T., Sewatanon, J., Memoli, M. J., Wrammert, J., Bournazos, S., Bhaumik, S. K., Pinsky, B. A., Chokephaibulkit, K., Onlamoon, N., Pattanapanyasat, K., Taubenberger, J. K., Ahmed, R., Ravetch, J. V. 2017; 355 (6323): 395-398

  Abstract

  Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor FcγRIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

  View details for DOI 10.1126/science.aai8128

  View details for PubMedID 28126818

• Clinical characteristics and outcomes of pediatric patients with CMV DNA detection in bronchoalveolar lavage fluid. Pediatric pulmonology Burgener, E. B., Waggoner, J., Pinsky, B. A., Chen, S. F. 2017; 52 (1): 112-118

  Abstract

  Cytomegalovirus (CMV) infection can cause severe pulmonary disease in immunocompromised patients. There are no standard diagnostic criteria for CMV pulmonary disease beyond histopathology findings on lung tissue, which is challenging to obtain in pediatric patients. Bronchoalveolar lavage (BAL) fluid is easier to obtain. Since CMV remains latent after primary infection and can potentially reactivate due to any inflammatory response, CMV detection in BAL specimen may not indicate acute CMV pulmonary disease. Thus, we describe the clinical manifestations and outcomes of pediatric patients with CMV detection in BAL fluid.We reviewed the clinical, radiologic, and laboratory data of patients <19 years old with a BAL specimen positive for CMV during a 5-year period.Thirty-four encounters in 29 patients were found with CMV detected in their BAL specimen. Half (17/34) of the encounters were in immunocompromised patients. CMV, polymerase chain reaction (PCR) was the most common positive test. Forty-seven percent of the patients had other infections detected in BAL specimens. The majority of patients were never treated for CMV and resolved their acute respiratory illness. Only one patient had probable CMV pulmonary disease.CMV is frequently recovered from BAL specimens but does not usually indicate acute CMV pulmonary disease. We would suggest that other diagnoses be considered first, even if CMV is recovered. Pediatr Pulmonol. 2016; 9999:XX-XX. © 2016 Wiley Periodicals, Inc.

  View details for DOI 10.1002/ppul.23494

  View details for PubMedID 27280337

• Development of a Real-Time Reverse Transcription Polymerase Chain Reaction for O'nyong-nyong Virus and Evaluation with Clinical and Mosquito Specimens from Kenya AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Waggoner, J., Heath, C., Ndenga, B., Mutuku, F., Sahoo, M. K., Mohamed-Hadley, A., Vulule, J., Mukoko, D., LaBeaud, A., Pinsky, B. A. 2017; 97 (1): 121–24

  Abstract

  O'nyong-nyong virus (ONNV), an alphavirus closely related to chikungunya virus (CHIKV), has been the documented cause of two large outbreaks in east Africa; however, little is known about the contribution of ONNV to cases of acute febrile illness during interepidemic periods. An ONNV real-time reverse transcription polymerase chain reaction (rRT-PCR) was developed and evaluated using clinical and mosquito pool samples. The ONNV rRT-PCR linear range extended from 8.0 to 2.0 log10 copies/μL, and the lower limit of 95% detection was 22.4 copies/μL. No cases of ONNV infection were identified in serum from 385 Kenyan children who presented with an acute febrile illness. Additionally, ONNV was not detected in 120 mosquito pools collected in coastal and western Kenya. The ONNV rRT-PCR demonstrated good analytical sensitivity when performed in monoplex or as a component of an ONNV-CHIKV duplex assay. This assay should provide a useful diagnostic for the detection of ONNV in surveillance studies.

  View details for PubMedID 28719301

• Closing the Brief Case: Confirmed Positive HIV-1 Serological Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D. n., Aziz, N. n., Pinsky, B. A. 2017; 55 (12): 3566–67

  View details for PubMedID 29180505

• The Brief Case: Confirmed Positive HIV-1 Serologic Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D. n., Aziz, N. n., Pinsky, B. A. 2017; 55 (12): 3316–20

  View details for PubMedID 29180504

• Zika Virus, Chikungunya Virus, and Dengue Virus in Cerebrospinal Fluid from Adults with Neurological Manifestations, Guayaquil, Ecuador. Frontiers in microbiology Acevedo, N., Waggoner, J., Rodriguez, M., Rivera, L., Landivar, J., Pinsky, B., Zambrano, H. 2017; 8: 42-?

  Abstract

  Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) have been associated with clinical presentations that involve acute neurological complaints. In the current study, we identified ZIKV, CHIKV, and DENV in cerebrospinal fluid (CSF) samples from patients admitted to the Hospital Luis Vernaza (Guayaquil, Ecuador) to the Emergency Room or the Intensive Care Unit, with neurological symptoms and/or concern for acute arboviral infections. Viral RNA from one or more virus was detected in 12/16 patients. Six patients were diagnosed with meningitis or encephalitis, three with Guillain-Barré Syndrome, and one with CNS vasculitis. Two additional patients had a systemic febrile illness including headache that prompted testing of CSF. Two patients, who were diagnosed with encephalitis and meningoencephalitis, died during their hospitalizations. These cases demonstrate the breadth and significance of neurological manifestations associated with ZIKV, CHIKV, and DENV infections.

  View details for DOI 10.3389/fmicb.2017.00042

  View details for PubMedID 28174559

  View details for PubMedCentralID PMC5258761

• The Human Virome - Implications for Clinical Practice in Transplantation Medicine. Journal of clinical microbiology Tan, S. K., Relman, D. A., Pinsky, B. A. 2017

  Abstract

  Advances in DNA sequencing technology have provided an unprecedented opportunity to study the human virome. Transplant recipients and other immunocompromised hosts are at particular risk for developing virus-related pathology; thus, the impact of the virome on health and disease may be even more relevant in this population. Here we discuss technical considerations in studying the human virome, the current literature on the virome in transplant recipients, and near future applications of sequence-based findings that can further our understanding of viruses in transplantation medicine.

  View details for PubMedID 28724557

• Prevalence of Drug-Resistant Minority Variants in Untreated HIV-1-Infected Individuals With and Those Without Transmitted Drug Resistance Detected by Sanger Sequencing. The Journal of infectious diseases Clutter, D. S., Zhou, S. n., Varghese, V. n., Rhee, S. Y., Pinsky, B. A., Jeffrey Fessel, W. n., Klein, D. B., Spielvogel, E. n., Holmes, S. P., Hurley, L. B., Silverberg, M. J., Swanstrom, R. n., Shafer, R. W. 2017; 216 (3): 387–91

  Abstract

  Minority variant human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are associated with an increased risk of virological failure during treatment with NNRTI-containing regimens. To determine whether individuals to whom variants with isolated NNRTI-associated drug resistance were transmitted are at increased risk of virological failure during treatment with a non-NNRTI-containing regimen, we identified minority variant resistance mutations in 33 individuals with isolated NNRTI-associated transmitted drug resistance and 49 matched controls. We found similar proportions of overall and nucleoside reverse transcriptase inhibitor-associated minority variant resistance mutations in both groups, suggesting that isolated NNRTI-associated transmitted drug resistance may not be a risk factor for virological failure during treatment with a non-NNRTI-containing regimen.

  View details for PubMedID 28859436

• Zika Virus and Chikungunya Virus CoInfections: A Series of Three Cases from a Single Center in Ecuador. American journal of tropical medicine and hygiene Zambrano, H., Waggoner, J. J., Almeida, C., Rivera, L., Benjamin, J. Q., Pinsky, B. A. 2016; 95 (4): 894-896

  Abstract

  Zika virus (ZIKV) and chikungunya virus (CHIKV) cocirculate throughout much of the tropical Western Hemisphere; however, few cases of coinfection with these two pathogens have been reported. Herein, we describe three cases of ZIKV-CHIKV coinfection detected at a single center in Ecuador: a patient who developed symptoms on postoperative day 5 from an orthopedic procedure, a woman who had traveled to Ecuador for fertility treatment, and a woman who was admitted for Guillain-Barré syndrome and had ZIKV and CHIKV detected in serum and cerebrospinal fluid. All cases were diagnosed using a multiplex real-time reverse transcription polymerase chain reaction, and ZIKV viremia was detected as late as 16 days after symptom onset. These cases demonstrate the varied clinical presentation of ZIKV-CHIKV coinfections as well as the importance of multiplexed arboviral testing for these pathogens.

  View details for PubMedID 27402518

• Homotypic Dengue Virus Reinfections in Nicaraguan Children. journal of infectious diseases Waggoner, J. J., Balmaseda, A., Gresh, L., Sahoo, M. K., Montoya, M., Wang, C., Abeynayake, J., Kuan, G., Pinsky, B. A., Harris, E. 2016; 214 (7): 986-993

  Abstract

   Infection with one of four related dengue virus serotypes (DENV-1-4) is thought to result in life-long immunity to re-infection with the same serotype (homotypic DENV re-infection). Archived serum samples, collected as part of an ongoing pediatric dengue cohort study in Nicaragua, were tested for DENV by real-time RT-PCR. Samples were collected from 2,892 children who presented with an acute febrile illness clinically attributed to a non-dengue cause ("C" cases). Test results were added to a database of previously-identified symptomatic dengue cases in the cohort to identify repeat infections. Four patients with homotypic DENV re-infections were identified and confirmed among 29 repeat DENV infections with serotype confirmation (13.8% of repeat symptomatic infections). Homotypic re-infections with DENV-1, -2, and -3 occurred 325-621 days after the initial infection. Each patient experienced one symptomatic dengue case and one DENV-positive C case, and two patients presented with symptomatic dengue during their second infection. These DENV-positive C cases did not elicit long-lived humoral immune responses, despite viremia up to 6.44 log10 copies/mL of serum. We describe the first set of virologically confirmed homotypic DENV re-infections. Such cases challenge the current understanding of DENV immunity and have important implications for modeling DENV transmission.

  View details for DOI 10.1093/infdis/jiw099

  View details for PubMedID 26984144

• Molecular diagnostics for human leptospirosis. Current opinion in infectious diseases Waggoner, J. J., Pinsky, B. A. 2016; 29 (5): 440-445

  Abstract

  The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods.Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described.Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

  View details for DOI 10.1097/QCO.0000000000000295

  View details for PubMedID 27537829

• Evaluation of the Aptima HIV-1 Quant Dx Assay Using Plasma and Dried Blood Spots. Journal of clinical microbiology Sahoo, M. K., Varghese, V., White, E., Winslow, M., Katzenstein, D. A., Shafer, R. W., Pinsky, B. A. 2016; 54 (10): 2597-2601

  Abstract

  HIV-1 RNA quantitation in plasma, or virus load testing, is the primary method by which the response to antiretroviral therapy is monitored. Here we describe evaluation of the Aptima HIV-1 Quant Dx assay (Aptima) performed on the automated Panther system. The clinical performance of Aptima was compared to that of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test v2.0 (CAP/CTM) using 162 EDTA plasma samples collected from patients undergoing HIV-1 monitoring. Overall agreement was 84.0% (136/162), with a kappa statistic of 0.723 (standard error, 0.047; 95% confidence interval [CI], 0.630 to 0.815), indicating substantial agreement. Using the 86 clinical samples quantifiable by both methods, Passing-Bablok regression revealed a regression line of Y = (1.069 × X) - 0.346 (95% CI of the slope [1.003 to 1.139] and intercept [-0.666 to -0.074]), and Bland-Altman analysis demonstrated a mean difference (Aptima-CAP/CTM) of -0.075 log10 copies/ml (95% limits of agreement of -0.624 to 0.475), consistent with negative bias. Comparison of Aptima results for paired dried blood spot (DBS) and plasma specimens archived from participants in the Peninsula AIDS Research Cohort Study (PARC) demonstrated an overall agreement of 94.7% (90/95) when 1,000 copies/ml was used as the threshold. In conclusion, the Aptima HIV-1 Quant Dx assay provides a suitable alternative for HIV-1 monitoring in plasma and DBS.

  View details for DOI 10.1128/JCM.01569-16

  View details for PubMedID 27535684

  View details for PubMedCentralID PMC5035416

• Q148N, a Novel Integrase Inhibitor Resistance Mutation Associated with Low-Level Reduction in Elvitegravir Susceptibility AIDS RESEARCH AND HUMAN RETROVIRUSES Varghese, V., Pinsky, B. A., Smith, D. S., Klein, D., Shafer, R. W. 2016; 32 (7): 702-704

  Abstract

  The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ∼2.4-4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy.

  View details for DOI 10.1089/aid.2016.0038

  View details for Web of Science ID 000379609100012

  View details for PubMedID 27009474

• Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses EMERGING INFECTIOUS DISEASES Waggoner, J. J., Gresh, L., Mohamed-Hadley, A., Ballesteros, G., Vargas Davila, M. J., Tellez, Y., Sahoo, M. K., Balmaseda, A., Harris, E., Pinsky, B. A. 2016; 22 (7): 1295-1297

  Abstract

  Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  View details for DOI 10.3201/eid2207.160326

  View details for Web of Science ID 000378563900030

  View details for PubMedID 27184629

  View details for PubMedCentralID PMC4918162

• Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection JOURNAL OF CLINICAL VIROLOGY Waggoner, J. J., Ballesteros, G., Gresh, L., Mohamed-Hadley, A., Tellez, Y., Sahoo, M. K., Abeynayake, J., Balmaseda, A., Harris, E., Pinsky, B. A. 2016; 78: 57-61

  Abstract

  Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR).From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/μL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.

  View details for DOI 10.1016/j.jcv.2016.01.007

  View details for Web of Science ID 000374480000013

  View details for PubMedID 26991052

• Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis. Open forum infectious diseases Tan, S. K., Burgener, E. B., Waggoner, J. J., Gajurel, K., Gonzalez, S., Chen, S. F., Pinsky, B. A. 2016; 3 (1): ofv212-?

  Abstract

  Background.  Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods.  Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results.  Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions.  Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.

  View details for DOI 10.1093/ofid/ofv212

  View details for PubMedID 26885542

• Respiratory Viruses CLINICAL VIROLOGY MANUAL, 5TH EDITION Robinson, C., Loeffelholz, M. J., Pinsky, B. A., Loeffelholz, M. J., Hodinka, R. L., Young, S. A., Pinsky, B. A. 2016: 257-276

  View details for DOI 10.1128/9781555819156.ch19

  View details for Web of Science ID 000703170200018

• Current and future molecular diagnostics for ocular infectious diseases. Current opinion in ophthalmology Doan, T. n., Pinsky, B. A. 2016; 27 (6): 561–67

  Abstract

  Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections.Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection.Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

  View details for PubMedID 27585214

• Delayed Diagnosis of Tuberculous Meningitis Misdiagnosed as Herpes Simplex Virus-1 Encephalitis With the FilmArray Syndromic Polymerase Chain Reaction Panel Open Forum Infectious Diseases Gomez, C. A., Pinsky, B. A., Liu, A., Banaei, N. 2016: ofw245

  Abstract

  The FilmArray meningitis/encephalitis (ME) panel is a novel syndromic, nucleic acid amplification test for diagnosis of acute meningitis and encephalitis. Emerging data on its performance are concerning for false-positive results. We present a case of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray ME panel. Strategies to mitigate erroneous results are discussed.

  View details for DOI 10.1093/ofid/ofw245

  View details for PubMedCentralID PMC5437853

• Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus (vol 10, e0142216, 2015) PLOS ONE Pinsky, B. A., Sahoo, M. K., Sandlund, J., Kleman, M., Kulkarni, M., Grufman, P., Nygren, M., Kwiatkowski, R., Baron, E., Tenover, F., Denison, B., Higuchi, R., Van Atta, R., Beer, N., Carrillo, A., Naraghi-Arani, P., Mire, C. E., Ranadheera, C., Grolla, A., Lagerqvist, N., Persing, D. H. 2015; 10 (12): e0145896

  View details for DOI 10.1371/journal.pone.0145896

  View details for Web of Science ID 000367092300097

  View details for PubMedID 26690909

  View details for PubMedCentralID PMC4686324

• Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus PLOS ONE Pinsky, B. A., Sahoo, M. K., Sandlund, J., Kleman, M., Kulkarni, M., Grufman, P., Nygren, M., Kwiatkowski, R., Baron, E. J., Tenover, F., Denison, B., Higuchi, R., van Atta, R., Beer, N. R., Carrillo, A. C., Naraghi-Arani, P., Mire, C. E., Ranadheera, C., Grolla, A., Lagerqvist, N., Persing, D. H. 2015; 10 (11)

  Abstract

  The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples.This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs.In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

  View details for DOI 10.1371/journal.pone.0142216

  View details for Web of Science ID 000364480900020

  View details for PubMedID 26562786

  View details for PubMedCentralID PMC4643052

• Next-Generation Sequencing for Infectious Disease Diagnosis and Management A Report of the Association for Molecular Pathology JOURNAL OF MOLECULAR DIAGNOSTICS Lefterova, M. I., Suarez, C. J., Banaei, N., Pinsky, B. A. 2015; 17 (6): 623-634

  Abstract

  Next-generation sequencing (NGS) technologies are increasingly being used for diagnosis and monitoring of infectious diseases. Herein, we review the application of NGS in clinical microbiology, focusing on genotypic resistance testing, direct detection of unknown disease-associated pathogens in clinical specimens, investigation of microbial population diversity in the human host, and strain typing. We have organized the review into three main sections: i) applications in clinical virology, ii) applications in clinical bacteriology, mycobacteriology, and mycology, and iii) validation, quality control, and maintenance of proficiency. Although NGS holds enormous promise for clinical infectious disease testing, many challenges remain, including automation, standardizing technical protocols and bioinformatics pipelines, improving reference databases, establishing proficiency testing and quality control measures, and reducing cost and turnaround time, all of which would be necessary for widespread adoption of NGS in clinical microbiology laboratories.

  View details for DOI 10.1016/j.jmoldx.2015.07.004

  View details for Web of Science ID 000363830000001

  View details for PubMedID 26433313

• Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients. Journal of clinical microbiology Waggoner, J. J., Okangba, C., Mohamed-Hadley, A., Lefterova, M. I., Banaei, N., Oyibo, W., Pinsky, B. A. 2015; 53 (11): 3596-3600

  Abstract

  Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n = 182) or plasma (n = 148) from 317 patients was tested; the average sample volume was 70 μl (range, 5 to 300 μl). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with ≥75 μl of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.

  View details for DOI 10.1128/JCM.01876-15

  View details for PubMedID 26354810

  View details for PubMedCentralID PMC4609704

• Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing. Journal of clinical microbiology Sahoo, M. K., Tan, S. K., Chen, S. F., Kapusinszky, B., Concepcion, K. R., Kjelson, L., Mallempati, K., Farina, H. M., Fernández-Viña, M., Tyan, D., Grimm, P. C., Anderson, M. W., Concepcion, W., Pinsky, B. A. 2015; 53 (10): 3226-3233

  Abstract

  BK virus (BKV) infection and end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep sequencing methodology and bioinformatics pipeline that identifies BKV variants across the genome and at BKV-specific HLA-A2, HLA-B0702, and HLA-B08 restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets and fragmentation libraries were sequenced on the Ion Torrent PGM. An error model and variant calling algorithm were developed to accurately identify rare variants. 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range 2-37, interquartile range 10), with the majority of variants (77%) detected at a frequency of less than 5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

  View details for DOI 10.1128/JCM.01385-15

  View details for PubMedID 26202116

• Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid Versus Serum or Plasma. American journal of tropical medicine and hygiene Waggoner, J. J., Soda, E. A., Seibert, R., Grant, P., Pinsky, B. A. 2015; 93 (2): 238-240

  Abstract

  Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia.

  View details for DOI 10.4269/ajtmh.15-0174

  View details for PubMedID 26033024

  View details for PubMedCentralID PMC4530740

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient JOURNAL OF CLINICAL MICROBIOLOGY Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015; 53 (8): 2749-2752

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for DOI 10.1128/JCM.00834-15

  View details for Web of Science ID 000358290200055

• Cytomegalovirus load at treatment initiation is predictive of time to resolution of viremia and duration of therapy in hematopoietic cell transplant recipients JOURNAL OF CLINICAL VIROLOGY Tan, S. K., Waggoner, J. J., Pinsky, B. A. 2015; 69: 179-183

  Abstract

  Preemptive antiviral therapy relies on viral load measurements and is the mainstay of cytomegalovirus (CMV) prevention in hematopoietic cell transplant (HCT) recipients. However, optimal CMV levels for the initiation of preemptive therapy have not been defined.The objectives of our work were to evaluate the relationship between plasma CMV DNA levels at initiation of preemptive therapy with time to resolution of viremia and duration of treatment.Retrospective analysis of HCT recipients undergoing serial CMV PCR testing between June 2011 and June 2014 was performed.221 HCT recipients underwent preemptive therapy for 305 episodes of CMV viremia. Median time to resolution was shorter when treatment was initiated at lower CMV levels (15 days at 135-440 international units (IU)/mL, 18 days at 441-1000IU/mL, and 21 days at >1000IU/mL, P<.001). Prolonged viremia lasting >30 days occurred less frequently when treatment was initiated at 135-440IU/mL compared to 441-1000IU/mL and >1000IU/mL (1%, 15%, 24%, P<.001). Median treatment duration was also shorter in the lower viral load groups (28, 34, 37 days, P<.001).Initiation of preemptive therapy at low CMV levels was associated with shorter episodes of viremia and courses of antiviral therapy. These data support the utility of initiating preemptive CMV therapy at viral loads as low as 135IU/mL in HCT recipients.

  View details for DOI 10.1016/j.jcv.2015.06.006

  View details for Web of Science ID 000358318400037

• Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing PLOS ONE Waggoner, J. J., Balassiano, I., Mohamed-Hadley, A., Vital-Brazil, J. M., Sahoo, M. K., Pinsky, B. A. 2015; 10 (7)

  Abstract

  Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.

  View details for DOI 10.1371/journal.pone.0132988

  View details for Web of Science ID 000358197600195

  View details for PubMedCentralID PMC4503744

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient. Journal of clinical microbiology Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for DOI 10.1128/JCM.00834-15

  View details for PubMedID 25994169

• How great is the threat of chikungunya virus? Expert review of anti-infective therapy Waggoner, J. J., Pinsky, B. A. 2015; 13 (3): 291-293

  Abstract

  In the last decade, chikungunya virus has emerged from an obscure arbovirus that caused limited outbreaks of disease in Africa and Asia to the cause of a pandemic affecting millions of people and spanning five continents. Two separate chikungunya virus genotypes have been responsible for outbreaks during this period, including strains adapted to transmission in Aedes albopictus mosquitoes. Further spread of this virus into new regions of the Western Hemisphere is predicted during the present rainy season in the tropics, and recurrent viral introductions and disease outbreaks, as occurred in Réunion in 2010, should be expected. Chikungunya virus no longer simply threatens; it has arrived as a significant, global pathogen.

  View details for DOI 10.1586/14787210.2015.995634

  View details for PubMedID 25537007

• Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR. Diagnostic microbiology and infectious disease Waggoner, J. J., Sahadeo, N. S., Brown, A., Mohamed-Hadley, A., Hadley, D., Carrington, L., Carrington, C. V., Pinsky, B. A. 2015; 81 (2): 105-106

  Abstract

  Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01).

  View details for DOI 10.1016/j.diagmicrobio.2014.10.001

  View details for PubMedID 25533614

  View details for PubMedCentralID PMC4297500

• Norovirus CLINICAL MICROBIOLOGY REVIEWS Robilotti, E., Deresinski, S., Pinsky, B. A. 2015; 28 (1): 134-164

  Abstract

  Norovirus, an RNA virus of the family Caliciviridae, is a human enteric pathogen that causes substantial morbidity across both health care and community settings. Several factors enhance the transmissibility of norovirus, including the small inoculum required to produce infection (<100 viral particles), prolonged viral shedding, and its ability to survive in the environment. In this review, we describe the basic virology and immunology of noroviruses, the clinical disease resulting from infection and its diagnosis and management, as well as host and pathogen factors that complicate vaccine development. Additionally, we discuss overall epidemiology, infection control strategies, and global reporting efforts aimed at controlling this worldwide cause of acute gastroenteritis. Prompt implementation of infection control measures remains the mainstay of norovirus outbreak management.

  View details for DOI 10.1128/CMR.00075-14

  View details for Web of Science ID 000347460400006

  View details for PubMedCentralID PMC4284304

• Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing. PloS one Waggoner, J. J., Balassiano, I., Mohamed-Hadley, A., Vital-Brazil, J. M., Sahoo, M. K., Pinsky, B. A. 2015; 10 (7)

  Abstract

  Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.

  View details for DOI 10.1371/journal.pone.0132988

  View details for PubMedID 26177295

  View details for PubMedCentralID PMC4503744

• Norovirus. Clinical microbiology reviews Robilotti, E., Deresinski, S., Pinsky, B. A. 2015; 28 (1): 134-164

  Abstract

  Norovirus, an RNA virus of the family Caliciviridae, is a human enteric pathogen that causes substantial morbidity across both health care and community settings. Several factors enhance the transmissibility of norovirus, including the small inoculum required to produce infection (<100 viral particles), prolonged viral shedding, and its ability to survive in the environment. In this review, we describe the basic virology and immunology of noroviruses, the clinical disease resulting from infection and its diagnosis and management, as well as host and pathogen factors that complicate vaccine development. Additionally, we discuss overall epidemiology, infection control strategies, and global reporting efforts aimed at controlling this worldwide cause of acute gastroenteritis. Prompt implementation of infection control measures remains the mainstay of norovirus outbreak management.

  View details for DOI 10.1128/CMR.00075-14

  View details for PubMedID 25567225

  View details for PubMedCentralID PMC4284304

• Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis PLOS ONE Waggoner, J. J., Balassiano, I., Abeynayake, J., Sahoo, M. K., Mohamed-Hadley, A., Liu, Y., Vital-Brazil, J. M., Pinsky, B. A. 2014; 9 (11)

  Abstract

  Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  View details for DOI 10.1371/journal.pone.0112356

  View details for Web of Science ID 000344863100077

  View details for PubMedCentralID PMC4224423

• Commutability of the Epstein-Barr Virus WHO International Standard across Two Quantitative PCR Methods JOURNAL OF CLINICAL MICROBIOLOGY Abeynayake, J., Johnson, R., Libiran, P., Sahoo, M. K., Cao, H., Bowen, R., Chan, K. C., Quynh-Thu Le, Q. T., Pinsky, B. A. 2014; 52 (10): 3802-3804

  Abstract

  The commutability of international reference standards is critical for ensuring quantitative agreement across different viral load assays. Here, we demonstrate the commutability of the Epstein-Barr virus (EBV) WHO international standard for the BamHI-W and artus EBV assays.

  View details for DOI 10.1128/JCM.01676-14

  View details for Web of Science ID 000342371700042

  View details for PubMedCentralID PMC4187779

• Encephalitis caused by chikungunya virus in a traveler from the kingdom of tonga. Journal of clinical microbiology Nelson, J., Waggoner, J. J., Sahoo, M. K., Grant, P. M., Pinsky, B. A. 2014; 52 (9): 3459-3461

  Abstract

  Febrile travelers from countries with unique endemic pathogens pose a significant diagnostic challenge. In this report, we describe the case of a Tongan man presenting with fever, rash, and altered mental status. The diagnosis of Chikungunya encephalitis was made using a laboratory-developed real-time RT-PCR and serologic testing.

  View details for DOI 10.1128/JCM.01288-14

  View details for PubMedID 24958800

• Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing. Antimicrobial agents and chemotherapy Chou, S., Ercolani, R. J., Sahoo, M. K., Lefterova, M. I., Strasfeld, L. M., Pinsky, B. A. 2014; 58 (8): 4697-4702

  Abstract

  In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.

  View details for DOI 10.1128/AAC.03214-14

  View details for PubMedID 24890586

• Multiplex nucleic Acid amplification test for diagnosis of dengue Fever, malaria, and leptospirosis. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Balassiano, I., Lefterova, M., Sahoo, M. K., Liu, Y., Vital-Brazil, J. M., Gresh, L., Balmaseda, A., Harris, E., Banaei, N., Pinsky, B. A. 2014; 52 (6): 2011-2018

  Abstract

  Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  View details for DOI 10.1128/JCM.00341-14

  View details for PubMedID 24671788

• Reply to "Inconclusive reverse transcription-PCR assay comparison for dengue virus detection and serotyping". Journal of clinical microbiology Waggoner, J. J., Pinsky, B. A. 2014; 52 (5): 1801-1802

  View details for DOI 10.1128/JCM.00013-14

  View details for PubMedID 24744403

• Evaluation of serial urine viral cultures for the diagnosis of cytomegalovirus infection in neonates and infants. Pediatric and developmental pathology Chisholm, K. M., Aziz, N., McDowell, M., Guo, F. P., Srinivas, N., Benitz, W. E., Norton, M. E., Gutierrez, K., Folkins, A. K., Pinsky, B. A. 2014; 17 (3): 176-180

  Abstract

  Cytomegalovirus (CMV) is the most common cause of congenital infection worldwide. Urine viral culture is the standard for CMV diagnosis in neonates and infants. The objectives of this study were to compare the performance of serial paired rapid shell vial cultures (SVC) and routine viral cultures (RVC), and to determine the optimal number of cultures needed to detect positive cases. From 2001 to 2011, all paired CMV SVC and RVC performed on neonates and infants less than 100 days of age were recorded. Testing episodes were defined as sets of cultures performed within 7 days of one another. A total of 1264 neonates and infants underwent 1478 testing episodes; 68 (5.4%) had at least one episode with a positive CMV culture. In episodes where CMV was detected before day 21 of life, the first specimen was positive in 100% (16/16) of cases. When testing occurred after 21 days of life, the first specimen was positive in 82.7% (43/52) of cases, requiring three cultures to reach 100% detection. The SVC was more prone to assay failure than RVC. Overall, when RVC was compared to SVC, there was 86.0% positive agreement and 99.9% negative agreement. In conclusion, three serial urine samples are necessary for detection of CMV in specimens collected between day of life 22 and 99, while one sample may be sufficient on or before day of life 21. Though SVC was more sensitive than RVC, the risk of SVC failure supports the use of multimodality testing to optimize detection.

  View details for DOI 10.2350/14-01-1432-OA.1

  View details for PubMedID 24617645

• Human Papilloma Virus is not Prevalent in Nevus Sebaceus PEDIATRIC DERMATOLOGY Kim, D., Benjamin, L. T., Sahoo, M. K., Kim, J., Pinsky, B. A. 2014; 31 (3): 326-330

  Abstract

  Nevus sebaceus (NS) is a common congenital cutaneous hamartoma that typically presents on the scalp and face at birth or in early childhood. Occasionally NS can be associated with the Schimmelpenning-Feuerstein-Mims syndrome, which presents with concomitant severe neurologic, skeletal, cardiovascular, ophthalmic, and genitourologic disorders. In a previous study, maternal transmission of the human papillomavirus (HPV) and infection of ectodermal stem cells by HPV was postulated to result in the development of NS. In this study we aimed to determine the incidence of HPV infection in pediatric NS samples to further clarify the potential link between HPV and the pathogenesis of NS. NS tissue samples (N = 16) were analyzed for HPV DNA using type-specific, real-time polymerase chain reaction (PCR) targeting HPV 6, 11, 16, and 18 and conventional PCR with modified general primers designed for broad-range HPV detection. The tissues were also histologically evaluated for evidence of HPV infection. HPV DNA was not detected in any of the NS tissue samples using PCR and HPV-associated histopathologic changes were absent in all 16 NS tissues. HPV infection is an unlikely etiologic cause of NS.

  View details for DOI 10.1111/pde.12249

  View details for PubMedID 24224641

• Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues. PeerJ Waggoner, J. J., Pinsky, B. A. 2014; 2

  Abstract

  Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types.

  View details for DOI 10.7717/peerj.334

  View details for PubMedID 24765569

  View details for PubMedCentralID PMC3994632

• Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis. PloS one Waggoner, J. J., Balassiano, I., Abeynayake, J., Sahoo, M. K., Mohamed-Hadley, A., Liu, Y., Vital-Brazil, J. M., Pinsky, B. A. 2014; 9 (11)

  Abstract

  Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  View details for DOI 10.1371/journal.pone.0112356

  View details for PubMedID 25379890

  View details for PubMedCentralID PMC4224423

• Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues. PeerJ Waggoner, J. J., Pinsky, B. A. 2014; 2: e334

  Abstract

  Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types.

  View details for DOI 10.7717/peerj.334

  View details for PubMedID 24765569

  View details for PubMedCentralID PMC3994632

• BK Polyomavirus Subtype III in a Pediatric Renal Transplant Patient with Nephropathy. Journal of clinical microbiology Kapusinszky, B., Chen, S. F., Sahoo, M. K., Lefterova, M. I., Kjelson, L., Grimm, P. C., Kambham, N., Concepcion, W., Pinsky, B. A. 2013; 51 (12): 4255-4258

  Abstract

  BK polyomavirus (BKV) is an emerging pathogen in immunocompromised individuals. BKV subtype III is rarely identified and has not previously been associated with disease. Here we provide the whole-genome sequence of a subtype III BKV from a pediatric kidney transplant patient with polyomavirus-associated nephropathy.

  View details for DOI 10.1128/JCM.01801-13

  View details for PubMedID 24048534

  View details for PubMedCentralID PMC3838085

• Detection of cytomegalovirus drug resistance mutations by next-generation sequencing. Journal of clinical microbiology Sahoo, M. K., Lefterova, M. I., Yamamoto, F., Waggoner, J. J., Chou, S., Holmes, S. P., Anderson, M. W., Pinsky, B. A. 2013; 51 (11): 3700-3710

  Abstract

  Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations.

  View details for DOI 10.1128/JCM.01605-13

  View details for PubMedID 23985916

• Profound plasmacytosis in a patient with dengue. International journal of hematology Ewalt, M. D., Abeynayake, J., Waggoner, J. J., Pinsky, B. A., Ohgami, R. S. 2013; 98 (5): 518-519

  View details for DOI 10.1007/s12185-013-1452-3

  View details for PubMedID 24114365

• Comparison of the FDA-Approved CDC DENV-1-4 Real-Time Reverse Transcription-PCR with a Laboratory-Developed Assay for Dengue Virus Detection and Serotyping. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Guo, F. P., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 51 (10): 3418-3420

  Abstract

  Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.

  View details for DOI 10.1128/JCM.01359-13

  View details for PubMedID 23903549

• Diagnosis of Congenital CMV Using PCR Performed on Formalin-fixed, Paraffin-embedded Placental Tissue. American journal of surgical pathology Folkins, A. K., Chisholm, K. M., Guo, F. P., McDowell, M., Aziz, N., Pinsky, B. A. 2013; 37 (9): 1413-1420

  Abstract

  Congenital cytomegalovirus (CMV) infection may be asymptomatic until hearing loss manifests in childhood. Because diagnosis of congenital CMV requires viral detection within an infant's first 21 days of life, CMV polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded (FFPE) placental tissue provides a unique opportunity to identify congenital exposure in cases in which CMV is not initially suspected. To assess the utility of this approach, a database of all CMV cultures performed from July 2001 to March 2012 was used to identify infants in whom urine CMV cultures were obtained within 100 days of life. Corresponding placentas were then identified through the pathology database. The database was also queried to identify placentas in which CMV immunohistochemical analysis had been performed. CMV PCR was positive in FFPE placental tissue from 100% (5/5) of cases in which the first urine culture collected before the first 21 days of life was positive. Placentas from 20 infants with negative CMV urine cultures were CMV PCR negative. Interestingly, CMV was detected in 12.5% (1/8) of placentas in which the first CMV-positive urine culture was collected after the first 21 days of life. Furthermore, 4% (1/26) of placentas with chronic villitis by histology (no urine cultures available) were CMV PCR positive. In the 10 CMV PCR-positive placentas, including 3 cases of fetal demise, CMV immunohistochemistry was positive in just 6 cases. These results suggest that the confirmation of CMV exposure in utero by PCR of FFPE placental tissue provides a useful adjunct to histologic evaluation and may identify infants requiring close clinical follow-up.

  View details for DOI 10.1097/PAS.0b013e318290f171

  View details for PubMedID 23797721

• Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 51 (7): 2172-2181

  Abstract

  A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic-acid amplification tests (NAATs). However, reports describing the direct comparison of different NAATs are limited. In this study, we report the design of an internally-controlled, real-time reverse-transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two-hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay; a commercially-produced, internally-controlled DENV rRT-PCR (the Altona assay); a widely-used hemi-nested RT-PCR; and a serotype-specific, multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 7.0 to 1.0 log10 complimentary DNA (cDNA) equivalents/μL and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μL depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved more clinically sensitive than either the Altona or hemi-nested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (p≤0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day five of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed, molecular diagnosis of DENV infection can be provided.

  View details for DOI 10.1128/JCM.00548-13

  View details for PubMedID 23637298

• A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue. American journal of surgical pathology Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. 2013; 37 (7): 995-1000

  Abstract

  Cytomegalovirus (CMV) can precipitate and exacerbate gastrointestinal (GI) mucosal injury. The gold standard for CMV detection in formalin-fixed, paraffin-embedded (FFPE) tissue is immunohistochemistry (IHC). Although CMV polymerase chain reaction (PCR) on fresh tissue may be a valuable adjunct to IHC, its utility is unknown for FFPE tissues. We therefore evaluated quantitative, real-time CMV PCR in a total of 102 FFPE GI biopsy specimens from 74 patients with a history of hematopoietic stem cell or solid organ transplant, inflammatory bowel disease, human immunodeficiency virus infection, or unspecified colitis. CMV DNA was detected by PCR in 90.9% (30/33) of IHC-positive, 14.5% (8/55) of IHC-negative, and 20.0% (1/5) of IHC-equivocal FFPE tissues. Quantitation of CMV DNA copies normalized to β-globin demonstrated a wide range of values (median 0.276; range, 0.0004 to 144.50). Importantly, 93.3% (14/15) of patients with IHC-positive, active colitis showed no evidence of CMV in matched concurrent, histologically normal biopsies tested by PCR. These results suggest that CMV PCR on FFPE GI biopsies complements IHC and has the potential to identify additional patients who may benefit from anti-CMV therapy.

  View details for DOI 10.1097/PAS.0b013e31827fcc33

  View details for PubMedID 23648457

• p16 Is Superior to ProEx C in Identifying High-grade Squamous Intraepithelial Lesions (HSIL) of the Anal Canal AMERICAN JOURNAL OF SURGICAL PATHOLOGY Bala, R., Pinsky, B. A., Beck, A. H., Kong, C. S., Welton, M. L., Longacre, T. A. 2013; 37 (5): 659-668

  Abstract

  Although the incidence of human papillomavirus (HPV)-associated anal neoplasia is increasing, interobserver and intraobserver reproducibility in the grading of biopsy specimens from this area remains unacceptably low. Attempts to produce a more reproducible grading scheme have led to the use of biomarkers for the detection of high-risk HPV (HR-HPV). We evaluated the performance of standard morphology and biomarkers p16, ProEx C, and Ki-67 in a set of 75 lesions [17 nondysplastic lesions, 23 low-grade squamous intraepithelial lesions (LSIL)/condyloma, 20 high-grade squamous intraepithelial lesions (HSIL), 15 invasive squamous cell carcinomas] from the anal and perianal region in 65 patients and correlated these findings with HPV subtype on the basis of a type-specific multiplex real-time polymerase chain reaction assay designed to detect HR-HPV. A subset of cases with amplifiable HPV DNA was also sequenced. HSIL was typically flat (15/20), and only a minority (4/20) had koilocytes. In contrast, only 1 LSIL was flat (1/23), and the remainder were exophytic. The majority of LSIL had areas of koilocytic change (20/23). HR-HPV DNA was detected in the majority (89%) of invasive carcinomas and HSIL biopsies, 86% and 97% of which were accurately labeled by strong and diffuse block-positive p16 and ProEx C, respectively. LSIL cases, however, only infrequently harbored HR-HPV (13%); most harbored low-risk HPV (LR-HPV) types 6 and 11. Within the LSIL group, p16 outperformed ProEx C, resulting in fewer false-positive cases (5% vs. 75%). Ki-67 was also increased in HR-HPV-positive lesions, although biopsies with increased inflammation and reactive changes also showed higher Ki-67 indices. These data suggest that strong and diffuse block-positive nuclear and cytoplasmic labeling with p16 is a highly specific biomarker for the presence of HR-HPV in anal biopsies and that this finding correlates with high-grade lesions.

  View details for DOI 10.1097/PAS.0b013e31828706c0

  View details for PubMedID 23552383

• An international collaboration to harmonize the quantitative plasma Epstein-Barr virus DNA assay for future biomarker-guided trials in nasopharyngeal carcinoma. Clinical cancer research Le, Q., Zhang, Q., Cao, H., Cheng, A., Pinsky, B. A., Hong, R., Chang, J. T., Wang, C., Tsao, K., Lo, Y. D., Lee, N., Ang, K. K., Chan, A. T., Chan, K. C. 2013; 19 (8): 2208-2215

  Abstract

  Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation.Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples.The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA.Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.

  View details for DOI 10.1158/1078-0432.CCR-12-3702

  View details for PubMedID 23459720

  View details for PubMedCentralID PMC3630245

• CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients JOURNAL OF CLINICAL VIROLOGY Cardenoso, L., Pinsky, B. A., Lautenschlager, I., Aslam, S., Cobb, B., Vilchez, R. A., Hirsch, H. H. 2013; 56 (2): 108-112

  Abstract

  Sensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice. OBJECTIVES/STUDY DESIGN: Using 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS(®) AMPLICOR MONITOR CMV tests.The median time between transplantation and testing samples was 57 days (range, 0-207 days). The median CMV load (log(10)) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (-0.15 [95% CI, -0.18 to -0.13] copies/mL), but slope differences indicated only limited co-linearity.The CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.

  View details for DOI 10.1016/j.jcv.2012.10.001

  View details for Web of Science ID 000313565100004

  View details for PubMedID 23146665

• An International Multicenter Performance Analysis of Cytomegalovirus Load Tests CLINICAL INFECTIOUS DISEASES Hirsch, H. H., Lautenschlager, I., Pinsky, B. A., Cardenoso, L., Aslam, S., Cobb, B., Vilchez, R. A., Valsamakis, A. 2013; 56 (3): 367-373

  Abstract

  Quantification of cytomegalovirus (CMV) load is central to the management of CMV infections in immunocompromised patients, but quantitative results currently differ significantly across methods and laboratories.The COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV test), developed using the first World Health Organization CMV standard in the calibration process, was compared to local assays used by 5 laboratories at transplant centers in the United States and Europe. Blinded plasma panels (n = 90) spiked with 2.18-6.7 log(10) copies/mL and clinical plasma samples from immunocompromised patients (n = 660) were tested.Observed mean panel member concentrations by site and 95% confidence intervals (CIs) of the data combined across sites were narrower for CAP/CTM CMV test compared with local assays. The 95% CI in log(10) copies/mL of the combined data per panel member for CAP/CTM CMV test vs comparator assays was .17 vs 1.5 at 2.18 log(10) copies/mL; .14 vs .52 at 2.74 log(10) copies/mL; .16 vs .6 at 3.3 log(10) copies/mL; .2 vs 1.11 at 4.3 log(10) copies/mL; .21 vs 1.13 at 4.7 log(10) copies/mL; and .18 vs 1.4 at 6.7 log(10) copies/mL. In clinical specimens, constant and variable quantification differences between the CAP/CTM CMV test and comparator assays were observed.High interlaboratory agreement and precision of CAP/CTM CMV test results across 5 different laboratories over 4 orders of magnitude suggest that this assay could be valuable in prospective studies identifying clinical viral load thresholds for CMV treatment.

  View details for DOI 10.1093/cid/cis900

  View details for Web of Science ID 000313617400014

  View details for PubMedID 23097587

  View details for PubMedCentralID PMC3540041

• Severe Hepatitis Associated with an Echovirus 18 Infection in an Immune-Compromised Adult JOURNAL OF CLINICAL MICROBIOLOGY Lefterova, M. I., Rivetta, C., George, T. I., Pinsky, B. A. 2013; 51 (2): 684-687

  Abstract

  Enteroviruses are recognized as important pathogens in pediatric patients; however, they are often overlooked as etiologic agents of disease in adults. Here, we report a case of echovirus 18-associated severe systemic infection and acute liver failure in an adult hematopoietic stem cell transplant recipient. Additionally, we illustrate the utility of molecular methods for the detection and typing of enteroviral infections.

  View details for DOI 10.1128/JCM.02405-12

  View details for PubMedID 23175267

• Laboratory-Developed L1 Sequencing and Type-Specific, Real-Time Polymerase Chain Reaction for the Detection and Typing of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Mills, A., Balasubramaniam, R., Longacre, T. A., Kong, C. S., Pinsky, B. A. 2013; 137 (1): 50-54

  Abstract

  The detection and typing of high-risk and low-risk human papillomavirus (HPV) in archival formalin-fixed, paraffin-embedded tissues by nucleic acid amplification testing is an important adjunct to immunohistochemical staining in evaluation of squamous cell proliferations of the oropharynx, larynx, and anal canal.To evaluate semiautomated, xylene-free extraction from formalin-fixed, paraffin-embedded tissues combined with laboratory-developed HPV L1 sequencing and type-specific HPV 6, 11, 16, and 18 real-time polymerase chain reaction for identification and typing of HPV in the clinical laboratory.We evaluated the adequacy of extraction using β-globin amplification and compared L1 sequencing and real-time polymerase chain reaction methods for typing accuracy using 68 formalin-fixed, paraffin-embedded tissues, including 56 anorectal biopsy or surgical resection specimens and 12 laryngeal papilloma specimens from patients with recurrent respiratory papillomatosis.Adequate DNA was obtained from 68 of 68 specimens analyzed and all were HPV positive. In 47 cases where L1 sequencing demonstrated that the predominant HPV type was 6, 11, 16, or 18, type-specific, real-time polymerase chain reaction provided concordant results. Sequencing revealed additional low-risk (HPV 40) and high-risk HPV types (HPV 31, 33, 56, and 58) in anorectal specimens, whereas HPV 6 or 11 were the types found in laryngeal papillomas.Both L1 sequencing and type-specific, real-time polymerase chain reaction are suitable methods for routine HPV testing of formalin-fixed, paraffin-embedded tissues in a clinical laboratory setting.

  View details for DOI 10.5858/arpa.2011-0392-OA

  View details for PubMedID 23276174

• Profound plasmacytosis in a patient with dengue International Journal of Hematology Ewalt, M. D., Abeynayake, J., Waggoner, J. ., Pinsky, B. ., Ohgami, R. S. 2013: 518–19
• Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses. PLoS neglected tropical diseases Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Pierro, A. M., Gaibani, P., Guo, F. P., Sambri, V., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 7 (4)

  Abstract

  Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log₁₀ cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

  View details for DOI 10.1371/journal.pntd.0002116

  View details for PubMedID 23638191

  View details for PubMedCentralID PMC3630127

• Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B JOURNAL OF VIROLOGICAL METHODS DiMaio, M. A., Sahoo, M. K., Waggoner, J., Pinsky, B. A. 2012; 186 (1-2): 137-140

  Abstract

  Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B.

  View details for DOI 10.1016/j.jviromet.2012.07.023

  View details for Web of Science ID 000312763600024

  View details for PubMedID 22841669

• Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma JOURNAL OF CLINICAL MICROBIOLOGY Waggoner, J., Ho, D. Y., Libiran, P., Pinsky, B. A. 2012; 50 (7): 2378-2383

  Abstract

  The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log(10) copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to ≥ 200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

  View details for DOI 10.1128/JCM.06800-11

  View details for Web of Science ID 000307360800033

  View details for PubMedID 22518866

  View details for PubMedCentralID PMC3405616

• Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Cao, H., Banh, A., Kwok, S., Shi, X., Wu, S., Krakow, T., Khong, B., Bavan, B., Bala, R., Pinsky, B. A., Colevas, D., Pourmand, N., Koong, A. C., Kong, C. S., Quynh-Thu Le, Q. T. 2012; 82 (3): E351-E358

  Abstract

  To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy.We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16(INK4a) staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy.HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using (18)F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis.Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  View details for DOI 10.1016/j.ijrobp.2011.05.061

  View details for PubMedID 21985946

• A Synonymous Change in the Influenza A Virus Neuraminidase Gene Interferes with PCR-Based Subtyping and Oseltamivir Resistance Mutation Detection JOURNAL OF CLINICAL MICROBIOLOGY Trevino, C., Bihon, S., Pinsky, B. A. 2011; 49 (8): 3101-3102

  View details for DOI 10.1128/JCM.00642-11

  View details for Web of Science ID 000293221900067

  View details for PubMedID 21697334

  View details for PubMedCentralID PMC3147745

• Ultrasensitive Detection of Drug-Resistant Pandemic 2009 (H1N1) Influenza A Virus by Rare-Variant-Sensitive High-Resolution Melting-Curve Analysis JOURNAL OF CLINICAL MICROBIOLOGY Chen, N., Pinsky, B. A., Lee, B. P., Lin, M., Schrijver, I. 2011; 49 (7): 2602-2609

  Abstract

  Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.

  View details for DOI 10.1128/JCM.00277-11

  View details for Web of Science ID 000292276200035

  View details for PubMedID 21543559

  View details for PubMedCentralID PMC3147852

• The Persistence of Influenza Infection Response EMERGING INFECTIOUS DISEASES Pinsky, B., Baron, E. J. 2010; 16 (11): 1818-1819

  View details for DOI 10.3201/eid1611.101431

  View details for Web of Science ID 000283699700043

• Long-term Shedding of Influenza A Virus in Stool of Immunocompromised Child EMERGING INFECTIOUS DISEASES Pinsky, B. A., Mix, S., Rowe, J., Ikemoto, S., Baron, E. J. 2010; 16 (7): 1165-1167

  Abstract

  In immunocompromised patients, influenza infection may progress to prolonged viral shedding from the respiratory tract despite antiviral therapy. We describe chronic influenza A virus infection in an immunocompromised child who had prolonged shedding of culturable influenza virus in stool.

  View details for DOI 10.3201/eid1607.091248

  View details for Web of Science ID 000279522200024

  View details for PubMedID 20587197

  View details for PubMedCentralID PMC3321893

• Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci JOURNAL OF CLINICAL MICROBIOLOGY Nguyen, D. T., Yeh, E., Perry, S., Luo, R. F., Pinsky, B. A., Lee, B. P., Sisodiya, D., Baron, E. J., Banaei, N. 2010; 48 (3): 785-790

  Abstract

  Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to beta-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.

  View details for DOI 10.1128/JCM.02150-09

  View details for Web of Science ID 000274996200016

  View details for PubMedID 20071556

  View details for PubMedCentralID PMC2832423

• Preferential Lower Respiratory Tract Infection in Swine-Origin 2009 A(H1N1) Influenza CLINICAL INFECTIOUS DISEASES Yeh, E., Luo, R. F., Dyner, L., Hong, D. K., Banaei, N., Baron, E. J., Pinsky, B. A. 2010; 50 (3): 391-394

  Abstract

  We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.

  View details for DOI 10.1086/649875

  View details for Web of Science ID 000273500300014

  View details for PubMedID 20047483

• Comparison of Real-Time PCR and Conventional Biochemical Methods for Identification of Staphylococcus lugdunensis JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Samson, D., Ghafghaichi, L., Baron, E. J., Banaei, N. 2009; 47 (11): 3472-3477

  Abstract

  Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory.

  View details for DOI 10.1128/JCM.00342-09

  View details for Web of Science ID 000271373000013

  View details for PubMedID 19741081

  View details for PubMedCentralID PMC2772579

• First documentation of isoniazid reversion in Mycobacterium tuberculosis INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE Richardson, E. T., Lin, S. G., Pinsky, B. A., Desmond, E., Banaei, N. 2009; 13 (11): 1347-1354

  Abstract

  Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure.To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost.Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress.Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG codon 300 which reverted from GGG (glycine, G) to a wild-type codon, TGG (tryptophan, W). The H37Rv DeltakatG::katG W300G mutant was resistant to INH, but also showed a deficiency in coping with oxidative stress.This study confirms that, in the absence of INH pressure, some INH-resistant mutants will revert to a drug-susceptible phenotype. This finding may have broader implications for INH-resistant strains and for the clinically useful lifespan of INH.

  View details for Web of Science ID 000271883400007

  View details for PubMedID 19861005

• Protein Phosphatase 1 Regulates Exit from the Spindle Checkpoint in Budding Yeast CURRENT BIOLOGY Pinsky, B. A., Nelson, C. R., Biggins, S. 2009; 19 (14): 1182-1187

  Abstract

  Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores, although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled. The target of the checkpoint is the Cdc20 protein, which initiates the anaphase-promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement. However, less is known about the mechanisms that silence the checkpoint after kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase 1 (PP1) homolog, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation as a result of a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.

  View details for DOI 10.1016/j.cub.2009.06.043

  View details for Web of Science ID 000268530200024

  View details for PubMedID 19592248

  View details for PubMedCentralID PMC2731492

• Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests PLOS ONE Yeh, E., Pinsky, B. A., Banaei, N., Baron, E. J. 2009; 4 (7)

  Abstract

  Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

  View details for DOI 10.1371/journal.pone.0006141

  View details for Web of Science ID 000267806300010

  View details for PubMedID 19578541

  View details for PubMedCentralID PMC2700971

• Bartholin's abscess caused by hypermucoviscous Klebsiella pneumoniae JOURNAL OF MEDICAL MICROBIOLOGY Pinsky, B. A., Baron, E. J., Janda, J. M., Banaei, N. 2009; 58 (5): 671-673

  Abstract

  Klebsiella pneumoniae serogroups displaying the hypermucoviscosity phenotype are associated with a distinct clinical syndrome characterized by liver abscesses, bacteraemia and metastatic lesions. We describe here what we believe to be the first reported case of hypermucoviscous K. pneumoniae causing a superficial Bartholin's abscess in the absence of systemic involvement.

  View details for DOI 10.1099/jmm.0.006734-0

  View details for Web of Science ID 000266018900019

  View details for PubMedID 19369531

• Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Banaei, N. 2008; 46 (7): 2241-2246

  Abstract

  The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.

  View details for DOI 10.1128/JCM.00347-08

  View details for Web of Science ID 000258906800016

  View details for PubMedID 18508937

  View details for PubMedCentralID PMC2446918

• Glc7/protein phosphatase 1 regulatory subunits can oppose the Ipl1/aurora protein kinase by redistributing Glc7 MOLECULAR AND CELLULAR BIOLOGY Pinsky, B. A., Kotwaliwale, C. V., Tatsutani, S. Y., Breed, C. A., Biggins, S. 2006; 26 (7): 2648-2660

  Abstract

  Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.

  View details for DOI 10.1128/MCB.26.7.2648-2660.2006

  View details for Web of Science ID 000236312200017

  View details for PubMedID 16537909

  View details for PubMedCentralID PMC1430313

• The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores NATURE CELL BIOLOGY Pinsky, B. A., Kung, C., Shokat, K. M., Biggins, S. 2006; 8 (1): 78-U28

  Abstract

  The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.

  View details for DOI 10.1038/ncb1341

  View details for Web of Science ID 000234651500015

  View details for PubMedID 16327780

• The spindle checkpoint: tension versus attachment TRENDS IN CELL BIOLOGY Pinsky, B. A., Biggins, S. 2005; 15 (9): 486-493

  Abstract

  The spindle checkpoint ensures the fidelity of chromosome segregation by preventing cell-cycle progression until all the chromosomes make proper bipolar attachments to the mitotic spindle and come under tension. Despite significant advances in our understanding of spindle checkpoint function, the primary signal that activates the spindle checkpoint remains unclear. Whereas some experiments indicate that the checkpoint recognizes the lack of microtubule attachment to the kinetochore, others indicate that the checkpoint senses the absence of tension generated on the kinetochore by microtubules. The interdependence between tension and microtubule attachment make it difficult to determine whether these signals are separable. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we consider recent evidence that supports and opposes the hypothesis that defects in tension act as the primary checkpoint signal.

  View details for DOI 10.1016/j.tcb.2005.07.005

  View details for Web of Science ID 000232333700006

  View details for PubMedID 16084093

• An Mtw1 complex promotes kinetochore biorientation that is monitored by the lpl1/Aurora protein kinase DEVELOPMENTAL CELL Pinsky, B. A., Tatsutani, S. Y., Collins, K. A., Biggins, S. 2003; 5 (5): 735-745

  Abstract

  Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.

  View details for Web of Science ID 000186544400011

  View details for PubMedID 14602074

• Top-SUMO wrestles centromeric cohesion DEVELOPMENTAL CELL Pinsky, B. A., Biggins, S. 2002; 3 (1): 4-6

  Abstract

  Sister chromatid cohesion at the centromere is distinct from cohesion at the chromosome arms. In the June issue of Molecular Cell, Bachant et al. have shown that centromeric cohesion in budding yeast is specifically regulated by SUMO-1 modification of Topoisomerase II.

  View details for Web of Science ID 000176769500003

  View details for PubMedID 12110161

• Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brott, B. K., Pinsky, B. A., Erikson, R. L. 1998; 95 (3): 963-968

  Abstract

  Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase, Nlk, that is a murine homolog of the Drosophila nemo (nmo) gene. The Nlk amino acid sequence is 54. 5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4. 0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60-70% of Nlk is localized to the nucleus, whereas 30-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.

  View details for Web of Science ID 000071878500031

  View details for PubMedID 9448268

  View details for PubMedCentralID PMC18639