Current Role at Stanford

Basic Life Science Research Scientist
Lab manager

Education & Certifications

  • PhD, Central Drug Research Institute, Lucknow, India and, Jawaharlal Nehru University, New Delhi, India, Life Sciences (2010)

All Publications

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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

  • 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


    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

  • 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

  • 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


    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

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

  • 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


    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

  • 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


    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


    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

  • 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


     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

  • 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


    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

  • 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


     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

  • 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


    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


    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

  • 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)


    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

  • 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)


    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

  • 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)


    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

  • 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)


    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


    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


    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


    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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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): e2116


    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


    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