Niaz Banaei received his medical education from Stanford University and completed residency training in laboratory medicine at the University of California, San Francisco. He then completed a postdoctoral fellowship in infectious diseases at the New York University. He is currently an Associate Professor of Pathology and Medicine (Division of Infectious Diseases & Geographic Medicine) at Stanford University and is the Medical Director of the Clinical Microbiology Laboratory at Stanford Health Care. In addition, he is the Director of Stanford Clinical Microbiology Fellowship. He serves on the board of advisors for the Center for Disease Control Tuberculosis Epidemiologic Studies Consortium II (TBESC). His research interests include (1) development, assessment, and improvement of novel infectious diseases diagnostics, (2) enhancing the quality of C. difficile diagnostic results, and (3) characterization of M. tuberculosis virulence determinants. He was the recipient of Kenneth L. Vosti Infectious Diseases and Stanford University clinical pathology junior faculty teaching awards.
- Clinical Pathology
- Mycobacterium Infections
- Clinical Laboratory Techniques
Associate Program Director, Clinical Pathology Residency Training, Stanford University (2016 - Present) (2016 - Present)
Medical Director, Clinical Microbiology Laboratory, Stanford University (2007 - Present) (2007 - Present)
Director, Clinical Microbiology Fellowship, Stanford University (2016 - Present)
Residency:UCSF Clinical Pathology Residency (2003) CA
Internship:UCSF Clinical Pathology Residency (2002) CA
Fellowship:New York University (2007) NY
Board Certification: Clinical Pathology, American Board of Pathology (2004)
Medical Education:Stanford University School of Medicine (2001) CA
MD, Stanford University, Medicine (2001)
Current Research and Scholarly Interests
His research interests include (1) development, assessment, and improvement of novel infectious diseases diagnostics, (2) enhancing the quality of C. difficile diagnostic results, and (3) characterization of M. tuberculosis virulence determinants.
- Independent Studies (5)
Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe
SCIENCE TRANSLATIONAL MEDICINE
2018; 10 (454)
Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the β-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients' sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.
View details for DOI 10.1126/scitranslmed.aar4470
View details for Web of Science ID 000441579200005
View details for PubMedID 30111644
- Spiking of intravenous bags does not cause time-dependent microbial contamination: a preliminary report. Infection control and hospital epidemiology 2018: 1–2
Development of colorimetric sensor array for diagnosis of tuberculosis through detection of urinary volatile organic compounds.
Diagnostic microbiology and infectious disease
BACKGROUND: Top priorities for tuberculosis control and elimination include a simple, low-cost screening test using sputum and a non-sputum-based test in patients that do not produce sputum. The aim of this study was to evaluate the performance of a colorimetric sensor array (CSA) test, for analysis of volatile organic compounds in urine, in the diagnosis of pulmonary TB.MATERIAL AND METHODS: Urine samples were collected from individuals suspected of having pulmonary TB in Western Kenya. Reference methods included MGIT culture and/or Xpert MTB/RIF nucleic acid amplification test on sputa. Fresh urine samples were tested with the CSA, with acid and base and without an additive. The CSA were digitally imaged, and the resulting colorimetric response patterns were used for chemometric analysis. Sensitivity, specificity, and negative (NPV) and positive predictive (PPV) values were determined for HIV-positive and HIV-negative patients.RESULTS: In HIV-negative patients, the highest accuracy was obtained in urine samples pre-treated with a base, yielding a sensitivity, specificity, PPV, and NPV of 78.3% (65/83), 69.2% (54/78), 73.0% (n/89) and 75.0% (n/72). The highest sensitivity of 79.5% was achieved using sensor data from all three test conditions at a specificity of 65.4%. In HIV-positive subjects, the sensor performance was substantially lower with sensitivity, specificity, PPV, and NPV ranging from 48.3% to 62.3%, 54.1% to 74.0%, 55.9% to 64.2%, and 60.6% to 64.9%, respectively.CONCLUSION: The CSA fingerprint of urine headspace volatiles showed moderate accuracy in diagnosing TB in HIV-negative patients, but the sensor performance dropped substantially in HIV-coinfected patients. Further development of TB-responsive CSA indicators may improve the accuracy of CSA urine assay.
View details for DOI 10.1016/j.diagmicrobio.2018.06.014
View details for PubMedID 30025968
Evaluation of the Xpert MTB/RIF Performance on Tissues: Potential Impact on Airborne Infection Isolation at a Tertiary Cancer Care Center
INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY
2018; 39 (4): 462–66
OBJECTIVES In this study, we sought to evaluate the performance of the Xpert MTB/RIF (Cepheid) assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA on fresh and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from oncology patients in an area with a low prevalence of tuberculosis. We also aimed to retrospectively assess the potential impact of Xpert MTB/RIF on the duration of airborne infection isolation (AII). SETTING A 473-bed, tertiary-care cancer center in New York City. DESIGN A total of 203 tissue samples (101 FFPE and 102 fresh) were tested using Xpert MTB/RIF, including 133 pulmonary tissue samples (65.5%) and 70 extrapulmonary tissue samples (34.5%). Acid-fast bacilli (AFB) culture was used as the diagnostic gold standard. The limit of detection (LOD) and reproducibility were also evaluated for both samples types using contrived specimens. The potential impact of the Xpert MTB PCR assay on tissue samples from AII patients on AII duration was retrospectively assessed. RESULTS Using the Xpert MTB/RIF for fresh tissue specimens, the sensitivity was 50% (95% CI, 1.3%-98.7%) and the specificity was 99% (95% CI, 94.5%-99.9%). For FFPE tissue specimens, the sensitivity was 100% (95% CI, 63.1%-100%) and the specificity was 98.3% (95% CI, 95.5%-100%. The LOD was 103 colony-forming units (CFU)/mL for both fresh and FFPE tissue specimens, and the Xpert MTB/RIF was 100% reproducible at concentrations 10 times that of the LOD. With an expected turnaround time of 24 hours, the Xpert MTB PCR could decrease the duration of AII from a median of 8 days to a median of 1 day. CONCLUSIONS The Xpert MTB/RIF assay offers a valid option for ruling out Mycobacterium tuberculosis complex (MTBC) on tissue samples from oncology patients and for minimizing AII resource utilization. Infect Control Hosp Epidemiol 2018;39:462-466.
View details for DOI 10.1017/ice.2018.7
View details for Web of Science ID 000428538000012
View details for PubMedID 29444723
- Answer to the letter to the editor of MN Capoor et al. concerning "Ribosomal PCR assay of excised intervertebral discs from patients undergoing single-level primary lumbar microdiscectomy'' by Alamin TF, Munoz M, Zagel A, et al.: Eur Spine J; 2017 EUROPEAN SPINE JOURNAL 2018; 27 (2): 518–19
- Trypanosoma cruzi Reactivation in the Brain. The New England journal of medicine 2018; 378 (19): 1824
Low Yield of FilmArray GI Panel in Hospitalized Patients with Diarrhea: an Opportunity for Diagnostic Stewardship Intervention.
Journal of clinical microbiology
2018; 56 (3)
The FilmArray GI panel (BioFire Diagnostics, Salt Lake City, UT) is a multiplex, on-demand, sample-to-answer, real-time PCR assay for the syndromic diagnosis of infectious gastroenteritis that has become widely adopted and, in some instances, has replaced conventional stool culture and parasite exams. Conventional testing has historically been restricted among hospitalized patients due to low diagnostic yield, but it is not known whether use of the FilmArray GI panel should be circumscribed. Cary-Blair stool samples submitted for FilmArray GI panel in adult patients admitted to an academic hospital from August 2015 to January 2017 were included in this study. Of 481 tests performed >72 h after admission, 29 (6.0%) were positive, all for a single target, excludingClostridium difficileWhen follow-up tests beyond the first positive per hospitalization were excluded, 20 (4.8%) of 414 tests were positive. There was no difference in yield by immune status. Most targets detected were viral (79% of all positives [n= 23] and 70% in unique patients [n= 14]). All four cases positive for a bacterial target could not be confirmed and presentation was atypical, suggesting possible false positives. After removing potential false positives and chronic viral shedders, the yield was 3.0% (12/406). Repeat testing performed >72 h after admission and following a negative result within the first 72 h was done in 19 patients and 100% (22/22) remained negative. The FilmArray GI panel has low yield in adult patients hospitalized for >72 h, similar to conventional stool microbiology tests, and it is reasonable to restrict its use in this population.
View details for DOI 10.1128/JCM.01558-17
View details for PubMedID 29237784
Microfluidics for Combating Antimicrobial Resistance
TRENDS IN BIOTECHNOLOGY
2017; 35 (12): 1129–39
The ever-growing threat of antimicrobial resistance (AMR) demands immediate countermeasures. With its novelty and enabling features including downscaled analysis, precisely controlled local environment, and enhanced speed, accuracy, and cost-efficiency, microfluidics has demonstrated potential in several key areas, including furthering our understanding of bacteria, developing better susceptibility testing tools, and overcoming obstacles in discovery and research of new antibiotics. While ample research results in the field of microfluidics are available, their transformation into practical application is still lagging far behind. We believe that the challenge of AMR will give microfluidics a much-needed opportunity to leap from research papers to true productivity, and gain wider acceptance as a mature technology.
View details for DOI 10.1016/j.tibtech.2017.07.008
View details for Web of Science ID 000415306900005
View details for PubMedID 29153761
Clostridium difficile PCR Cycle Threshold Predicts Free Toxin
JOURNAL OF CLINICAL MICROBIOLOGY
2017; 55 (9): 2651–60
There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.
View details for DOI 10.1128/JCM.00563-17
View details for Web of Science ID 000408223700012
View details for PubMedID 28615471
View details for PubMedCentralID PMC5648702
- Detecting New Mycobacterium tuberculosis Infection Time for a More Nuanced Interpretation of QuantiFERON Conversions AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 2017; 196 (5): 546–47
Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting.
Journal of clinical microbiology
2017; 55 (6): 1650-1657
Although launched in 2015, little is known about the accuracy of QuantiFERON-TB Gold-Plus (QFT-Plus) for diagnosis of latent M. tuberculosis infection (LTBI). Unlike its predecessor, QFT-Plus utilizes two antigen tubes to elicit an immune response from CD4(+) and CD8(+) T lymphocytes. We conducted a cross-sectional study in low-risk health care workers (HCWs) at a single U.S. center to compare QFT-Plus to QuantiFERON-TB Gold in-tube (QFT). A total of 989 HCWs were tested with both QFT and QFT-Plus. Risk factors for LTBI were obtained from a questionnaire. QFT-Plus was considered positive if either antigen tube 1 (TB1) or TB2 tested positive, per the manufacturer's recommendations, or if both TB1 and TB2 tested positive, using a conservative definition. Results were compared using Cohen's kappa and linear regression, respectively. Agreement of QFT with QFT-Plus was high, at 95.6% (95% confidence interval [CI], 94.3 to 96.9; kappa, 0.57). The majority of discordant results between QFT and QFT-Plus TB1 (84.8%) and QFT and QFT-Plus TB2 (88.6%) fell within the range of 0.2 to 0.7 IU/ml. The positivity rate in 626 HCWs with no identifiable risk factors and no self-reported history of positive LTBI tests was 2.1% (CI, 1.0 to 3.2) and 3.0% (CI, 1.7 to 4.3) with QFT and QFT-Plus, respectively. A conservative definition of a QFT-Plus-positive result yielded a positivity rate of 1.0% (CI, 0.2 to 1.7; P value of 0.0002 versus QFT-Plus and 0.07 versus QFT). On follow-up testing, of 11 HCWs with discordant QFT-Plus results, 90.9% (10/11) had a negative QFT result. The QFT-Plus assay showed a high degree of agreement with QFT in U.S. HCWs. A conservative interpretation of QFT-Plus eliminated nearly all nonreproducible positive results in low-risk HCWs. Larger studies are needed to validate the latter finding and to more clearly define conditions under which a conservative interpretation can be used to minimize nonreproducible positive results in low-risk populations.
View details for DOI 10.1128/JCM.02498-16
View details for PubMedID 28298455
- Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections. Ophthalmology 2017
Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing.
European urology focus
2017; 3 (2-3): 293–99
BACKGROUND: Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship.OBJECTIVE: To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin.DESIGN, SETTING, AND PARTICIPANTS: Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth.MEASUREMENTS: Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports.RESULTS AND LIMITATIONS: For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use.CONCLUSIONS: The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI.PATIENT SUMMARY: We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections.
View details for DOI 10.1016/j.euf.2015.12.010
View details for PubMedID 28753748
Clostridium difficile rates in asymptomatic and symptomatic hospitalized patients using nucleic acid testing.
Diagnostic microbiology and infectious disease
2017; 87 (4): 365-370
The Clostridium difficile rate in symptomatic patients represents both those with C. difficile infection (CDI) and those with colonization. To predict the extent of CDI overdiagnosis, we compared the asymptomatic colonization rate to the symptomatic positivity rate in hospitalized patients using nucleic acid testing.Between July 2014 and April 2015, formed stool samples were collected from asymptomatic patients after admission to 3 hospital wards at the Stanford Hospital. Stool samples from symptomatic patients with suspected CDI in the same wards were collected for testing per provider order. The GeneXpert C. difficile tcdB polymerase chain reaction (PCR) assay (Cepheid, Sunnyvale, CA, USA) was performed on all stool samples and PCR cycle threshold was used as a measure of genomic equivalents. Chart review was performed to obtain clinical history and medication exposure.We found an asymptomatic C. difficile carriage rate of 11.8% (43/365) (95% confidence interval [CI], 8.5-15.1%) and a positivity rate in symptomatic patients of 15.4% (54/351) (95% CI, 11.6-19.2%; P=0.19). The median PCR cycle thresholds was not significantly different between asymptomatic carriers and symptomatic positives (29.5 versus 27.3; P=0.07). Among asymptomatic patients, 11.6% (5/43) of carriers and 8.4% (27/322; P=0.56) of noncarriers subsequently became symptomatic CDI suspects within the same hospitalization. Single and multivariate analysis did not identify any demographic or clinical factors as being significantly associated with C. difficile carriage.Asymptomatic C. difficile carriage rate was similar to symptomatic positivity rate. This suggests the majority of PCR-positive results in symptomatic patients are likely due to C. difficile colonization. Disease-specific biomarkers are needed to accurately diagnose patients with C. difficile disease.
View details for DOI 10.1016/j.diagmicrobio.2016.12.014
View details for PubMedID 28087170
- Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas MYCOPATHOLOGIA 2017; 182 (3-4): 315-318
New and developing diagnostic technologies for urinary tract infections.
Nature reviews. Urology
Timely and accurate identification and determination of the antimicrobial susceptibility of uropathogens is central to the management of UTIs. Urine dipsticks are fast and amenable to point-of-care testing, but do not have adequate diagnostic accuracy or provide microbiological diagnosis. Urine culture with antimicrobial susceptibility testing takes 2-3 days and requires a clinical laboratory. The common use of empirical antibiotics has contributed to the rise of multidrug-resistant organisms, reducing treatment options and increasing costs. In addition to improved antimicrobial stewardship and the development of new antimicrobials, novel diagnostics are needed for timely microbial identification and determination of antimicrobial susceptibilities. New diagnostic platforms, including nucleic acid tests and mass spectrometry, have been approved for clinical use and have improved the speed and accuracy of pathogen identification from primary cultures. Optimization for direct urine testing would reduce the time to diagnosis, yet these technologies do not provide comprehensive information on antimicrobial susceptibility. Emerging technologies including biosensors, microfluidics, and other integrated platforms could improve UTI diagnosis via direct pathogen detection from urine samples, rapid antimicrobial susceptibility testing, and point-of-care testing. Successful development and implementation of these technologies has the potential to usher in an era of precision medicine to improve patient care and public health.
View details for DOI 10.1038/nrurol.2017.20
View details for PubMedID 28248946
Journal of clinical microbiology
Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [≥3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days (P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days (P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days (P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates.
View details for DOI 10.1128/JCM.02319-16
View details for PubMedID 28250001
Is Follow-up Testing with FilmArray Gastrointestinal Multiplex PCR Panel Necessary?
Journal of clinical microbiology
FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT) is a simple, sample-to-answer, on-demand, multiplex, nucleic acid amplification test for syndromic diagnosis of infectious gastroenteritis. The aim of this study was to measure the yield of follow-up testing with FilmArray GI Panel within 4 weeks of an initial test. Consecutive adult and pediatric patients tested at an academic institution between August 2015 and June 2016 were included in this study. Of 145 follow-up tests in 106 unique patients with an initial negative result, 134 (92.4%) tests and 98 (92.5%) patients remained negative upon follow-up testing. Excluding targets that are not reported at our institution (Clostridium difficile, enteroaggregative Escherichia coli, enteropathogenic E. coli, and enterotoxigenic E. coli), 137 (94.5%) follow-up tests and 101 (95.3%) patients remained negative. Weekly conversion rates were not significantly different across the 4-week follow-up interval. No epidemiological or clinical factors were significantly associated with a negative to positive conversion. Of 80 follow-up tests in patients with an initial positive result, 43 (53.8%) remained positive for the same target, 34 (42.5%) were negative, and 3 were positive for a different target (3.8%). Follow-up testing with FilmArray GI Panel within 4 weeks of a negative result rarely changed the initial result, and the follow-up test reverted to negative less than half the time after an initial positive result. In the absence of clinical or epidemiological evidence for a new infection, follow-up testing should be limited and FilmArray GI Panel should not be used as a test of cure.
View details for DOI 10.1128/JCM.02354-16
View details for PubMedID 28122874
- Determining the cause of recurrent Clostridium difficile infection using whole genome sequencing DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE 2017; 87 (1): 11-16
Intramolecular substitution uncages fluorogenic probes for detection of metallo-carbapenemase-expressing bacteria
2017; 8 (11): 7669-7674
This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of β-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group-dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.
View details for DOI 10.1039/C7SC02416A
View details for PubMedCentralID PMC5849144
Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease
Clinical Infectious Diseases
Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD.Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified.Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases.The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.
View details for DOI 10.1093/cid/cix728
Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas.
Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.
View details for PubMedID 27822731
Adenosine triphosphate bioluminescence for bacteriological surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes.
Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriologic/biologic residue. In this prospective study we evaluate the utility of ATP in bacteriologic surveillance and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing.ATP bioluminescence was measured after precleaning, manual cleaning, and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was remeasured in duodenoscopes (1) after re-education and competency testing of endoscopy staff and subsequently (2) after 2 cycles of precleaning and manual cleaning and single cycle of HLD or (3) after 2 cycles of precleaning, manual cleaning, and HLD.The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after precleaning (23,218.0 vs 1340.5 RLUs, P < .01) and HLD (177.0 vs 12.0 RLUs, P < .01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures.ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decreased elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes.
View details for DOI 10.1016/j.gie.2016.10.035
View details for PubMedID 27818222
Determining the cause of recurrent Clostridium difficile infection using whole genome sequencing.
Diagnostic microbiology and infectious disease
Understanding the contribution of relapse and reinfection to recurrent Clostridium difficile infection (CDI) has implications for therapy and infection prevention, respectively. We used whole genome sequencing to determine the relation of C. difficile strains isolated from patients with recurrent CDI at an academic medical center in the United States. Thirty-five toxigenic C. difficile isolates from 16 patients with 19 recurrent CDI episodes with median time of 53.5days (range, 13-362) between episodes were whole genome sequenced on the Illumina MiSeq platform. In 84% (16) of recurrences, the cause of recurrence was relapse with prior strain of C. difficile. In 16% (3) of recurrent episodes, reinfection with a new strain of C. difficile was the cause. In conclusion, the majority of CDI recurrences at our institution were due to infection with the same strain rather than infection with a new strain.
View details for DOI 10.1016/j.diagmicrobio.2016.09.023
View details for PubMedID 27771207
Serial testing for latent tuberculosis using QuantiFERON-TB Gold In-Tube: A Markov model
Healthcare workers (HCWs) in low-incidence settings are often serially tested for latent TB infection (LTBI) with the QuantiFERON-TB Gold In-Tube (QFT) assay, which exhibits frequent conversions and reversions. The clinical impact of such variability on serial testing remains unknown. We used a microsimulation Markov model that accounts for major sources of variability to project diagnostic outcomes in a simulated North American HCW cohort. Serial testing using a single QFT with the recommended conversion cutoff (IFN-g > 0.35 IU/mL) resulted in 24.6% (95% uncertainty range, UR: 23.8-25.5) of the entire population testing false-positive over ten years. Raising the cutoff to >1.0 IU/mL or confirming initial positive results with a (presumed independent) second test reduced this false-positive percentage to 2.3% (95%UR: 2.0-2.6%) or 4.1% (95%UR: 3.7-4.5%), but also reduced the proportion of true incident infections detected within the first year of infection from 76.5% (95%UR: 66.3-84.6%) to 54.8% (95%UR: 44.6-64.5%) or 61.5% (95%UR: 51.6-70.9%), respectively. Serial QFT testing of HCWs in North America may result in tremendous over-diagnosis and over-treatment of LTBI, with nearly thirty false-positives for every true infection diagnosed. Using higher cutoffs for conversion or confirmatory tests (for initial positives) can mitigate these effects, but will also diagnose fewer true infections.
View details for DOI 10.1038/srep30781
View details for Web of Science ID 000380659100001
View details for PubMedID 27469388
- Rapid Diagnosis of Tuberculosis from Analysis of Urine Volatile Organic Compounds ACS SENSORS 2016; 1 (7): 852-856
Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability?
Journal of clinical microbiology
2016; 54 (4): 845-850
Interferon-γ release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI). IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies in low risk individuals have revealed higher false conversion rates with IGRAs compared with TST. Reproducibility studies have identified various sources of variability that contribute to non-reproducible results. Sources of variability can be broadly classified as pre-analytical, analytical, post-analytical, manufacturing, and immunological. In this review, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability.
View details for DOI 10.1128/JCM.02803-15
View details for PubMedID 26763969
Organism burden, toxin concentration, and lactoferrin concentration do not distinguish between clinically significant and nonsignificant diarrhea in patients with Clostridium difficile
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2016; 84 (4): 343-346
Clostridium difficile infection is often overdiagnosed in patients with mild diarrhea. We evaluated 4 biomarkers as surrogates for clinically significant diarrhea (≥3 episodes in 24hours) in 59 PCR-positive patients with and 59 PCR-positive patients without clinically significant diarrhea. Organism burden (median tcdB cycle threshold value, 26.9 versus 27.1, P=0.25) and toxin A and B concentrations (toxin A, median, 0 versus 0ng/mL, P=0.42; toxin B, median, 0 versus 0ng/mL, P=0.25) were not significantly different between patients with and without clinically significant diarrhea. Fecal lactoferrin concentrations were significantly increased in patients with clinically significant diarrhea (median, 99.0 versus 55.1μg/mL, P=0.05); however, lactoferrin could not sufficiently classify patients into those with and without clinically significant diarrhea. Interventions that limit C. difficile testing to patients with clinically significant diarrhea are needed to improve the positive predictive value of C. difficile diagnostics.
View details for DOI 10.1016/j.diagmicrobio.2015.11.022
View details for Web of Science ID 000372768800014
Bacterial culture detection and identification in blood agar plates with an optoelectronic nose.
2016; 141 (3): 918-925
Clinical microbiology automation is currently limited by the lack of an in-plate culture identification system. Using an inexpensive, printed, disposable colorimetric sensor array (CSA) responsive to the volatiles emitted into plate headspace by microorganisms during growth, we report here that not only the presence but the species of bacteria growing in plate was identified before colonies are visible. In 1894 trials, 15 pathogenic bacterial species cultured on blood agar were identified with 91.0% sensitivity and 99.4% specificity within 3 hours of detection. The results indicate CSAs integrated into Petri dish lids present a novel paradigm to speciate microorganisms, well-suited to integration into automated plate handling systems.
View details for DOI 10.1039/c5an01990g
View details for PubMedID 26753182
First case of infectious endocarditis caused by Parvimonas micra.
2015; 36: 53-55
P. micra is an anaerobic Gram-positive cocci, and a known commensal organism of the human oral cavity and gastrointestinal tract. Although it has been classically described in association with endodontic disease and peritonsillar infection, recent reports have highlighted the role of P. micra as the primary pathogen in the setting of invasive infections. In its most recent taxonomic classification, P. micra has never been reported causing infectious endocarditis in humans. Here, we describe a 71 year-old man who developed severe native valve endocarditis complicated by aortic valvular destruction and perivalvular abscess, requiring emergent surgical intervention. Molecular sequencing enabled identification of P. micra.
View details for DOI 10.1016/j.anaerobe.2015.10.007
View details for PubMedID 26485192
- Next-Generation Sequencing for Infectious Disease Diagnosis and Management A Report of the Association for Molecular Pathology JOURNAL OF MOLECULAR DIAGNOSTICS 2015; 17 (6): 623-634
- 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 2015; 53 (11): 3596-3600
First case of mesh infection due to Coccidioides spp. and literature review of fungal mesh infections after hernia repair.
2015; 58 (10): 582-587
Fungal mesh infections are a rare complication of hernia repairs with mesh. The first case of Coccidioides spp. mesh infection is described, and a systematic literature review of all known fungal mesh infections was performed. Nine cases of fungal mesh infection are reviewed. Female and male patients are equally represented, median age is 49.5 years, and critical illness and preinfection antibiotic use were common. Fungal mesh infections are rare, but potentially fatal, complications of hernias repaired with mesh.
View details for DOI 10.1111/myc.12364
View details for PubMedID 26293423
- A small-molecule antivirulence agent for treating Clostridium difficile infection SCIENCE TRANSLATIONAL MEDICINE 2015; 7 (306)
A small-molecule antivirulence agent for treating Clostridium difficile infection.
Science translational medicine
2015; 7 (306): 306ra148-?
Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.
View details for DOI 10.1126/scitranslmed.aac9103
View details for PubMedID 26400909
In vitro immunomodulation for enhancing T cell-based diagnosis of Mycobacterium tuberculosis infection
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2015; 83 (1): 41-45
Interferon-gamma release assays have limited sensitivity for detecting latent tuberculosis infection. In this study, we determine if the addition of immunomodulators to the QuantiFERON-TB Gold In-Tube (QFT-GIT) increased test sensitivity without compromising specificity. We prospectively compared QFT-GIT results with and without incubation with 2 immunomodulators (lipopolysaccharide [LPS] and polyinosine-polycytidylic acid [PolyIC]) in 2 cohorts-113 culture-confirmed tuberculosis (TB) subjects in Hanoi, Vietnam, and 226 documented QFT-GIT-negative, low TB risk health care workers undergoing annual TB screening at a US academic institution. Sensitivity of the tests in TB subjects was 84.1% with the standard QFT-GIT and 85.8% and 74.3% after incubation with LPS and PolyIC, respectively. Specificity in low TB risk health care workers was 100% with the standard QFT-GIT by design and 86.7% with LPS and 63.3% with PolyIC. In conclusion, use of the 2 immunomodulators did not improve sensitivity of the QFT-GIT in TB patients and reduced specificity in low-risk health care workers.
View details for DOI 10.1016/j.diagmicrobio.2015.05.007
View details for Web of Science ID 000359754000010
View details for PubMedID 26081239
- Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium PLOS ONE 2015; 10 (8)
Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient
JOURNAL OF CLINICAL MICROBIOLOGY
2015; 53 (8): 2749-2752
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
Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing
JOURNAL OF CLINICAL MICROBIOLOGY
2015; 53 (7): 2329-2331
Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform.
View details for DOI 10.1128/JCM.00956-15
View details for Web of Science ID 000358287700044
View details for PubMedCentralID PMC4473243
- Molecular epidemiology of Aspergillus collected from cystic fibrosis patients JOURNAL OF CYSTIC FIBROSIS 2015; 14 (4): 474-481
Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood
JOURNAL OF CLINICAL MICROBIOLOGY
2015; 53 (7): 2251-2257
Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.
View details for DOI 10.1128/JCM.00542-15
View details for Web of Science ID 000358287700034
Rapid Detection of Acquired and Inducible Clarithromycin Resistance in Mycobacterium abscessus Group by a Simple Real-Time PCR Assay
JOURNAL OF CLINICAL MICROBIOLOGY
2015; 53 (7): 2337-2339
By targeting the erm(41) and rrl genes in the Mycobacterium abscessus group, a multiplex real-time PCR assay for clarithromycin resistance showed 95% (38/40) concordance with nucleic acid testing and 95% (37/39) concordance with phenotypic testing. This assay provides a simple and rapid alternative to extended incubation or erm(41) sequencing.
View details for DOI 10.1128/JCM.00132-15
View details for Web of Science ID 000358287700046
- Burden of Clostridium difficile infection in the United States. New England journal of medicine 2015; 372 (24): 2368-2369
Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium.
2015; 10 (8)
Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.
View details for DOI 10.1371/journal.pone.0134692
View details for PubMedID 26252384
Reproducibility of interferon gamma (IFN-?) release Assays. A systematic review.
Annals of the American Thoracic Society
2014; 11 (8): 1267-1276
Interferon gamma (IFN-γ) release assays for latent tuberculosis infection result in a larger-than-expected number of conversions and reversions in occupational screening programs, and reproducibility of test results is a concern.Knowledge of the relative contribution and extent of the individual sources of variability (immunological, preanalytical, or analytical) could help optimize testing protocols.We performed a systematic review of studies published by October 2013 on all potential sources of variability of commercial IFN-γ release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB). The included studies assessed test variability under identical conditions and under different conditions (the latter both overall and stratified by individual sources of variability). Linear mixed effects models were used to estimate within-subject SD.We identified a total of 26 articles, including 7 studies analyzing variability under the same conditions, 10 studies analyzing variability with repeat testing over time under different conditions, and 19 studies reporting individual sources of variability. Most data were on QuantiFERON (only three studies on T-SPOT.TB). A considerable number of conversions and reversions were seen around the manufacturer-recommended cut-point. The estimated range of variability of IFN-γ response in QuantiFERON under identical conditions was ±0.47 IU/ml (coefficient of variation, 13%) and ±0.26 IU/ml (30%) for individuals with an initial IFN-γ response in the borderline range (0.25-0.80 IU/ml). The estimated range of variability in noncontrolled settings was substantially larger (±1.4 IU/ml; 60%). Blood volume inoculated into QuantiFERON tubes and preanalytic delay were identified as key sources of variability.This systematic review shows substantial variability with repeat IFN-γ release assays testing even under identical conditions, suggesting that reversions and conversions around the existing cut-point should be interpreted with caution.
View details for DOI 10.1513/AnnalsATS.201405-188OC
View details for PubMedID 25188809
Environmental Sampling for Clostridium difficile on Alcohol-Based Hand Rub Dispensers in an Academic Medical Center
2014; 15 (5): 581-584
Clostridum difficile is a gram-positive, spore-forming anaerobic bacillus that has substantial associated morbidity, mortality, and associated healthcare burdens. Clostridium difficile spores are not destroyed by alcohol. Alcohol gel dispensers are used commonly as the hand sanitization method of choice in hospitals. It is possible that gel dispensers are fomites for C. difficile.Thirty alcohol-based gel dispenser handles outside of rooms of patients with active C. difficile infection were sampled. The samples were assessed for C. difficile by both culture and polymerase chain reaction (PCR). The samples were also assessed for other organisms by culture.No C. difficile was cultured or detected by PCR on any of the gel dispensers. Coagulase-negative Staphyloccus spp., diptheroids, and Bacillus spp. were the organisms detected most commonly.At our institution, C. difficile is not present on alcohol-based gel dispensers, but other potentially pathogenis are.
View details for DOI 10.1089/sur.2013.102
View details for Web of Science ID 000343224800018
Mycobacterium tuberculosis Lipoprotein LprG Binds Lipoarabinomannan and Determines Its Cell Envelope Localization to Control Phagolysosomal Fusion
2014; 10 (10)
Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.
View details for DOI 10.1371/journal.ppat.1004471
View details for Web of Science ID 000344548800055
View details for PubMedID 25356793
- Using cerebrospinal fluid for the diagnosis of tuberculous meningitis with GeneXpert. European respiratory journal 2014; 44 (4): 1094-1095
- LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis PLOS PATHOGENS 2014; 10 (9)
LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis.
2014; 10 (9)
Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.
View details for DOI 10.1371/journal.ppat.1004376
View details for PubMedID 25232742
Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria.
Angewandte Chemie (International ed. in English)
2014; 53 (31): 8113-8116
Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.
View details for DOI 10.1002/anie.201402012
View details for PubMedID 24764125
Multiplex nucleic Acid amplification test for diagnosis of dengue Fever, malaria, and leptospirosis.
Journal of clinical microbiology
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
Adult and Pediatric Intra-Institutional Trends of Ciprofloxacin Susceptibility in E. coli Positive Urinary Cultures.
Antibiotics (Basel, Switzerland)
2014; 3 (2): 163-173
Antimicrobial drug resistance in treatment of urinary tract infection (UTI) continues to rise worldwide. To examine contributions of physician prescribing patterns to fluoroquinolone (ciprofloxacin, CP) resistance, we examined Escherichia coli (E. coli) resistance patterns in urinary cultures. Since CP usage is limited in children, we compared CP resistance trends in adults and children to those of more commonly used trimethoprim-sulfamethoxazole (TMP-SMX) and nitrofurantoin (NF). Our data show that although the general pediatric population has lower resistance to ciprofloxacin, resistance levels are rising with increased usage. While NF susceptibility is historically stable, TMP-SMX resistance is slightly higher in children compared to adults. In both adults and children, antimicrobial resistance patterns vary according to clinical practice site, with ambulatory urology patients showing the highest resistance. This suggests that physician's prescribing patterns contribute to antimicrobial resistance.
View details for DOI 10.3390/antibiotics3020163
View details for PubMedID 27025742
- Occupational Screening for Tuberculosis. A Testing Time for Interferon-? Release Assays. Annals of the American Thoracic Society 2014; 11 (3): 399-401
Colorimetric Sensor Array Allows Fast Detection and Simultaneous Identification of Sepsis-Causing Bacteria in Spiked Blood Culture
JOURNAL OF CLINICAL MICROBIOLOGY
2014; 52 (2): 592-598
Sepsis is a medical emergency demanding early diagnosis and tailored antimicrobial therapy. Every hour of delay in initiating effective therapy measurably increases patient mortality. Blood culture is currently the reference standard for detecting bloodstream infection, a multistep process which may take one to several days. Here, we report a novel paradigm for earlier detection and the simultaneous identification of pathogens in spiked blood cultures by means of a metabolomic "fingerprint" of the volatile mixture outgassed by the organisms. The colorimetric sensor array provided significantly faster detection of positive blood cultures than a conventional blood culture system (12.1 h versus 14.9 h, P < 0.001) while allowing for the identification of 18 bacterial species with 91.9% overall accuracy within 2 h of growth detection. The colorimetric sensor array also allowed for discrimination between unrelated strains of methicillin-resistant Staphylococcus aureus, indicating that the metabolomic fingerprint has the potential to track nosocomial transmissions. Altogether, the colorimetric sensor array is a promising tool that offers a new paradigm for diagnosing bloodstream infections.
View details for DOI 10.1128/JCM.02377-13
View details for Web of Science ID 000330444200030
View details for PubMedID 24478493
Economic Evaluation of Laboratory Testing Strategies for Hospital-Associated Clostridium difficile Infection.
Journal of clinical microbiology
2014; 52 (2): 489-496
Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.
View details for DOI 10.1128/JCM.02777-13
View details for PubMedID 24478478
Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection
CLINICAL MICROBIOLOGY REVIEWS
2014; 27 (1): 3-20
Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers.
View details for DOI 10.1128/CMR.00034-13
View details for Web of Science ID 000329324800001
View details for PubMedID 24396134
Traveler's encounter with nymphs in a hotel bed.
2014; 1 (2): 24-25
This case illustrates skin lesions in a traveler staying in a hotel bed infested with tics. Although infestation of hotels with bedbugs belonging to the Cimex genus is a growing problem worldwide, tick infestation has never been reported before.
View details for DOI 10.1016/j.idcr.2014.03.002
View details for PubMedID 26839772
- False-Positive Quantiferon Results at a Large Healthcare Institution. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2014
Pretreatment of sinus aspirates with dithiothreitol improves yield of fungal cultures in patients with chronic sinusitis
INTERNATIONAL FORUM OF ALLERGY & RHINOLOGY
2013; 3 (12): 992-996
Mold pathogens are a leading cause of chronic rhinosinusitis. Successful isolation of mold on culture is helpful in establishing a diagnosis and guiding therapy. Though mucolytic agents are commonly used in European countries, they are not part of everyday use in North America. In this case-control prospective study, we investigated the yield of fungal culture before and after treatment of sinus aspirates with the mucolytic agent dithiothreitol in a United States hospital.Over a 5-month period during 2011-2012, 359 sinus aspirates from 294 patients with symptoms suspicious for chronic sinusitis or allergic fungal sinusitis were collected. Aspirates were cultured on fungal medium before and after treatment with dithiothreitol.Of the 359 pairs of cultures, 62 (17.3%) demonstrated mold growth on at least 1 of the plates, 9 (14.5%) of which grew more than 1 species of mold. A total of 75 molds were identified, 41 (54.7%) of which were successfully cultured only when the mucus was pretreated with dithiothreitol (p < 0.0001). Quantitatively, more colonies grew from dithiothreitol-treated mucus than from direct-inoculation (p < 0.0001).This study confirms improved recovery of mold from sinus cultures after pretreatment of samples with dithiothreitol. Further studies are needed to correlate these findings with clinical outcome.
View details for DOI 10.1002/alr.21230
View details for Web of Science ID 000328300500008
View details for PubMedID 24124079
A Pediatric Case of New Delhi Metallo-ß-Lactamase-1-Producing Enterobacteriaceae in The United States.
Pediatric infectious disease journal
2013; 32 (11): 1291-1294
We report the second pediatric case of New Delhi metallo-beta-lactamase (NDM-1)-producing Enterobacteriaceae in the United States in a girl from India who presented to a teaching hospital in Northern California with cystitis due to NDM-1-producing E. coli and K. pneumoniae. Laboratory methods included various phenotypic antimicrobial susceptibility testing assays, as well as PCR assays for carbapenemase-encoding genes. Laboratory challenges included a false negative modified Hodge test and reversion of carbapenem resistance in the E. coli strain. The limited number of effective antimicrobial agents and the lack of pediatric-specific safety and efficacy data for these drugs presented significant therapeutic challenges.
View details for DOI 10.1097/INF.0b013e31829eca34
View details for PubMedID 23743543
Impact of Blood Volume, Tube Shaking, and Incubation Time on Reproducibility of QuantiFERON-TB Gold In-Tube Assay.
Journal of clinical microbiology
2013; 51 (11): 3521-3526
Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Agvigorous minus nilgentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.
View details for DOI 10.1128/JCM.01627-13
View details for PubMedID 23966505
Alerting Physicians during Electronic Order Entry Effectively Reduces Unnecessary Repeat PCR Testing for Clostridium difficile.
Journal of clinical microbiology
2013; 51 (11): 3872-3874
Hospital information systems (HIS) alerts restricting repeat Clostridium difficile PCR ordering by physicians in patients with a prior result within 7 days eliminated 91% of repeat tests, from 14.5% (282/1,949) repeats preintervention to 1.3% (135/10,285) postintervention. HIS alerting is an effective, targeted, patient-specific tool for improving the quality and utilization of C. difficile results.
View details for DOI 10.1128/JCM.01724-13
View details for PubMedID 23985918
Challenges with QuantiFERON-TB Gold Assay for Large-Scale, Routine Screening of U.S. Healthcare Workers
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
2013; 188 (8): 1005-1010
North American occupational health programs that switched from the tuberculin skin test (TST) to IFN-γ release assays for latent tuberculosis screening are reporting challenges with interpretation of serial testing results in healthcare workers (HCWs). However, limited data exist on the reproducibility of serial IFN-γ release assay results in low-risk HCWs.To evaluate the short-term reproducibility of QuantiFERON-TB Gold In-Tube (QFT) in a large cohort of HCWs and to define a QFT cutoff yielding a conversion rate equivalent to historical TST rates.We retrospectively evaluated the QFT results from HCWs with two or more QFT tests performed between June 2008 and July 2010 at an academic institution. Outcome measures were proportions of reproducibility, quantitative results, and conversion rates with alternate QFT cutoffs.A total of 9,153 HCWs with two or more QFT tests were included in the analysis. Of 8,227 individuals with a negative result, 4.4% (n = 361) converted their QFT result over 2 years. A total of 261 (72.3%) of the HCWs with conversions underwent repeat short-term testing after the first positive result with 64.8% reverting (n = 169). An IFN-γ cutoff of 5.3 IU/ml or higher (manufacturer's cutoff is ≥0.35 IU/ml) yielded a conversion rate of 0.4%, equal to our institution's historical TST conversion rate.The manufacturer's definition of QFT conversion results in an inflated conversion rate that is incompatible with our low-risk setting. A significantly higher QFT cutoff value is needed to match the historical TST conversion rate. Nonreproducible conversions in most converters suggested false-positive results.
View details for DOI 10.1164/rccm.201305-0831OC
View details for Web of Science ID 000325789700021
View details for PubMedID 23978270
- Occupational screening of health care workers for tuberculosis infection: tuberculin skin testing or interferon-gamma release assays? OCCUPATIONAL MEDICINE-OXFORD 2013; 63 (7): 458-460
A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California.
Journal of clinical microbiology
2013; 51 (9): 3000-3005
Rapid and accurate detection of Shiga-toxin producing E. coli of all serotypes from patients with diarrhea is critical for medical management and for prevention of ongoing transmissions. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx1 and stx2 for detection of O157 and non-O157 Shiga-toxin producing E. coli from diarrheal stool samples enriched in GN broth. We show that the assay is 100% sensitive (95% confidence interval (CI), 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%), based on a panel of 40 known STEC-positive and 65 known negative specimens. During a two-year post-validation period, the assay detected a greater number of positive samples from patients in Northern California compared to culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains with 5.8% (1/17) of patients with non-O157 strains and 42.9% (6/14) with O157 strains (P=0.03) developing hemolytic uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to O157 serotype using a sensitive assay. Additionally, a survey of 17 clinical laboratories in Northern California demonstrated that nearly 50% do not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins recommended in the 2009 CDC guidelines.
View details for DOI 10.1128/JCM.00991-13
View details for PubMedID 23843484
Molecular Approaches and Biomarkers for Detection of Mycobacterium tuberculosis.
Clinics in laboratory medicine
2013; 33 (3): 553-566
Tuberculosis (TB) continues to be a public health emergency, compounded by the lack of adequate diagnostic testing in many regions of the world. New advances in the molecular detection of Mycobacterium tuberculosis, including faster and simpler nucleic acid amplification tests, have resulted in rapid and cost-effective methods to diagnose TB and test for drug resistance. Ongoing research on biomarkers for TB infection may lead to new tests for blood, urine, breath, and sputum. Sustained investment in the development and dissemination of diagnostic tests for TB is critical for increasing TB case finding, placing patients on appropriate treatment, and reducing transmission.
View details for DOI 10.1016/j.cll.2013.03.012
View details for PubMedID 23931838
Utility of DNA Sequencing for Direct Identification of Invasive Fungi From Fresh and Formalin-Fixed Specimens
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
2013; 140 (2): 203-208
Objectives: To describe and discuss the utility and potential pitfalls of ribosomal RNA locus sequencing for direct identification of invasive fungi from fresh and formalin-fixed, paraffin-embedded specimens. Methods: DNA was extracted from fresh and formalin-fixed, paraffin-embedded tissue and subjected to real-time polymerase chain reaction (PCR) targeting ITS2 and D2 regions of fungal ribosomal RNA locus. Cycle sequencing was performed on PCR products, and the identity of sequences was determined using a public database. Results: Four clinical cases of invasive fungal infection are presented to illustrate the utility of DNA sequencing for determining etiology when microbiological culture is negative, for shortening the time to identification of slow-growing fungi, for guiding antifungal therapy, and for shedding light on the pathogenesis of disseminated fungal infection. Conclusions: Fungal ribosomal RNA locus sequencing from fresh or formalin-fixed, paraffin-embedded specimens is a powerful tool for rapid and accurate diagnosis of patients with culture-negative or uncultured invasive mycosis.
View details for DOI 10.1309/AJCPNSU2SDZD9WPW
View details for Web of Science ID 000322149600010
View details for PubMedID 23897255
Sorting Inactivated Cells Using Cell-Imprinted Polymer Thin Films
2013; 7 (7): 6031-6036
Previous work showed that cell imprinting in a polydimethylsiloxane (PDMS) film produced artificial receptors to cells by template-assisted rearrangement of functional groups on the surface of the polymer thin film which facilitated cell capture in the polymer surface indentations by size, shape, and most importantly chemical recognition. We report here that inactivation of cells by treatment with formaldehyde (4%), or glutaraldehyde (2%), or a combination of the two leads to markedly improved capture selectivity (a factor of 3) when cells to be analyzed are inactivated in the same manner. The enhanced capture efficiency compared to living cells results from two factors: (1) rigidification of the cell surface through crosslinking of amine groups by the aldehyde; and (2) elimination of chemicals excreted from living cells which interfere with the fidelity of the cell imprinting process. Moreover, cell inactivation has the advantage of removing biohazard risks associated with working with virulent bacteria. These results are demonstrated using different strains of mycobacterium tuberculosis.
View details for DOI 10.1021/nn401768s
View details for Web of Science ID 000322417400045
View details for PubMedID 23725546
- Images in clinical medicine. Strongyloides stercoralis embryonated ova in the lung. New England journal of medicine 2013; 368 (12)
Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak
2013; 8 (3)
Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods.To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina's sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case.Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.
View details for DOI 10.1371/journal.pone.0058235
View details for Web of Science ID 000315637900110
View details for PubMedID 23472164
Trends in incidence and susceptibility among methicillin-resistant Staphylococcus aureus isolated from intranasal cultures associated with rhinosinusitis.
American journal of rhinology & allergy
2013; 27 (2): 134-137
Reports regarding the incidence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in rhinosinusitis (RS) are limited. This study was designed to identify epidemiology and trends of MRSA incidence and antimicrobial resistance in the sinonasal cavities.This is a retrospective case series. All intranasal/sinus cultures obtained by otolaryngologists at Stanford over a 20-year period (1990-2010) were retrospectively reviewed by mining the microbiology database. Nested searches were then made for all S. aureus and MRSA cultures. Patterns of incidence and changes in antibiotic susceptibilities were tabulated and statistical analysis was performed.Our search retrieved 10,387 positive intranasal culture samples, with S. aureus found in 800 (7.7%), and MRSA comprising 110 (1.06%) of this subset. Between the years of 1990 and 1999, only 2/112 (1.7%) of S. aureus-positive nasal cultures were positive for MRSA, with a sharp rise in incidence to 86/606 (14.2%) from 2000 to 2005, and to 22/82, 26.8% from 2006 to 2010. On a percent basis, using logistic regression modeling, this represents a statistically significant increasing trend (p < 0.0001) for MRSA sinusitis. However, over the 20-year interval studied, the patterns of antibiotic resistance among MRSA remained unaltered, especially with regard to trimethoprim-sulfamethoxazole and vancomycin.S. aureus and MRSA isolates from intranasal cultures, which were essentially absent before the year 2000, became significantly more common earlier this decade. These data show the increased role of MRSA in sinusitis. MRSA antibiotic susceptibilities have remained, however, largely stable during this time period.
View details for DOI 10.2500/ajra.2013.27.3858
View details for PubMedID 23562203
- Interferon γ-Release Assays for Diagnosis of Latent Tuberculosis in Healthcare Workers in Low-Incidence Settings: Pros and Cons. Clinical chemistry 2013
In Vitro Immunomodulation of a Whole Blood IFN-gamma Release Assay Enhances T Cell Responses in Subjects with Latent Tuberculosis Infection
2012; 7 (10)
Activation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.
View details for DOI 10.1371/journal.pone.0048027
View details for Web of Science ID 000310705300033
View details for PubMedID 23144722
Investigation of False-Positive Results Given by the QuantiFERON-TB Gold In-Tube Assay
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (9): 3105-3107
We investigated a sudden increase in the rate of positive QuantiFERON-TB Gold In-Tube results from 10% to 31% at a U.S. academic institution. Direct comparison of the TB antigen tubes with tubes from a different lot number identified that a potential problem with the TB antigen vials in a certain tube lot was the likely cause of the elevated positive rate. The underlying defect remains unknown. This finding warrants refinement of quality control programs by the manufacturer and users.
View details for DOI 10.1128/JCM.00730-12
View details for Web of Science ID 000307941900046
View details for PubMedID 22785197
Performance of BinaxNOW for Diagnosis of Malaria in a US Hospital
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (9): 2877-2880
Microscopic diagnosis and species identification of Plasmodium in areas of nonendemicity provide a robust method for malaria diagnosis but are technically challenging. A prospective study was conducted to measure the performance of BinaxNOW compared to microscopy (the gold standard) in a U.S. teaching hospital. Overall, BinaxNOW was 84.2% sensitive and 99.8% specific. Excluding patients on antimalarial therapy, the sensitivity was 92.9%. Importantly, BinaxNOW initially misclassified a case of Plasmodium falciparum malaria as non-falciparum. These results support the judicious use of BinaxNOW in screening of individuals suspected of having malaria in areas of nonendemicity.
View details for DOI 10.1128/JCM.01013-12
View details for Web of Science ID 000307941900007
View details for PubMedID 22718936
Sensitivity of QuantiFERON-TB GOLD In-Tube for diagnosis of recent versus remote M.tuberculosis infection
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2012; 73 (3): 257-259
The sensitivity of QuantiFERON-TB GOLD In-Tube was measured in 104 subjects with recent (≤2 years) and remote Mycobacterium tuberculosis infection using tuberculin skin test conversion as the reference standard. The sensitivity was not significantly different between the 2 groups (33% versus 20%, P = 0.3). This finding suggests interferon-γ release assays may not be more sensitive for diagnosis of recent than remote infection. Longitudinal studies are needed to validate this finding.
View details for DOI 10.1016/j.diagmicrobio.2012.03.018
View details for Web of Science ID 000305546300010
View details for PubMedID 22521052
Can a Simple Flotation Method Lower the Limit of Detection of Mycobacterium tuberculosis in Extrapulmonary Samples Analyzed by the GeneXpert MTB/RIF Assay?
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (7): 2272-2276
The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.
View details for DOI 10.1128/JCM.01012-12
View details for Web of Science ID 000307360800017
View details for PubMedID 22553234
First Isolation of Cryptococcus uzbekistanensis from an Immunocompromised Patient with Lymphoma
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (3): 1125-1127
Cryptococcus species are known agents of opportunistic infections in healthy and immunocompromised hosts. Here we describe the first case of Cryptococcus uzbekistanensis causing bone marrow infection in an elderly Asian man with undiagnosed T cell lymphoma presenting with fever of unknown origin, pancytopenia, and exposure to chicken manure.
View details for DOI 10.1128/JCM.05678-11
View details for Web of Science ID 000300997800099
View details for PubMedID 22189126
IMP-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States
JOURNAL OF CLINICAL MICROBIOLOGY
2011; 49 (12): 4239-4245
The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) producing acquired carbapenemases have created a global public health crisis. In the United States, CRE producing the Klebsiella pneumoniae carbapenemase (KPC) are increasingly common and are endemic in some regions. Metallo-β-lactamase (MBL)-producing CRE have recently been reported in the United States among patients who received medical care in countries where such organisms are common. Here, we describe three carbapenem-resistant K. pneumoniae isolates recovered from pediatric patients at a single U.S. health care facility, none of whom had a history of international travel. The isolates were resistant to carbapenems but susceptible to aztreonam, trimethoprim-sulfamethoxazole, and fluoroquinolones. The three isolates were closely related to each other by pulsed-field gel electrophoresis and contained a common plasmid. PCR and sequence analysis confirmed that these isolates produce IMP-4, an MBL carbapenemase not previously published as present among Enterobacteriaceae in the United States.
View details for DOI 10.1128/JCM.05297-11
View details for Web of Science ID 000298113400036
View details for PubMedID 21998425
Preanalytical Delay Reduces Sensitivity of QuantiFERON-TB Gold In-Tube Assay for Detection of Latent Tuberculosis Infection
JOURNAL OF CLINICAL MICROBIOLOGY
2011; 49 (8): 3061-3064
The effects of incubation delays on the accuracy of the QuantiFERON-TB gold in-tube assay (QFT-GIT) were measured. Compared to immediate incubation, 6- and 12-hour delays resulted in positive-to-negative reversion rates of 19% (5/26) and 22% (5/23), respectively. These findings underscore the need for standardizing QFT-GIT preanalytical practices.
View details for DOI 10.1128/JCM.01136-11
View details for Web of Science ID 000293221900054
View details for PubMedID 21697332
Suboptimal Activation of Antigen-Specific CD4(+) Effector Cells Enables Persistence of M. tuberculosis In Vivo
2011; 7 (5)
Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.
View details for DOI 10.1371/journal.ppat.1002063
View details for Web of Science ID 000291014000043
View details for PubMedID 21637811
Clinical Application and Limitations of Interferon-gamma Release Assays for the Diagnosis of Latent Tuberculosis Infection
CLINICAL INFECTIOUS DISEASES
2011; 52 (8): 1031-1037
Interferon-release assays (IGRAs) represent advances in tuberculosis immunology and evolutionary biology. IGRAs were designed to replace tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection because of their logistical advantages and enhanced specificity over TST. Although IGRAs and TST have been useful in epidemiologic studies, they lack the sensitivity and reproducibility normally expected from diagnostic tests in clinical practice. In this review, we present an overview of the current recommendations and knowledge in the field and discuss practical approaches in areas of uncertainty related to discordant IGRA results.
View details for DOI 10.1093/cid/cir068
View details for Web of Science ID 000289300100014
View details for PubMedID 21460320
Brain Abscess Caused by Phaeoacremonium parasiticum in an Immunocompromised Patient
JOURNAL OF CLINICAL MICROBIOLOGY
2011; 49 (3): 1171-1174
Phaeoacremonium parasiticum is an environmental fungus usually associated with subcutaneous infections. We report the first documented case of central nervous system involvement with brain abscess formation in a patient with chronic granulomatous disease and review the literature on Phaeoacremonium parasiticum infections.
View details for DOI 10.1128/JCM.00830-10
View details for Web of Science ID 000287967100072
View details for PubMedID 21191052
View details for PubMedCentralID PMC3067714
Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites
2011; 47 (41): 11435-11437
Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.
View details for DOI 10.1039/c1cc14871k
View details for Web of Science ID 000295696300011
View details for PubMedID 21935547
Is Repeat PCR Needed for Diagnosis of Clostridium difficile Infection?
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (10): 3738-3741
Patients with diarrhea, defined as loose or watery stool, and two or more Clostridium difficile tcdB PCR tests within 14 days of each other were investigated. Repeat PCR for 293 patients with a prior negative result yielded negative results in 396 (97.5%) of 406 tests. Ten new positives were detected, including one false positive. Repeat PCR within 7 days appears rarely useful, except for patients with evidence of a new infection.
View details for DOI 10.1128/JCM.00722-10
View details for Web of Science ID 000282544700042
View details for PubMedID 20686078
Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (8): 2672-2676
In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37 degrees C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.
View details for DOI 10.1128/JCM.00482-10
View details for Web of Science ID 000280550500002
View details for PubMedID 20519472
Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae-Mycobacterium abscessus group to the species level
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2010; 67 (4): 333-336
The Mycobacterium chelonae-Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.
View details for DOI 10.1016/j.diagmicrobio.2010.03.011
View details for Web of Science ID 000280468700004
View details for PubMedID 20638600
Comparison of Single-Copy and Multicopy Real-Time PCR Targets for Detection of Mycobacterium tuberculosis in Paraffin-Embedded Tissue
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (7): 2569-2570
Real-time PCR can rapidly identify Mycobacterium tuberculosis in paraffin-embedded tissue in the absence of microbiological culture. In a comparison of single-copy and multicopy PCR targets in 70 tissue samples, the sensitivities were 26% and 54%, respectively, with 100% specificity. Sensitivity was 75% for newer samples and was not decreased for acid-fast bacillus (AFB) stain-negative specimens.
View details for DOI 10.1128/JCM.02449-09
View details for Web of Science ID 000279318700040
View details for PubMedID 20463168
View details for PubMedCentralID PMC2897508
Mixed infection involving Actinomyces, Aggregatibacter, and Fusobacterium species presenting as perispinal tumor
2010; 16 (2): 174-178
A representative case in which a polymicrobial infection involving Fusobacterium nucleatum, Actinomyces israelii and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans was initially diagnosed as malignancy in an edentulous patient. Additional history obtained after the nature of the syndrome was elucidated revealed that he had had his two remaining teeth extracted four months prior to this episode.
View details for DOI 10.1016/j.anaerobe.2009.07.003
View details for Web of Science ID 000277734600018
View details for PubMedID 19628046
Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (3): 785-790
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
Preferential Lower Respiratory Tract Infection in Swine-Origin 2009 A(H1N1) Influenza
CLINICAL INFECTIOUS DISEASES
2010; 50 (3): 391-394
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
First documentation of isoniazid reversion in Mycobacterium tuberculosis
INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE
2009; 13 (11): 1347-1354
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
Comparison of Real-Time PCR and Conventional Biochemical Methods for Identification of Staphylococcus lugdunensis
JOURNAL OF CLINICAL MICROBIOLOGY
2009; 47 (11): 3472-3477
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
Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
2009; 53 (9): 3799-3802
Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.
View details for DOI 10.1128/AAC.00647-09
View details for Web of Science ID 000270014200025
View details for PubMedID 19596883
Nontuberculous Mycobacteria Infections in Immunocompromised Patients Single Institution Experience
JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY
2009; 31 (8): 556-560
Disseminated infection due to nontuberculous Mycobacterium (NTM) species is rare in pediatrics. Here we report 6 infections affecting 5 patients at a single institution in an immunocompromised population of pediatric oncology and stem cell transplant recipients. The patients presented within a 1-year period with catheter-associated bacteremia. New pulmonary nodules were noted in 4 of the 5 patients. All of the infections were due to rapidly growing NTM. Patients were successfully treated with removal of the infected catheter and combination antibiotic therapy. There are currently no consensus guidelines for treatment of NTM infections in this population, and a therapeutic approach is presented here.
View details for Web of Science ID 000268815000006
View details for PubMedID 19641470
Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests
2009; 4 (7)
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
Bartholin's abscess caused by hypermucoviscous Klebsiella pneumoniae
JOURNAL OF MEDICAL MICROBIOLOGY
2009; 58 (5): 671-673
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
Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR
JOURNAL OF CLINICAL MICROBIOLOGY
2009; 47 (5): 1497-1502
The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n = 96), MAC (n = 97), MCAG (n = 68), and M. mucogenicum (n = 9) and 95% and 100%, respectively, for MFG (n = 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.
View details for DOI 10.1128/JCM.01868-08
View details for Web of Science ID 000265641000032
View details for PubMedID 19297596
Challenges and Pitfalls of Morphologic Identification of Fungal Infections in Histologic and Cytologic Specimens A Ten-Year Retrospective Review at a Single Institution
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
2009; 131 (3): 364-375
Despite the advantages of providing an early presumptive diagnosis, fungal classification by histopathology can be difficult and may lead to diagnostic error. To assess the accuracy of histologic diagnosis of fungal infections vs culture ("gold standard"), we performed a 10-year retrospective review at our institution. Of the 47 of 338 positive mold and yeast cultures with concurrent surgical pathology evaluation without known history of a fungal infection, 37 (79%) were correctly identified based on morphologic features in histologic and/or cytologic specimens. The 10 discrepant diagnoses (21%) included misidentification of septate and nonseptate hyphal organisms and yeast forms. Errors resulted from morphologic mimics, use of inappropriate terminology, and incomplete knowledge in mycology. The accuracy did not correlate with preceding antifungal therapy (P = .14) or use of special stains (P = .34) and was not operator-dependent. Among 8 discrepancies with clinical follow-up available, 2 potential adverse clinical consequences resulted. While histopathologic identification of fungi in tissue sections and cytologic preparations is prone to error, implementation of a standardized reporting format should improve diagnostic accuracy and prevent adverse outcomes.
View details for DOI 10.1309/AJCP99OOOZSNISCZ
View details for Web of Science ID 000263427400008
View details for PubMedID 19228642
RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins
CELL HOST & MICROBE
2009; 5 (1): 35-46
RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.
View details for DOI 10.1016/j.chom.2008.12.002
View details for Web of Science ID 000262885700007
View details for PubMedID 19154986
Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level
JOURNAL OF CLINICAL MICROBIOLOGY
2008; 46 (7): 2241-2246
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
Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs
JOURNAL OF EXPERIMENTAL MEDICINE
2008; 205 (1): 105-115
The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4(+) T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4(+) T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4(+) T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.
View details for DOI 10.1084/jem.20071367
View details for Web of Science ID 000252507100012
View details for PubMedID 18158321
Evaluation of a semi-automated reporter phage assay for susceptibility testing Myobacterium tuberculosis isolates in South Africa
2008; 88 (1): 64-68
In a prospective study conducted by laboratory technologists in a diagnostic laboratory in Cape Town, South Africa, a semi-automated phage-based antibiotic susceptibility assay was implemented and the performance of the luciferase reporter mycobacteriophage (LRP) system for susceptibility testing of clinical Mycobacterium tuberculosis complex (MTC) isolates against rifampin and isoniazid was evaluated. Two hundred consecutive clinical MGIT cultures of MTC species were included in this study. Antibiotic susceptibility assays were set up manually for the LRP and BACTEC radiometric systems (BACTEC) and read in a plate luminometer and the BACTEC 460 instrument, respectively. Discrepant susceptibility results were resolved by the conventional agar proportion method. Of the 200 secondary cultures prepared for this study, 9 (4.5%) were lost to contamination (LRP 4, BACTEC 1, both 4). All of the remaining 191 cultures underwent susceptibility testing by both methods and the overall agreement between the LRP and BACTEC was 98.4% (rifampin 100%; isoniazid 96.9%). Of the 6 discrepant cultures tested by the agar proportion method, 2 gave results in agreement with the LRP. The sensitivity of the LRP for detection of drug-resistant isolates was 100% for both rifampin (n=9) and isoniazid (n=12). The median turnaround time for susceptibility testing was 2 days with the LRP and 9 days with BACTEC. In conclusion, the semi-automated LRP-based assay offers a rapid and practical approach for accurate susceptibility testing of M. tuberculosis cultures in diagnostic laboratories with limited financial resources, but with competent technologists.
View details for DOI 10.1016/j.tube.2007.08.006
View details for Web of Science ID 000252573900008
View details for PubMedID 17980664
LspA-independent action of globomycin on Mycobacterium tuberculosis
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
2007; 60 (2): 414-416
The objective of this study was to investigate the antimicrobial activity and specificity of globomycin, an inhibitor of lipoprotein signal peptidase II (LspA), against Mycobacterium tuberculosis.The mycobactericidal and mycobacteriostatic activity of globomycin was determined by optical density and cfu plating. The specificity of globomycin was determined by western immunoblotting using anti-MPT83 antibody.Globomycin is mycobactericidal at concentrations>or=40 mg/L. However, at 80 mg/L, the processing of the lipoprotein MPT-83 is unaffected and growth-inhibitory effect of globomycin is unchanged in an lspA null mutant (DeltalspA::lspAmut) lacking the putative drug target.Globomycin kills M. tuberculosis through a mechanism that is independent of LspA.
View details for DOI 10.1093/jac/dkm223
View details for Web of Science ID 000248986500031
View details for PubMedID 17579235
Genornics and the evolution, pathogenesis, and diagnosis of tuberculosis
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (7): 1738-1745
Tuberculosis kills nearly 2 million people annually, and current approaches to tuberculosis control are expensive, have limited efficacy, and are vulnerable to being overcome by extensively drug-resistant strains of Mycobacterium tuberculosis. Determination of the genome sequence of M. tuberculosis has revolutionized tuberculosis research, contributed to major advances in the understanding of the evolution and pathogenesis of M. tuberculosis, and facilitated development of new diagnostic tests with increased specificity for tuberculosis. In this review, we describe some of the major progress in tuberculosis research that has resulted from knowledge of the genome sequence and note some of the problems that remain unsolved.
View details for DOI 10.1172/JCI31810
View details for Web of Science ID 000247837700002
View details for PubMedID 17607348
Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages
INFECTION AND IMMUNITY
2006; 74 (11): 6449-6457
Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.
View details for DOI 10.1128/IAI.00190-06
View details for Web of Science ID 000241600500048
View details for PubMedID 16923787
Potent inhibition of macrophage responses to IFN-gamma by live virulent Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2
JOURNAL OF IMMUNOLOGY
2006; 176 (5): 3019-3027
Mycobacterium tuberculosis is a highly successful pathogen that can persist and cause disease despite an immune response. One potential mechanism for resisting elimination is by inhibiting the action of IFN-gamma. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-gamma, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN-gamma-responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN-gamma by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-gamma, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN-gamma to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-gamma. M. tuberculosis inhibition of responses to IFN-gamma requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-gamma. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-gamma.
View details for Web of Science ID 000238768000040
View details for PubMedID 16493060
alpha-Defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes
JOURNAL OF LEUKOCYTE BIOLOGY
2004; 75 (2): 332-341
Alpha-defensins are antimicrobial peptides that contribute to innate-immune functions of neutrophils and intestinal Paneth cells. Transcription of alpha-defensin genes occurs early in neutrophilic myelopoeisis. To examine the mechanisms that regulate alpha-defensin gene expression, we analyzed transcription of rat neutrophil alpha-defensin NP-3 in D4 cells, a subclone of the promyelocytic cell line IPC-81. Northern blot analysis showed that D4 cells express fivefold higher levels of alpha-defensin mRNA than the parental cell line in a manner relatively independent of passage number. Increased levels of steady-state mRNA in D4 cells correlated with markedly elevated peptide levels detected by immunocytochemical staining. To identify the cis-acting DNA elements involved in tissue-specific expression, D4 cells were transfected with luciferase reporter constructs containing NP-3 gene 5'-flanking sequences. Analyses of transfected D4 cells demonstrated that the proximal 87 base pair (bp) sequence contained cis-acting DNA elements necessary for optimal promoter activity. Mutational analyses within the 87-bp region suggested the involvement of the CAAT box and a putative polyoma enhancer-binding protein 2/core-binding factor (PEBP2/CBF) site in defensin gene transcription. Transient transfection analyses using tandem repeats of oligonucleotides containing these sequences demonstrated that proximity of the CAAT box and PEBP2/CBF site was important for defensin promoter activity. Electrophoretic mobility shift assays indicated that PEBP2/CBF or a PEBP2/CBF-related protein was involved in a specific protein-DNA interaction occurring within a DNA fragment containing the CAAT and PEBP2/CBF sequences. These data identify functional trans- and cis-elements that regulate rat defensin gene expression in high defensin-expressing promyelocytic cells.
View details for DOI 10.1189/jlb.0803384
View details for Web of Science ID 000188791700022
View details for PubMedID 14634060
Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages
JOURNAL OF MEDICAL MICROBIOLOGY
2003; 52 (7): 557-561
In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.
View details for DOI 10.1099/jmm.0.05149-0
View details for Web of Science ID 000184233400005
View details for PubMedID 12808076
Detection and drug-susceptibility testing of M-tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2003; 45 (1): 53-61
Rapid diagnosis of drug-resistant M.tuberculosis (Mtb) is desirable worldwide. We (i) describe a new luciferase reporter phage (LRP), phAE142 for this purpose; (ii) compare it to the automated MGIT 960 for time-to-detection of Mtb in clinical specimens; and (iii) evaluate its use for species confirmation and antibiotic susceptibility testing(AST) of Mtb. Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After "positives" were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST. The LRP method proved comparably efficient to MGIT 960 at detecting Mtb. Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of 3 days, with a sensitivity of 97%. Overall agreement in susceptibility results was 96%. Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16d) with LRP applied to sputum samples.
View details for Web of Science ID 000181116700007
View details for PubMedID 12573551
Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico
JOURNAL OF CLINICAL MICROBIOLOGY
2001; 39 (11): 3883-3888
The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.
View details for Web of Science ID 000171934200011
View details for PubMedID 11682502
View details for PubMedCentralID PMC88459
RAT NEUTROPHIL DEFENSINS - PRECURSOR STRUCTURES AND EXPRESSION DURING NEUTROPHILIC MYELOPOIESIS
JOURNAL OF IMMUNOLOGY
1995; 155 (9): 4476-4484
Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.
View details for Web of Science ID A1995TB46800045
View details for PubMedID 7594610