Bio

Clinical Focus


  • Clinical Laboratory Techniques
  • Clinical Pathology
  • Mycobacterium Infections

Academic Appointments


Administrative Appointments


  • Director, Clinical Microbiology (2007 - Present)

Professional Education


  • Fellowship:New York University (2007) NY
  • Residency:UCSF - Departments of Pathology and Laboratory Medicine (2003) CA
  • Internship:UCSF - Departments of Pathology and Laboratory Medicine (2002) CA
  • Board Certification: Clinical Pathology, American Board of Pathology (2004)
  • Medical Education:Stanford University School of Medicine (2001) CA
  • MD, Stanford University, Medicine (2001)

Research & Scholarship

Current Research and Scholarly Interests


Development and implementation of rapid diagnostic assays for the detection, identification, and susceptibility testing of clinically important mycobacteria. Understanding the role of M. tuberculosis lipoproteins in the pathogenesis of tuberculosis.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Colorimetric Sensor Array Allows Fast Detection and Simultaneous Identification of Sepsis-Causing Bacteria in Spiked Blood Culture JOURNAL OF CLINICAL MICROBIOLOGY Lim, S. H., Mix, S., Xu, Z., Taba, B., Budvytiene, I., Berliner, A. N., Queralto, N., Churi, Y. S., Huang, R. S., Eiden, M., Martino, R. A., Rhodes, P., Banaei, N. 2014; 52 (2): 592-598

    Abstract

    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 Schroeder, L. F., Robilotti, E., Peterson, L. R., Banaei, N., Dowdy, D. W. 2014; 52 (2): 489-496

    Abstract

    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 Pai, M., Denkinger, C. M., Kik, S. V., Rangaka, M. X., Zwerling, A., Oxlade, O., Metcalfe, J. Z., Cattamanchi, A., Dowdy, D. W., Dheda, K., Banaei, N. 2014; 27 (1): 3-20

    Abstract

    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-?) 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

  • False-Positive Quantiferon Results at a Large Healthcare Institution. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Slater, M., Dubose, A., Banaei, N. 2014

    View details for DOI 10.1093/cid/ciu139

    View details for PubMedID 24610428

  • Pretreatment of sinus aspirates with dithiothreitol improves yield of fungal cultures in patients with chronic sinusitis INTERNATIONAL FORUM OF ALLERGY & RHINOLOGY Chisholm, K. M., Getsinger, D., Vaughan, W., Hwang, P. H., Banaei, N. 2013; 3 (12): 992-996

    Abstract

    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

  • Alerting Physicians during Electronic Order Entry Effectively Reduces Unnecessary Repeat PCR Testing for Clostridium difficile. Journal of clinical microbiology Luo, R. F., Spradley, S., Banaei, N. 2013; 51 (11): 3872-3874

    Abstract

    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

  • A Pediatric Case of New Delhi Metallo-▀-Lactamase-1-Producing Enterobacteriaceae in The United States. Pediatric infectious disease journal Green, D. A., Srinivas, N., Watz, N., Tenover, F. C., Amieva, M., Banaei, N. 2013; 32 (11): 1291-1294

    Abstract

    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 Gaur, R. L., Pai, M., Banaei, N. 2013; 51 (11): 3521-3526

    Abstract

    Gamma interferon (IFN-?) 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-? 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-? 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

  • Challenges with QuantiFERON-TB Gold Assay for Large-Scale, Routine Screening of U.S. Healthcare Workers AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Slater, M. L., Welland, G., Pai, M., Parsonnet, J., Banaei, N. 2013; 188 (8): 1005-1010

    Abstract

    North American occupational health programs that switched from the tuberculin skin test (TST) to IFN-? 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-? 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-? 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 Pai, M., Banaei, N. 2013; 63 (7): 458-460

    View details for DOI 10.1093/occmed/kqt105

    View details for Web of Science ID 000327542700002

    View details for PubMedID 24097956

  • 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 Lefterova, M. I., Slater, K. A., Budvytiene, I., Dadone, P. A., Banaei, N. 2013; 51 (9): 3000-3005

    Abstract

    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 Luo, R. F., Banaei, N. 2013; 33 (3): 553-566

    Abstract

    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 Moncada, P. A., Budvytiene, I., Ho, D. Y., Deresinski, S. C., Montoya, J. G., Banaei, N. 2013; 140 (2): 203-208

    Abstract

    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 ACS NANO Ren, K., Banaei, N., Zare, R. N. 2013; 7 (7): 6031-6036

    Abstract

    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 Schroeder, L., Banaei, N. 2013; 368 (12)

    View details for DOI 10.1056/NEJMicm1204579

    View details for PubMedID 23514310

  • Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak PLOS ONE Kato-Maeda, M., Ho, C., Passarelli, B., Banaei, N., Grinsdale, J., Flores, L., Anderson, J., Murray, M., Rose, G., Kawamura, L. M., Pourmand, N., Tariq, M. A., Gagneux, S., Hopewell, P. C. 2013; 8 (3)

    Abstract

    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 Rujanavej, V., Soudry, E., Banaei, N., Baron, E. J., Hwang, P. H., Nayak, J. V. 2013; 27 (2): 134-137

    Abstract

    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 ?-Release Assays for Diagnosis of Latent Tuberculosis in Healthcare Workers in Low-Incidence Settings: Pros and Cons. Clinical chemistry Pollock, N. R., McAdam, A. J., Pai, M., Nardell, E. A., Bernardo, J., Banaei, N., Mobo, J. 2013

    View details for DOI 10.1373/clinchem.2012.201178

    View details for PubMedID 24100806

  • In Vitro Immunomodulation of a Whole Blood IFN-gamma Release Assay Enhances T Cell Responses in Subjects with Latent Tuberculosis Infection PLOS ONE Gaur, R. L., Suhosk, M. M., Banaei, N. 2012; 7 (10)

    Abstract

    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-?) 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-? 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 Slater, M., Parsonnet, J., Banaei, N. 2012; 50 (9): 3105-3107

    Abstract

    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 DiMaio, M. A., Pereira, I. T., George, T. I., Banaei, N. 2012; 50 (9): 2877-2880

    Abstract

    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 Bautista, J., Banaei, N. 2012; 73 (3): 257-259

    Abstract

    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-? 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 Taylor, N., Gaur, R. L., Baron, E. J., Banaei, N. 2012; 50 (7): 2272-2276

    Abstract

    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 Powel, M. S., Alizadeh, A. A., Budvytiene, I., Schaenman, J. M., Banaei, N. 2012; 50 (3): 1125-1127

    Abstract

    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 Limbago, B. M., Rasheed, J. K., Anderson, K. F., Zhu, W., Kitchel, B., Watz, N., Munro, S., Gans, H., Banaei, N., Kallen, A. J. 2011; 49 (12): 4239-4245

    Abstract

    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-?-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 Doberne, D., Gaur, R. L., Banaei, N. 2011; 49 (8): 3061-3064

    Abstract

    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 PLOS PATHOGENS Bold, T. D., Banaei, N., Wolf, A. J., Ernst, J. D. 2011; 7 (5)

    Abstract

    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-? 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-? production. During the early phase of infection, ?10% of P25TCRTh1 cells produced IFN-? 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 Herrera, V., Perry, S., Parsonnet, J., Banaei, N. 2011; 52 (8): 1031-1037

    Abstract

    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 McNeil, C. J., Luo, R. F., Vogel, H., Banaei, N., Ho, D. Y. 2011; 49 (3): 1171-1174

    Abstract

    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

  • Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites CHEMICAL COMMUNICATIONS Koo, C., Wang, S., Gaur, R. L., Samain, F., Banaei, N., Kool, E. T. 2011; 47 (41): 11435-11437

    Abstract

    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 Luo, R. F., Banaei, N. 2010; 48 (10): 3738-3741

    Abstract

    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 Herrera, V., Yeh, E., Murphy, K., Parsonnet, J., Banaei, N. 2010; 48 (8): 2672-2676

    Abstract

    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 Guarin, N., Budvytiene, I., Ghafghaichi, L., Banaei, N. 2010; 67 (4): 333-336

    Abstract

    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 Luo, R. F., Scahill, M. D., Banaei, N. 2010; 48 (7): 2569-2570

    Abstract

    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

  • Mixed infection involving Actinomyces, Aggregatibacter, and Fusobacterium species presenting as perispinal tumor ANAEROBE Ghafghaichi, L., Troy, S., Budvytiene, I., Banaei, N., Baron, E. J. 2010; 16 (2): 174-178

    Abstract

    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 Nguyen, D. T., Yeh, E., Perry, S., Luo, R. F., Pinsky, B. A., Lee, B. P., Sisodiya, D., Baron, E. J., Banaei, N. 2010; 48 (3): 785-790

    Abstract

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

    View details for DOI 10.1128/JCM.02150-09

    View details for Web of Science ID 000274996200016

    View details for PubMedID 20071556

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

    Abstract

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

    View details for DOI 10.1086/649875

    View details for Web of Science ID 000273500300014

    View details for PubMedID 20047483

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

    Abstract

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

    View details for Web of Science ID 000271883400007

    View details for PubMedID 19861005

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

    Abstract

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

    View details for DOI 10.1128/JCM.00342-09

    View details for Web of Science ID 000271373000013

    View details for PubMedID 19741081

  • Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Banaei, N., Kincaid, E. Z., Lin, S. G., Desmond, E., Jacobs, W. R., Ernst, J. D. 2009; 53 (9): 3799-3802

    Abstract

    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 Wei, M. C., Banaei, N., Yakrus, M. A., Stoll, T., Gutierrez, K. M., Agarwal, R. 2009; 31 (8): 556-560

    Abstract

    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 PLOS ONE Yeh, E., Pinsky, B. A., Banaei, N., Baron, E. J. 2009; 4 (7)

    Abstract

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

    View details for DOI 10.1371/journal.pone.0006141

    View details for Web of Science ID 000267806300010

    View details for PubMedID 19578541

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

    Abstract

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

    View details for DOI 10.1099/jmm.0.006734-0

    View details for Web of Science ID 000266018900019

    View details for PubMedID 19369531

  • Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR JOURNAL OF CLINICAL MICROBIOLOGY Richardson, E. T., Samson, D., Banaei, N. 2009; 47 (5): 1497-1502

    Abstract

    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 Sangoi, A. R., Rogers, W. M., Longacre, T. A., Montoya, J. G., Baron, E. J., Banaei, N. 2009; 131 (3): 364-375

    Abstract

    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 Blumenthal, A., Kobayashi, T., Pierini, L. M., Banaei, N., Ernst, J. D., Miyake, K., Ehrt, S. 2009; 5 (1): 35-46

    Abstract

    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 Pinsky, B. A., Banaei, N. 2008; 46 (7): 2241-2246

    Abstract

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

    View details for DOI 10.1128/JCM.00347-08

    View details for Web of Science ID 000258906800016

    View details for PubMedID 18508937

  • 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 Wolf, A. J., Desvignes, L., Linas, B., Banaiee, N., Tamura, T., Takatsu, K., Ernst, J. D. 2008; 205 (1): 105-115

    Abstract

    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 TUBERCULOSIS Banaiee, N., January, V., Barthus, C., Lambrick, M., Roditi, D., Behr, M. A., Jacobs, W. R., Steyn, L. M. 2008; 88 (1): 64-68

    Abstract

    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 Banaiee, N., Jacobs, W. R., Ernst, J. D. 2007; 60 (2): 414-416

    Abstract

    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 Ernst, J. D., Trevejo-Nunez, G., Banaiee, N. 2007; 117 (7): 1738-1745

    Abstract

    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 Banaiee, N., Jacobs, W. R., Ernst, J. D. 2006; 74 (11): 6449-6457

    Abstract

    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 na´ve 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 Banaiee, N., Kincaid, E. Z., Buchwald, U., Jacobs, W. R., Ernst, J. D. 2006; 176 (5): 3019-3027

    Abstract

    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 Yamamoto, C. M., Banaiee, N., Yount, N. Y., Patel, B., Selsted, M. E. 2004; 75 (2): 332-341

    Abstract

    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 Banaiee, N., Bobadilla-del-Valle, M., Riska, P. F., Bardarov, S., Small, P. M., Ponce-de-Leon, A., Jacobs, W. R., Hatfull, G. F., Sifuentes-Osornio, J. 2003; 52 (7): 557-561

    Abstract

    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 Bardarov, S., Dou, H., Eisenach, K., Banaiee, N., Ya, S., Chan, J., Jacobs, W. R., Riska, P. F. 2003; 45 (1): 53-61

    Abstract

    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 Banaiee, N., Bodadilla-del-Valle, M., Bardarov, S., Riska, P. F., Small, P. M., Ponce-de-Leon, A., Jacobs, W. R., Hatfull, G. F., Sifuentes-Osornio, J. 2001; 39 (11): 3883-3888

    Abstract

    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 L÷wenstein-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

  • RAT NEUTROPHIL DEFENSINS - PRECURSOR STRUCTURES AND EXPRESSION DURING NEUTROPHILIC MYELOPOIESIS JOURNAL OF IMMUNOLOGY Yount, N. Y., WANG, M. S., Yuan, J., Banaiee, N., Ouellette, A. J., Selsted, M. E. 1995; 155 (9): 4476-4484

    Abstract

    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

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