The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD 660 ) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n ‫؍‬ 838) and nonrespiratory (n ‫؍‬ 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens. The Cobas Amplicor test is based on amplification of a 584-bp 5= part of the 16S rRNA gene (1, 2) using biotinylated primers, a capture probe, and photometric staining for quantification (3, 4). The Cobas Amplicor test was extensively evaluated for various clinical specimens and demonstrated high sensitivity and specificity, in particular for smear-positive samples (5). We reported previously on a substantial rate of false-positive samples, in particular when the Cobas Amplicor MTB test showed results with optical density at 660 nm (OD 660 ) values of Ͼ0.35 and Ͻ2.0 (6). The observed false-positive results were demonstrated to be due to cross-reactivity of the capture probe with closely related species, such as nontuberculous mycobacterial species and Corynebacterium spp. (6).Recently, Roche Diagnostics (Rotkreuz, Switzerland) replaced the Cobas Amplicor MTB test with the Cobas TaqMan MTB test. The Cobas TaqMan MTB test is a real-time PCR assay that amplifies part of the 16S rRNA gene with the use of a TaqMan probe for the detection of Mycobacterium tuberculosis complex DNA in clinical specimens (7). Here, we evaluated the Cobas TaqMan MTB assay and compared its performance with that of the Cobas Amplicor MTB assay. In a prospective study, we analyzed the performance of the Cobas TaqMan MTB assay for routine mycobacteriology laboratory use during a 6-month period in which 1,143 specimens were submitted for MTB PCR testing. MATERIALS AND METHODSPatient population. The Institute of Medical Microbiology (IMM) serves the 850-bed tertiary University Hospital of Zurich and smaller surrounding hospitals. The patients ...

Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology.Using 6875 clinical samples and a study period of 35 months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1–2 days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB.MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy.Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

We have recently developed a PCR assay for detection of Mycobacterium spp. at the genus level based on the Cobas Amplicor platform. The sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. The specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. In a follow-up study, we have systematically evaluated the Mycobacterium genus assay in parallel with the Cobas Amplicor Mycobacterium tuberculosis assay on 2,169 clinical specimens, including respiratory and nonrespiratory specimens. Based on the genus assay, nontuberculous mycobacteria were readily detected and identified to the species level by PCR-mediated sequencing. In addition, our data point to a limited specificity of the Cobas Amplicor M. tuberculosis assay.Nontuberculous mycobacteria (NTM) are frequently associated with human disease. Infections with NTM are increasingly observed in immunocompromised patients (10, 16), although patients without underlying medical conditions are also affected. Diseases caused by NTM include lung disease (e.g., Mycobacterium abscessus, Mycobacterium kansasii), cutaneous ulcers (e.g., Mycobacterium marinum), disseminated infections (e.g., Mycobacterium genavense), lymphadenitis (e.g., Mycobacterium avium), and joint infections (e.g., Mycobacterium haemophilum) (for a review, see references 5 and 16). Rapid and reliable laboratory detection of NTM is crucial for clinical management and proper antibiotic therapy. Molecular genetic assays for the detection of NTM in clinical specimens are only infrequently implemented in routine diagnostics. With a view to developing an assay that is capable of detecting a large number of nontuberculous mycobacteria in clinical specimens, we have recently developed a semiautomated PCR-based assay for NTM on the basis of the Roche Cobas Amplicor platform (8). The sensitivity of the genus assay was 100% for smearpositive specimens and 47.9% for smear-negative specimens. The specificity of the genus assay was 97.7%, the positive predictive value (PPV) 84.6%, and the negative predictive value 93.1% (8). These values are comparable to those published for the Cobas Amplicor Mycobacterium tuberculosis test (11; reviewed in reference 9). We have now extensively evaluated the Mycobacterium genus assay under routine laboratory conditions. MATERIALS AND METHODSDecontamination of specimens, microscopy, and culture. Clinical specimens were decontaminated using the sodium hydroxide method for samples from sterile sites and the N-acetyl-L-cysteine-sodium hydroxide method for respiratory samples (6). Auramine-rhodamine fluorochrome staining was used for microscopic examination; positive microscopy results were confirmed using ZiehlNeelsen staining (6). For the recovery of mycobacteria from culture, standard media were inoculated (7H11 plates and BBL MGIT [Becton, Dickinson and Company]) and maintained for 7 weeks at 37°C. Mycobacteria were identified by 16S rRNA gene sequence analysis as described previously (7)....

bThe Xpert MTB/RIF assay is a rapid and fully automated real-time PCR assay. The performance of the Xpert MTB/RIF assay as a primary screening test for urgent clinical specimens was evaluated during a 2-year period. The results showed that replacing smear microscopy with the Xpert MTB/RIF assay facilitates laboratory handling and improves the sensitivity and specificity of Mycobacterium tuberculosis detection. Rapid and accurate diagnosis of tuberculosis (TB) is indispensable to adequately manage the disease and control its transmission. Acid-fast bacilli (AFB) smear microscopy is an established low-cost screening procedure to identify patients with tuberculosis. However, the sensitivity of smear microscopy is low, varying between 22 and 80% (1). In general, the routine application of nucleic acid amplification techniques for detection of Mycobacterium tuberculosis results in accurate diagnosis of tuberculosis (2, 3, 4, 5, 6) but requires laborious processing time and dedicated biosafety conditions. The recently introduced Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, walk-away real-time PCR-based assay with a time to result of approximately 2 h. To detect M. tuberculosis and mutations associated with resistance to rifampin (RIF), the 81-bp core region of the rpoB gene is amplified and probed with five overlapping molecular beacons (7). M. tuberculosis is identified when at least 2 probes give a positive signal within a predefined number of cycles. A RIF mutation is detected by lack of or delayed onset of fluorescence of at least one molecular beacon. Disadvantages of the Xpert MTB/RIF are its exceeding costs and a reduced sensitivity in comparison with other PCR M. tuberculosis assays (8, 9).The objective of the present study was to evaluate the feasibility of the Xpert MTB/RIF assay to replace direct smear microscopy as a primary screening test for urgent clinical specimens in a setting of low TB prevalence.The Institute of Medical Microbiology (University of Zurich) is a tertiary care diagnostic center that receives clinical specimens 7 days/week for AFB microscopy to urgently confirm or rule out TB in newly identified suspect cases. Such samples are marked as urgent cases and are not to be confused with regular samples submitted to the mycobacteriology laboratory. All respiratory and nonrespiratory specimens submitted for urgent smear microscopy between July 2010 and June 2012 were included in this study; ethical approval was not needed. Specimens were adjusted to 5 ml with sterile distilled water when the specimen volume was Ͻ5 ml. The Xpert MTB/RIF assay was performed following the manufacturer's instructions for respiratory specimens, using a 1-ml aliquot. Nonrespiratory specimens were tested similarly. The remaining 4 ml of all unprocessed specimen with the exception of cerebrospinal fluid (CSF) was homogenized and decontaminated using an equal volume of N-acetyl-L-cysteine (NALC)-2% NaOH for 15 min (10). After neutralization with phosphate buffer (67 mM, pH 6.8) and centrifugation, ...

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