dProsthetic valve endocarditis (PVE) due to fast-growing nontuberculous mycobacteria (NTM) has been reported anecdotally. Reports of PVE with slowly growing NTM, however, are lacking. We present here one case of PVE and one case of bloodstream infection caused by Mycobacterium chimaera. Randomly amplified polymorphic DNA (RAPD)-PCR indicated a relatedness of the two M. chimaera strains. Both patients had heart surgery 2 years apart from each other. A nosocomial link was not detected. Infective endocarditis due to nontuberculous mycobacteria (NTM) is a rare complication after heart valve surgery. The reported cases in the literature are associated with the insertion of biological and also of mechanical valves (1). Cases of prosthetic valve endocarditis (PVE) due to NTM often involve rapidly growing mycobacteria. To date, there have been no concise reports on slowly growing mycobacteria, such as the Mycobacterium avium complex (MAC), as the agent causing PVE. However, the MAC is found occasionally on resected heart valves. A case series examining microbiological cultures from valves found slowly growing NTM in 5.5% of the cases without further clinical or histopathological evidence of infective endocarditis (IE) (2).MAC members are the most common cause of NTM infections in humans. M. intracellulare and M. avium are the main representatives of the MAC, but different MAC sequevars, e.g., M. chimaera, have been identified in recent years (3). Similar to other members of the MAC, M. chimaera has been reported to cause mainly pulmonary disease (3). In the summer of 2011, we encountered one fatal case of definite PVE and one fatal bloodstream infection due to M. chimaera. Randomly amplified polymorphic DNA (RAPD)-PCR was used to study the relatedness of the M. chimaera isolates from these two patients.(Part of this research was presented at the 101st Annual Meeting of the United States and Canadian Academy of Pathology, Vancouver, BC, Canada, 17 to 23 March 2012.) CASE REPORTS Patient 1. In June 2011, a 58-year-old male was admitted to the hospital for mitral and aortic valve replacement. In 2008, the patient had undergone aortic and mitral reconstruction with implantation of a mitral annuloplasty ring. Twelve months prior to the current admission, the patient experienced intermittent fever, weight loss, and respiratory distress. PVE was ruled out with repeated negative conventional blood cultures and a transesophageal echocardiogram that showed only moderate mitral and aortic insufficiency not suggestive of infective endocarditis. At that time, systemic sarcoidosis had been diagnosed based on unspecific granulomatous inflammation in liver and kidney biopsy specimens, a reticular pattern on the chest X ray together with a severely constrained CO diffusion capacity, and a bronchoalveolar lavage showing a predominance of lymphocytes but only a slightly elevated CD4/CD8 quotient. A Mycobacterium genus PCR from the preserved liver and kidney biopsy specimens was performed retrospectively 1 year later and showed negative re...
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 ...
c Xpert-MTB/Rif is one of the most frequently used molecular screening tests for multidrug-resistant tuberculosis worldwide. We report false-negative assay results in the presence of rpoB Leu533Pro, which is associated with low-level phenotypic rifampin resistance. Accurate and timely confirmation of rifampin susceptibility results obtained with Xpert-MTB/Rif is imperative.T he accurate diagnosis of drug-resistant and multidrug-resistant (MDR) tuberculosis (TB) is imperative to initiate adequate treatment, to avoid transmission of the disease, and to prevent the development of further drug resistance. Because of its worldwide rollout and rapid implementation, the Xpert-MTB/Rif assay (Cepheid) has become one of the most frequently used molecular screening tests for TB and MDR TB in both resource-poor and resource-rich countries (1). Recently, a 67-year-old Swissborn male patient was admitted to a secondary-care hospital in Switzerland with clinical and radiologic suspicion of pulmonary TB. Rapid testing by Xpert-MTB/Rif showed the presence of Mycobacterium tuberculosis complex in two sputum samples (Table 1; samples 1 and 2). In addition, these specimens showed indeterminate and definite rifampin (RMP) resistance, respectively (Table 1, samples 1 and 2). Since the patient had no history of TB and was from Switzerland (a low MDR TB incidence setting), based on previous reports of false RMP resistance assay results (2, 3), the clinician doubted the accuracy of the Xpert-MTB/ Rif RMP results. Therefore, an additional four sputum samples were submitted to the Swiss National Reference Center for Mycobacteria for rapid confirmation before initiation of MDR TB therapy. Further patient testing revealed HIV positivity.The four additional sputum specimens (Table 1, samples 3 to 6) were acid-fast smear positive by the Ziehl-Neelsen method (4), and Xpert-MTB/Rif testing detected the presence of M. tuberculosis complex. Surprisingly, all of the samples were scored RMP susceptible by the molecular assay (Table 1). In order to resolve the discrepant Xpert-MTB/Rif results and to rapidly confirm or rule out MDR TB, direct rpoB sequencing of the 81-bp core region and additional molecular screening for isoniazid (INH) resistance were performed as described previously (3, 5) and revealed rpoB Leu(CTG)533Pro(CCG) and katG Ser(AGC)315Thr(ACC) mutations, respectively, in all four specimens. Growth detection and quantitative phenotypic drug susceptibility testing (DST) for firstand second-line antituberculosis drugs with the MGIT 960 system and EpiCenter software with the TB eXiST module (Becton, Dickinson Microbiology Systems, Sparks, MD) were performed as described earlier (6). Quantitative DST identified resistance to RMP at 0.5 g/ml and susceptibility at 1.0, 4.0, and 20 g/ml, and resistance to rifabutin at 0.1 g/ml and susceptibility at 0.4 and 2.0 g/ml. DST for INH showed resistance at 0.1 and 1.0 g/ml and intermediate resistance at 3.0 and 10.0 g/ml. No drug resistance was identified by conventional DST for other first-and second...
The goal of the present study was to examine the transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Switzerland. Between 2006 and 2012, a total of 49 MDR-TB cases were reported to the Swiss Federal Office of Public Health, 46 of which were of foreign origin. All 49 initial strains were evaluated by molecular epidemiologic methods at the Swiss National Reference Centre for Mycobacteria. In 43 strains, unique DNA fingerprint patterns were identified. Twelve strains were grouped into six clusters. Data from contact tracing suggest likely in-country transmission in four clusters, mostly among close contacts. In the remaining two clusters, no contact tracing data were available, but the identified genotypes were known to be prevalent in the countries of origin of the patients, suggesting the possibility that the infection was acquired there. While most MDR-TB cases are imported to Switzerland, at least four of the 49 MDR-TB cases were due to transmission within the country. The imported cases, however, did not lead to secondary cases outside the circles of close contacts. The results also indicate that prevention of MDR-TB transmission among immigrants may require closer monitoring.
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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