Amplicor MTB Test Research Articles

Evaluation of a modified direct agar proportion method for testing susceptibility of Mycobacterium tuberculosis from MGIT samples

The emergence of resistance to anti-tuberculosis (TB) drugs has become an obstacle to effective TB control. Thus, there is an urgent need to identify patients and initiate adequate treatment for drug-resistant cases in a timely manner. The BACTEC MGIT 960 system is well known for its rapid culturing time, and is in widespread use in Taiwan. In this study, we evaluated the possibility of replacing the traditional indirect agar proportion method with a modified direct agar proportion method (MDAPM), as a technique for rapid testing the drug susceptibility of Mycobacterium tuberculosis without additional cost. In this study, 432 positive MGIT 960 samples that were identified as M. tuberculosis complex using the MeDiPro M. tuberculosis Antigen Rapid Test or the Cobas Amplicor MTB test were evaluated. Each sample was tested separately by the MDAPM and indirect agar proportion method, between July 2008 and December 2008, to compare the consistency and total turnaround time. Four first-line anti-TB drugs-rifampin, isoniazid, ethambutol, and streptomycin-were tested. For the MDAPM and indirect agar proportion method, the respective consistencies for each drug were 99.31%, 98.38%, 98.38%, and 97.22%. Our results also indicated that the MDAPM leads to an average saving in working time of 2 weeks, compared with the traditional indirect agar proportion method. In addition to having the potential to shorten turnaround time without compromising diagnostic quality, the MDAPM also provides a more efficient and cost-effective procedure. This modified procedure presents potential benefits for TB diagnosis in laboratories already equipped with the MGIT 960 system.

Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens

A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens. In this cross-sectional study (2008-2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases. The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5-95.4%), 76.6% (59/77 specimens; CI, 65.6-85.5%), 79.2% (61/77 specimens; CI, 68.5-87.6%) and 59.7% (46/77 specimens; CI, 47.9-70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens. The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings.

Assessment of the Amplicor PCR for the detection of Mycobacterium tuberculosis in smear negative respiratory and non respiratory specimens: a retrospective analysis

Commercial polymerase chain reaction (PCR) kits are widely accepted for analysis of smear positive respiratory specimens, but the sensitivity is variable for smear negative ones. To assess the PCR method usefulness in smear negative respiratory and non respiratory specimens. We compared the PCR results (AMPLICOR MTB test, Roche) of 235 specimens subjected to culture in Loewenstein-Jensen agar (as the gold standard). 181 (76%) were respiratory and 54 (24%) extra-respiratory specimens. The sensitivity was 88%) and 50%>, respectively, specificity and PPV was 100%> in both cases. NPV was 99.4%> in respiratory specimens and 96.1% in non-respiratory specimens. The good performance of this PCR in smear negative respiratory specimens allows the clinician to take decisions based on the result of this exam. In extra-respiratory specimens the contribution is important only when the PCR result is positive.

Análisis retrospectivo del rendimiento de Amplicor-PCR® para la detección de Mycobacterium tuberculosis en muestras respiratorias y no respiratorias con baciloscopia negativa: A retrospective analysis

Commercial polymerase chain reaction (PCR) kits are widely accepted for analysis of smear positive respiratory specimens, but the sensitivity is variable for smear negative ones.To assess the PCR method usefulness in smear negative respiratory and non respiratory specimens.We compared the PCR results (AMPLICOR MTB test, Roche) of 235 specimens subjected to culture in Loewenstein-Jensen agar (as the gold standard).181 (76%) were respiratory and 54 (24%) extra-respiratory specimens. The sensitivity was 88%) and 50%>, respectively, specificity and PPV was 100%> in both cases. NPV was 99.4%> in respiratory specimens and 96.1% in non-respiratory specimens.The good performance of this PCR in smear negative respiratory specimens allows the clinician to take decisions based on the result of this exam. In extra-respiratory specimens the contribution is important only when the PCR result is positive.

Nontuberculous mycobacterial infections among patients suspected of pulmonary tuberculosis

The purpose of this study was to present a retrospective analysis of the frequency of nontuberculous mycobacteria (NTM)-related pulmonary infections among the AFB-positive and/or culture-positive patients in the Warsaw region who were suspected of tuberculosis (TB) and hospitalized in the university hospital between 1999 and 2005. All the AFB-positive pulmonary samples were examined with a molecular method using the Amplicor MTB test (Roche) for detection of Mycobacterium tuberculosis complex, and all mycobacterial isolates were speciated by high performance liquid chromatography (HPLC) analysis of mycolic acids. Patients who met clinical, radiological, and bacteriological criteria of mycobacteriosis were classified according to the American Thoracic Society (ATS) guidelines for diagnosis of NTM related disease. Among the 445 smear-positive or/and culture-positive patients, 142 subjects (31.9%) were found to be infected with M. tuberculosis. Among 303 non-TB patients, mycobacteriosis was found in 27 (8.9%) subjects. The frequency of NTM-related lung disease as compared to the bacteriologically-confirmed lung TB was estimated at 1:5. The rapid, precise methods of NTM speciation are necessary for progress in diagnostics of NTM related diseases.

Sensitivity of Polymerase Chain Reaction for Pleural Tuberculosis according to the Amount of Pleural Effusion Specimens

Background: For the diagnosis of pleural tuberculosis, polymerase chain reaction (PCR) of pleural effusion specimens has shown very low sensitivity, which might be due to the small number of bacilli in the samples. The purpose of this investigation is to determine whether the sensitivity of PCR testing can be improved when increasing the amount of pleural effusion specimens. Methods: We prospectively analyzed pleural effusion specimens obtained from 53 patients for whom the exclusion of the possibility of tuberculous pleural effusion was necessary. We performed Mycobacterium tuberculosis PCR testing using the Cobas Amplicor MTB test (Roche Diagnostic Systems) with three different amounts (10ml, 25ml, and 50ml) of pleural effusion specimen in each patient. Pleural tuberculosis was defined as having one of the following: culture-positive pleural fluid sample, histopathologic finding consistent with tuberculosis on pleural biopsy, culture-positive sputum specimen, and/or positive response to anti-tuberculous medication without other possible causes of pleural effusion. Results: Of the 53 patients, 26 received the diagnosis of pleural tuberculosis. The sensitivities of AFB smearing, Mycobacterium tuberculosis culture of pleural effusion specimen, pleural biopsy, and measurement of ADA were 3.8%, 15.4%, 84.6%, and 88.5%, respectively. The results of PCR testing were positive for 3 (11.5%), 4 (15.4%), and 3 (11.5%) of the 26 patients when using 10ml, 25ml, and 50ml of pleural effusion specimens, respectively. These results did not show a statistically significant difference in the sensitivity of PCR testing when increasing the amount of pleural effusion samples (p>0.05, symmetry exact test). Conclusion: For specimens such as pleural effusion, in which the bacillary load is very low, the clinical utility of PCR testing seems highly limited with the kits designed for the diagnosis of pulmonary tuberculosis. An increased amount of pleural effusion sample does not improve the sensitivity of PCR testing.

Detection of Mycobacterium tuberculosis DNA in respiratory and nonrespiratory specimens by the Amplicor® MTB PCR

To evaluate the diagnostic performance of a commercially available Mycobacterium tuberculosis PCR assay (Amplicor® MTB-ROCHE), 2296 respiratory and nonrespiratory specimens from 2296 patients with suspected tuberculosis (TB) were collected prospectively in an 8-year period. Clinical data for each patient were abstracted, and all samples were examined blindly by direct microscopy, culture, and PCR. M. tuberculosis DNA was detected in 93 of 113 culture-positive samples and in 29 of 38 samples from patients with probable TB. The lowest sensitivity was observed in pleural fluid and abscess aspirates. The sensitivity, specificity, and positive predictive value of the assay were 97.2%, 100%, and 100% for smear-positive specimens and 75.3%, 97.0%, and 47.5% for smear-negative specimens, respectively. The PCR cost per additional correct clinical decision was €2826 but would have declined to €308 if the test was applied only to smear-positive specimens. The overall performance of Amplicor MTB test was excellent for smear-positive, but suboptimal for smear-negative specimens.

Comparison of the Sodium Hydroxide Specimen Processing Method with the C 18 -Carboxypropylbetaine Specimen Processing Method Using Independent Specimens with Auramine Smear, the MB/BacT Liquid Culture System, and the COBAS AMPLICOR MTB Test

A study was performed to diagnose tuberculosis by smear, culture, and nucleic acid amplification. The study was comprised of two independent arms. Each arm used a different specimen processing method; in one arm, all specimens were processed with N-acetyl-l-cysteine-sodium hydroxide, and in the other arm, all specimens were processed with C(18)-carboxypropylbetaine and lytic enzymes. In each arm, all processed sediments were split for analysis by auramine smear, by culture using the MB/BacT liquid culture system and solid media, and by nucleic acid amplification using the COBAS AMPLICOR MTB test. In the N-acetyl-l-cysteine-sodium hydroxide arm, 1,468 specimens were analyzed: 65 were smear positive; 88 and 42 were culture positive for Mycobacterium tuberculosis and nontuberculous mycobacteria, respectively; and 103 were PCR positive. Relative to cultures positive for M. tuberculosis, the sensitivity and specificity of the smear were 68.2% and 99.6%, respectively, and those of PCR were 75.0% and 97.3%, respectively. In the C(18)-carboxypropylbetaine study arm, 1,423 specimens were analyzed: 44 were smear positive; 82 and 31 were culture positive for M. tuberculosis and nontuberculous mycobacteria, respectively; and 91 were PCR positive. The sensitivity and specificity of the smear were 48.8% and 99.7%, respectively, and those of PCR were 78.0% and 98.0%, respectively. When the two arms were compared, C(18)-carboxypropylbetaine specimen processing significantly increased the number of smear-negative and culture-positive specimens and significantly increased the PCR sensitivity among this same group of specimens while at the same time significantly reducing the inhibition rate.

The Clinical Utility of Polymerase Chain Reaction for the Diagnosis of Pleural Tuberculosis

There is no exact consensus about the usefulness of the Mycobacterium tuberculosis polymerase chain reaction (PCR) testing for the diagnosis of tuberculous pleural effusion because of the diverse PCR methods and the different diagnostic criteria that are described in other studies. We analyzed pleural effusion specimens obtained from 111 patients for whom the exclusion of the possibility of tuberculous pleural effusion was necessary. We performed M. tuberculosis PCR testing using the Cobas Amplicor MTB test (Roche Diagnostic Systems), which is fully automated and commercially available. Results of the M. tuberculosis PCR test of pleural effusion specimens were positive for 7 (17.1%) of the 41 patients with confirmed pleural tuberculosis and for 3 (18.8%) of the 16 patients with probable pleural tuberculosis. The overall sensitivity and specificity of M. tuberculosis PCR testing of pleural effusion were 17.5% and 98.1%, respectively. The sensitivity of M. tuberculosis PCR testing for each group of patients with tuberculous pleural effusion detected by smear-positive results, smear-negative and culture-positive results, and culture-negative and pleural biopsy-positive results, was 100.0%, 33.3%, and 3.7%, respectively. Of the 57 patients with pleural tuberculosis, only 3 (5.3%) had positive results of M. tuberculosis PCR testing along with negative results of smearing, negative results of pleural pathological analysis, and a low level of adenosine deaminase. For specimens such as pleural effusion, in which the bacillary load is very low, the clinical utility of PCR testing seems highly limited.

Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference method. A total of 824 respiratory specimens, comprised of sputa, bronchoalveolar lavage fluid, and bronchial and tracheal aspirates from 580 patients, were evaluated. Out of 824 clinical specimens, 109 specimens from 43 patients were culture positive for M. tuberculosis. Of these 109 specimens, 67 were smear positive, 85 were positive by the COBAS AMPLICOR MTB test, and 94 were positive by the BDProbeTec ET. Of the 715 culture-negative specimens, 17 were positive by the auramine staining, 11 were positive by the COBAS AMPLICOR MTB test, and 12 were positive by the BDProbeTec ET. After discrepancy analysis and review of the patients' clinical data, 130 specimens from 50 patients were considered "true-positive" specimens. This resulted in the following sensitivities: microscopy, 61.5%; COBAS AMPLICOR MTB test, 78.0%; and BDProbeTec ET, 86.2%. The specificities of each system, based on the clinical diagnosis, were 99.7% for microscopy, 99.9% for the COBAS AMPLICOR MTB test, and 99.9% for the BDProbeTec ET. The data presented represent a considerable number of specimens evaluated with a considerable number of culture- and auramine-positive and culture-positive and auramine-negative results and therefore give a realistic view of how the data should be interpreted in a daily routine situation. Specifically, the data with regard to the culture-positive and auramine-negative specimens are useful, because in a routine situation, auramine-negative specimens are sometimes accepted, on clinical indications, to be analyzed by an amplification method.

Evaluation of the COBAS AMPLICOR MTB test for the detection of Mycobacterium tuberculosis complex

We evaluated the COBAS AMPLICOR polymerase chain reaction (PCR) based test for the detection of Mycobacterium tuberculosis complex in 866 respiratory and non-respiratory samples. Acid-fast staining and culture on Lowenstein-Jensen medium were also performed on all samples. Of the 866 samples tested, 87 (10.0%) were PCR-positive compared to 94 (10.9%) culture positive. There were no false positive results but 7 PCR-negative, culture-positive samples were, considered false negatives after reviewing medical records of patients. A PCR inhibitory rate of 2.0% (17/866) was observed in respiratory samples only. Sensitivity, specificity, and positive and negative predictive values for this test were 92.5%, 100%, 100% and 99.1% respectively. This test is a valuable diagnostic tool for today's mycobacteriology laboratory.

The Cobas Amplicor MTB test for detection of Mycobacterium tuberculosis complex from respiratory and non-respiratory clinical specimens.

The Cobas Amplicor MTB test is a polymerase chain reaction (PCR) technique commonly used for direct detection of Mycobacterium tuberculosis in clinical samples. This assay is only validated for respiratory specimens, but many physicians also request PCR analyses for non-respiratory ones. 877 respiratory and 564 non-respiratory specimens were analysed by this test. Using culture results as standard, the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the PCR were, respectively, 97.9%, 100%, 100% and 94.4% for smear-positive respiratory specimens, 68.8%, 99.2%, 87.5% and 97.5% for smear-negative respiratory samples, 57.8%, 98.6%, 78.8% and 96.4% for all non-respiratory specimens, and 42.4%, 98.6%, 66.7% and 96.4% for smear-negative non-respiratory specimens. 154 cerebrospinal fluid samples were analysed and the sensitivity, specificity, PPV and NPV were 55.6%, 97.2%, 55.6% and 97.2%, respectively. These results indicate that the Cobas Amplicor MTB test enables detection of tuberculosis in respiratory specimens, but does not perform well enough in non-respiratory specimens. The method fails particularly in cases where a reliable and rapid test is urgently needed, e.g. in tuberculous meningitis.

TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test

Setting: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa.Objective: To assess the role of the semi-automated Roche COBAS AMPLICORTMMycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM).Design: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICORTMTB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture.Results: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICORTM. The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICORTMPCR test. The comparative sensitivities of the TB COBAS AMPLICORTMPCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICORTM, giving a specificity of 100%.Conclusion: The COBAS AMPLICORTMTB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICORTMTB PCR test.

Transport of digested decontaminated sputum specimens to a central laboratory for testing for M. tuberculosis by Amplicor MTB test.

Rapid diagnosis of tuberculosis may improve management of infected patients and facilitate infection control procedures. The relatively slow growth rate of M. tuberculosis and the limited sensitivity and specificity of microscopy make rapid diagnosis difficult. Nucleic acid amplification techniques have been extensively studied for the detection of M. tuberculosis DNA and a number of commercial products for detection of M. tuberculosis nucleic acid in clinical specimens are now available. As performance of diagnostic PCR at central reference laboratories may be desirable, the impact of specimen transport on the performance of the Amplicor MTB PCR assay is of practical importance. We have assessed the performance of the Amplicor MTB PCR on specimens submitted and initially processed in laboratories in 3 cities and then transported to a single laboratory for PCR assay. The overall sensitivity of the PCR test was 97 per cent and the corrected specificity was 100 per cent. All of 23 culture positive specimens collected locally were PCR positive compared with 10 of 11 culture positive specimens transported from elsewhere. In this study transportation of digested decontaminated specimens to a central laboratory either frozen at -20 degrees, or overnight at room temperature had no apparent effect on the performance characteristic of the Amplicor MTB PCR assay.

Evaluation of Amplicor MTB test as adjunct to smears and culture for direct detection of Mycobacterium tuberculosis in the French Caribbean

A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.