JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2011, p. 173–176 Vol. 49, No.

0095-1137/11/$12.00 doi:10.1128/JCM.00694-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Evaluation of Cobas TaqMan MTB PCR for Detection
of Mycobacterium tuberculosis䌤
Jeong Hyun Kim,1† Young Jae Kim,1† Chang-Seok Ki,2* Ji-Youn Kim,3 and Nam Yong Lee2
Department of Laboratory Medicine, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon,
South Korea1; Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of
Medicine, Seoul, South Korea2; and Center for Clinical Medicine, Samsung Biomedical Research Institute,
Samsung Medical Center, Seoul, South Korea3
Received 6 April 2010/Returned for modification 1 June 2010/Accepted 25 October 2010

Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a
real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel,
Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the
Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously
tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with
other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24
specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs,
while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both
PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these
were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV)
predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the
Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous
mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/␮l. The Cobas
TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without dis-
turbing the specificity and NPV than the Amplicor MTB PCR test.

Tuberculosis is a global public health problem involving sig- The aim of this study was to evaluate the performance of the
nificant morbidity, and early diagnosis followed by adequate Cobas TaqMan MTB Test by comparing the results to those
treatment is essential for the prevention of morbidity and mor- from the Amplicor MTB PCR. The cross-reactivity with non-
tality (12, 17). The reemergence of tuberculosis due to the tuberculous mycobacterial species and the detection limit were
appearance of multidrug-resistant strains of Mycobacterium tu- also evaluated.
berculosis has intensified the need for rapid detection of M.
tuberculosis (19). Over the past several decades, diagnostic MATERIALS AND METHODS
methods for M. tuberculosis have improved, and nucleic acid- Study design. A total of 406 clinical specimens were prospectively collected
based amplification techniques now allow rapid and sensitive from 247 patients with suspected tuberculosis infection between June and August
detection in clinical settings (1, 7, 10). 2008 at a tertiary care hospital in Seoul, South Korea. Clinical data, including
history and radiologic and laboratory findings, were collected for each patient.
Currently, several assays are commercially available for the
All clinical specimens were examined blindly by direct microscopy, conventional
detection of the M. tuberculosis complex, including the Cobas culture, and Cobas Amplicor and TaqMan MTB PCR tests for the detection of
Amplicor MTB PCR test (Roche Diagnostics, Basel, Switzer- M. tuberculosis. To evaluate cross-reactivity between M. tuberculosis and nontu-
land), the RealArt Mycobact Diff Kit (Qiagen, Hamburg, Ger- berculous mycobacteria (NTM), 40 NTM and 2 M. tuberculosis colonies were
many) (2), AMTD (Gen-Probe, Inc. San Diego, California) (14), tested by Cobas TaqMan MTB PCR. The detection limit for the TaqMan MTB
PCR was measured by 1:10 serial dilutions of a cultured colony of M. tuberculosis.
the Inno-LiPA line probe assay (Innogenetics, Ghent, Belgium) To evaluate the sensitivity and specificity, the overall diagnosis of M. tuberculosis
(8), and the GenoType MTBC assay (Hain Lifescience, Nehren, was defined by positive culture results.
Germany) (15). These commercial assays are divided into two Processing of specimens. All specimens were decontaminated with N-acetyl-
methods: real-time PCR and probe hybridization. In particular, L-cysteine-sodium hydroxide and concentrated by 20 min of centrifugation at
3,000 ⫻ g. After resuspension of the sediments in phosphate buffer, smears with
real-time PCR technology has replaced the methodology of mi-
Ziehl-Neelsen staining were prepared and examined by an experienced labora-
crobiological diagnosis using an automated system based on in- tory technologist using the technique described by Stewart in 1953 (18). Myco-
creased sensitivity. More recently, the Cobas TaqMan MTB PCR bacterial cultures were prepared by the inoculation of a 200-␮l aliquot of the
test, which uses a real-time PCR technique, was introduced. decontaminated samples onto 3% Ogawa agar. The cultures were incubated for
8 weeks at 36°C and were examined for growth weekly. The culture results were
divided into the following 5 classifications: no growth; trace, ⬍50 colonies; 1⫹, 50
to 1,000 colonies; 2⫹, 100 to 200 colonies; 3⫹, nearly fused colonies; and 4⫹,
* Corresponding author. Mailing address: Department of Labora- fused colonies.
tory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan PCR test. All specimens were evaluated by both the Cobas Amplicor and
University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul TaqMan MTB PCR tests. The Cobas Amplicor and TaqMan MTB PCR tests
135-710, South Korea. Phone: 82-2-3410-2709. Fax: 82-2-3410-2719. were conducted in accordance with the manufacturer’s instructions. Positive and
E-mail: negative controls were included in each run. Briefly, 100-␮l aliquots of the
† J.H.K. and Y.J.K. contributed equally to this work. decontaminated samples were mixed with 500 ␮l specimen wash solution. The

Published ahead of print on 3 November 2010. mixtures were subjected to 10 min of centrifugation at 12,000 ⫻ g. The super-


formed using the statistical software SPSS for Windows (version 12. the mixture was incubated at 60°C for 45 min. RESULTS In the cross-reactivity test between cultured colonies of M.6% for Amplicor MTB PCR. and invalid results according to Cobas TaqMan MTB PCR results (n) sample types by TaqMan MTB PCR Species Negative Positive Invalid Sample No.8% higher (P ⫽ 0. the measured limit was 4 respiratory specimens. and the 98. and 1 BAL fluid. the sensitivity of the Taq- were culture negative and PCR positive were from patients Man MTB PCR was 20. Of these. avium 4 0 4 Pleural fluid 0 3 0 3 M. Twenty cases yielded invalid results: 14 tissue sam- Statistical analysis. none of the colonies showed false pos- A total of 406 samples from 247 patients were processed. 5 were identified as true positive by positive culture results. as positive. The corresponding values for specimens were culture negative. specificity. Cross-reactivity between NTM and M.174 KIM ET AL.3/99. intracellulare 10 0 2 Total 26 360 20 (4. and the nonrespiratory specimens were body fluids. The specimens included 96 respiratory and 310 non- limit of the TaqMan MTB PCR.1/98. SPSS. for the respiratory specimens. Positive. and invalid using software provided in the Cobas TaqMan The amplified patterns of the graphs were reviewed for all analyzer. tuberculosis infection. The respiratory specimens were sputum copies/␮l. and one body fluid sample. The sensitivity. natant was discarded.2) 170 M.7 Amplicor 14 10 2 380 58.8 86.3/98. The samples were processed using the three tissue samples. McNemar’s test was used.9) 406 M. and culture assessments. 3.8% lower than those for the Amplicor MTB PCR. Switzerland) with posi.1% and (20. However. 1 was regarded as true positive by a history of M.2% for TaqMan MTB PCR and 58.2 Amplicor 14 10 1 71 58. negative.5/97. The overall sensitivity and specificity were 79. and 4 were regarded as false positive. only by TaqMan MTB PCR (Table 1). 1.1/98. and 100 ␮l of neutralization TaqMan MTB PCR were detected in 22 samples of sputum.4 Respiratory TaqMan 19 5 3 69 79. An invalid result was displayed in cases where the internal control was out of range due to inhibitors or improperly prepared specimens. specimens that showed discrepant results between the TaqMan and Amplicor PCR tests. To evaluate the differences in sensitivity between the TaqMan and Amplicor PCR methods. A 50-␮l portion was added to 50 ␮l of the master mix solution. and pleural and bronchoalveolar lavage (BAL) fluids. 2 joint fluid samples. CLIN.7% for BAL fluid.8% for TaqMan positive results by both PCRs while five showed positive results MTB PCR and 58. 3 sputum samples. tuberculosis in cultured colonies TABLE 2. TABLE 1. szulgai 2 0 0 . including joint flu. fortuitum 1 0 5 M. NPV. respectively. (%) invalid Total M. negative.3% and 99. Among the Cobas TaqMan 48 Analyzer (Roche Diagnostics. and two of them showed respiratory specimens were 79. tuberculosis 0 2 0 Sputum 22 53 3 (3. The two specimens that Thus.9%) of the and all specimens were tested by culture. and the pellet was resuspended in 100 ␮l of lysis reagent. ples. nonrespiratory specimens. were as follows: 6.3%) sults for concordant specimens as reference standards (Table were positive by both TaqMan and Amplicor PCRs. tuberculosis infection.1% and 95. 13 (30. The results of the TaqMan PCR test were displayed was a true positive. and positive and negative predic- ids. and nucleic 42 colonies showed invalid results. negative predictive value. itives or false negatives (Table 3). and drainage and tissue specimens. including the internal controls.9%) were culture positive. The percentages of invalid results according to sample type Chicago. A total of 24 tive values were analyzed in comparison with the culture re- specimens (5. The other 382 plicor MTB PCR.8) 78 Tissue 3 123 14 (10. dictive values were calculated based on the results of concurrently performed All of these specimens showed negative results by Amplicor cultures.1/95. three were false negatives and one tive and negative controls.2 73. positive predictive value. 14 (58.6 93. The calculations were per- PCR. staining. With regard to the types of specimens. while five 1). specimens.7% for tissue samples (Table 2). IL).5% for Am- remaining five were negative by both PCR tests.4/93. The sensitivity. and 10.002) and the spec- with a clinical history of M. J.5 87. Of the 10 ificity was 2. acid-fast bacillus (AFB) stain. positive results by then. negative No.7) 140 NTM 27 0 13 Body fluid 1 167 2 (1. Performance of the Cobas TaqMan and Amplicor PCRs based on culture results Culture-positive (n) Culture-negative (n) Specimens Test Sensitivity/specificity (%) PPV/NPVa (%) ⫹ ⫺ PCR PCR PCR⫹ PCR⫺ All TaqMan 19 5 7 375 79.2% for body fluid.8% for sputum.6 a PPV.8%) were positive by only TaqMan MTB PCR. ascites. positive No. In tests for the detection acid tests.3% and 98. abscessus 10 0 2 BAL fluid 0 14 1 (6.3/87. reagent was added.7) 15 M. MICROBIOL. specificity. Basel. TABLE 3. tuberculosis and NTM. and positive and negative pre.

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