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Journal of Clinical Microbiology, March 2006, p. 716-719, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.716-719.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Performance Assessment of the DR. MTBC Screen Assay and the BD ProbeTec ET System for Direct Detection of Mycobacterium tuberculosis in Respiratory Specimens

Jann-Yuan Wang,¹ Li-Na Lee,^1,2 Hsiao-Leng Hsu,² Po-Ren Hsueh,^1,2* and Kwen-Tay Luh^1,2

Departments of Internal Medicine,¹ Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan²

Received 8 June 2005/ Returned for modification 5 September 2005/ Accepted 21 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The performance of the DR. MTBC PCR-based assay and the BD ProbeTec ET Mycobacterium tuberculosis Complex Direct Detection (DTB) assay for the direct detection of Mycobacterium tuberculosis was evaluated using 1,066 consecutive clinical respiratory samples collected from 494 patients who did not have old cases of pulmonary tuberculosis and were not receiving antituberculosis treatment at National Taiwan University Hospital from January to February 2005. The results of both assays were compared to the "gold standard" of combined culture results and clinical diagnosis. The overall sensitivity and specificity of the DR. MTBC Screen assay were 56.6% and 98.9%, respectively, and of the DTB assay were 63.2% and 98.4%, respectively. The positive and negative predictive values for the DR. MTBC Screen assay were 84.5% and 95.4%, respectively, and for the DTB assay were 81.7% and 96.0%, respectively. The DR. MTBC Screen assay produced 11 false-positive results for 11 patients, including three samples yielding non-M. tuberculosis mycobacteria (one each for M. abscessus, a mixture of M. abscessus and M. chelonae, and unidentified non-tuberculosis mycobacteria). The DTB assay produced 15 false-positive results for 13 patients, including five samples from four patients yielding non-tuberculosis mycobacteria (two for M. abscessus, one for a mixture of M. abscessus and M. chelonae, and two for unidentified non-tuberculosis mycobacteria). This study demonstrated that the DR. MTBC Screen assay has a similar diagnostic value but fewer false-positive results than the DTB assay for respiratory specimens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tuberculosis (TB) remains one of the important causes of morbidity and mortality worldwide. The World Health Organization estimated that in this decade, 300 million more people will become infected with tuberculosis, and 30 million people will die from this disease (22). In 2003, the incidence and mortality of tuberculosis in Taiwan was 62.38 and 5.80 per 100,000 people, respectively (5). Successful control of tuberculosis depends on rapid detection of Mycobacterium tuberculosis complex (MTB) to allow for early treatment and education of patients and thereby decrease the likelihood of dissemination to others. The conventional method for laboratory diagnosis of tuberculosis is based on acid-fast staining and culture. Staining and microscopy comprise a rapid screening method for detection of AFB in clinical specimens, but this method has low sensitivity and is labor dependent (3, 11). Moreover, this method does not discriminate MTB from non-tuberculous mycobacteria (NTM), which could be a colonizer in patients with chronic respiratory disease or a true pathogen in immunocompromised hosts, particularly in patients with AIDS. Culture has acceptable sensitivity and specificity but may take about 10 days on average to detect positive specimens, even when a radiometric procedure is used (12).

Newer diagnostic methods employing nucleic acid amplification and detection may provide very quick and specific tests for the identification of MTB (4, 6-10, 13, 15, 17-19, 21). Among them, the BD ProbeTec ET Mycobacterium tuberculosis Complex Direct Detection (DTB) assay, which uses an internal amplification control designed to detect the presence of inhibiting substances, has been consistently reported to have an excellent performance (7, 16, 17). As most currently available amplification methods do not distinguish live MTB bacilli from dead organisms or nontuberculous mycobacteria, false-positive results remain a problem (16). Recently, the DR. Chip Corporation in Taiwan developed a new assay for detection of MTB, the DR. MTBC Screen assay. This technique uses a PCR technique and is designed for semiautomated use with an overall processing time of <5 h.

The purpose of this study was to compare the value of the DTB assay and the DR. MTBC Screen assay for the identification of MTB from clinical respiratory samples.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Specimen collection and processing. A total of 1,206 consecutive clinical respiratory specimens were collected from 548 patients treated at National Taiwan University Hospital from January to February 2005. Of the 1,206 specimens, 117 specimens that were collected from 46 patients with old cases of pulmonary TB and 23 specimens that were collected from 8 patients under anti-TB treatment were excluded. Specimens that could not be processed on receipt were stored at 2 to 8°C for no longer than 48 h. All specimens were processed and pretreated as previously described (21). Briefly, each specimen was processed by the addition of an equal volume of NaOH-citrate-N-acetyl-L-cysteine at room temperature for 15 min. After centrifugation, the precipitate was resuspended in 1.5 ml phosphate-buffered saline (pH 7.4).

Smear and culture. Smears for acid-fast bacilli (AFB) of the processed specimens were stained with auramine-rhodamine fluorochrome and examined by standard procedures (12). Fluorochrome stain-positive smears were confirmed by the Kinyoun stain method (12). Cultures were performed by inoculating 0.5 ml of sediment onto Middlebrook 7H11 selective agar with antimicrobials (Remel, Inc., Lexena, Kans.) and by using the fluorometric BACTEC technique (BACTEC MGIT 960 system; Becton-Dickinson Diagnostic Instrument Systems, Sparks, Md.) as previously described (21).

DTB assay. The DTB assay was performed according to the instructions supplied by the manufacturer (Becton Dickinson). Briefly, a 500-µl sediment sample portion was added to sample wash buffer to remove possible inhibitors, centrifuged, and heated at 105°C for 30 min. The pellet was then resuspended in sample lysis buffer and sonicated for 45 min at 65°C in a water sonic bath (Branson Ultrasonic Corp., Danbury, Conn.). After the addition of sample neutralization buffer, samples and positive and negative controls were dispensed into priming microwells (containing amplification primers, fluorescence-labeled detector, an internal amplification control [IAC], and other reagents) that were incubated at room temperature for 20 min and then placed into a 71.5°C heating block. Meanwhile, amplification microwells (containing DNA polymerase and restriction endonuclease) were placed into a heating block at 53.5°C for prewarming. After 10 min, samples from the priming microwells were transferred into the corresponding amplification microwells, which were promptly sealed and immediately placed in the DTB instrument. When the amplification signal was >3,400 method-other-than-acceleration (MOTA) units, the result was considered positive regardless of the values of IAC. Values of <3,400 MOTA units were considered negative when the IAC value was >5,000 and indeterminate when the IAC value was <5,000. In the latter case, samples were retested, and the results of the repeated tests were used in the analysis (17).

DR. MTBC Screen assay. The DR. MTBC Screen assay was performed according to the instructions supplied by the manufacturer (DR. Chip Corp.). Briefly, a 500-µl sediment sample portion was added to a 0.5-ml portion of E1 (phosphate buffer solution) buffer and centrifuged for 5 min at 10,000 x g at room temperature. Then, the supernatant was removed. The pellet was then resuspended in 1 ml of E1 buffer and centrifuged again. The pellet was resuspended with 50 µl of E2 buffer (Tris-HCl solution with Tritonex = 100). After 20 min of heating in boiling water and 5 min of cooling on ice, followed by a 5-min centrifugation at 3,000 x g, the supernatant containing extracted DNA was transferred to a new microcentrifuge vial. Then, 5 µl of extracted DNA was transferred to the PCR tube on ice containing 20 µl of the amplification reagent (TSM and DNA polymerase) for amplification.

The primers for PCR were derived from the M. tuberculosis genome, encoding the insertion sequence IS6110 with the primer sequences 5'-CCGCAAAGTGTGGCTAACCC-3' and 5'-biotin GAGCGTAGGCGTCGGTGACA-3'.

For each assay, one PCR-positive control and two hybridization controls were prepared. The PCR was carried out in a thermal reactor. After a 5-min incubation at 95°C, the amplification was performed for 15 cycles, each cycle consisting of 95°C for 20 s, 55°C for 20 s, and 72°C for 40 s; and 25 cycles, each consisting of 95°C for 20 s, 60°C for 20 s, and 72°C for 40 s. After the last cycle, the samples were incubated for 5 min at 72°C. The PCR-positive control contained the control primers and PCR mixture.

In a hybridization tube, 2.5 µl of each amplified DNA sample and 97.5 µl of prewarmed DR. Hyb buffer were heated in boiling water for 5 min and cooled on ice for 5 min. After the 50 µl of hybridization mixture was transferred into a well and covered with a plastic membrane, the plate was incubated at 55°C and was vibrated for 1 h. With the use of the Auto-Washer machine (DR. Chip Corp.), prewarmed wash buffer and blocking and detection reagents were transferred to Wash 1, 2, and 3 bottles, respectively. The hybridization and colorimetric detection processes were then performed by running the TB-SC program of DR. Fluido software accompanying the DR. MTBC Screen system. Briefly, the well was washed with 200 µl of wash buffer a total of three times after hybridization, for 1 min each time. Then, 50 µl of blocking reagent and 0.05 µl of Strep-AP were added to the well and allowed to react at room temperature for 30 min. The well was then washed with 200 µl of wash buffer three times, for 1 min each time. After the well was rinsed with 200 µl of detection buffer, 49 µl of detection buffer and 1 µl of nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate were added into the well and allowed to react in the dark for 10 min at room temperature. After the reaction mixture was discarded, the well was washed with distilled water. For positive PCR control and hybridization control, the well was also coated with a PCR control-specific probe and a hybridization probe. The former reacts to the PCR-positive control products, whereas the latter reacts with the anti-sense DNA in the DR. Hyb Buffer. The result developed in the button of the well was read with the DR. AiM reader. The well also contained the PCR-positive control-specific probe.

The DR. MTBC Screen results were interpreted as follows. The image captured by reader was compared to template TB-SC600. A successful test should appear with a hybridization-positive control and a PCR-positive control. The threshold set for template TB-SC600 was 5. A positive result was determined when the difference of gray level calculated by the software was >5 between the spots of target probe and background. If the difference was 5, it was determined to be a negative result. Positive and negative results were shown on each well, and data were stored as a Microsoft EXCEL file.

Clinical evaluation of patients. Medical records, including history, symptoms, signs, radiology, pathology, microbiology results, and follow-up observations, were carefully reviewed to obtain the necessary data from the combination of culture results and observation of clinical condition to perform an accurate assessment, which served as the "gold standard" for diagnosis (resolved results). Clinically, two categories of samples were considered true positives: (i) samples that were culture positive for MTB and (ii) samples that were culture negative for MTB but which originated from a patient either whose other samples were culture positive or who had a clinical diagnosis of tuberculosis (17, 21). The clinical diagnosis of tuberculosis was established if the biopsy material demonstrated caseating granulomas or if the clinical and radiographic presentations were consistent with tuberculosis and showed marked improvement after antituberculosis therapy. After this analysis, amplification results were reclassified as appropriate.

Statistical analysis. Statistical comparisons were performed using the chi-square test; a P value of <0.05 was considered significant. Agreement between assays was measured by calculating the kappa coefficient.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The specimen and patient characteristics are summarized in Table 1.

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TABLE 1. Specimen and patient characterizations

DTB and DR. MTBC Screen assay results. The results of the two assays of respiratory specimens according to the culture results and gold standard diagnosis made on the basis of culture results and clinical findings are shown in Table 2 and Table 3. Of the 30 respiratory samples that were smear positive and culture positive for MTB, 25 were both DR. MTBC Screen positive and DTB positive. Fifty samples were smear negative for AFB but culture positive for MTB; 29 were DR. MTBC Screen positive and 33 were DTB positive (P = 0.410). There were 26 samples that were both smear and culture negative; those were collected from 14 patients with a clinical diagnosis of pulmonary tuberculosis. Five of these samples were both DR. MTBC Screen and DTB positive. The remaining 21 samples were negative by both assays. The cumulative difference for all MTB-positive specimens (60 positive by the DR. MTBC Screen assay and 67 positive by DTB) was not significant (P = 0.327). The kappa coefficient for all MTB-positive specimens was 0.785 (P < 0.001), suggesting that the results of the two assays were highly agreed.

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TABLE 2. Correlation between two amplification assays for detection of M. tuberculosis and culture results among 1,066 respiratory specimens

View this table: [in this window] [in a new window]

TABLE 3. Correlation between two amplification assays for detection of M. tuberculosis and resolved results (culture and clinical diagnosis) among 1,066 respiratory specimens

In a comparison of the findings of culture and clinical diagnosis which served as the gold standard, a total of 11 respiratory samples collected from 11 patients had false-positive results by the DR. MTBC Screen assay, including 3 samples yielding non-tuberculosis mycobacteria (1 sample each with M. abscessus, mixed M. abscessus and M. chelonae, and unidentified non-tuberculosis mycobacteria). The DTB assay produced 15 false-positive results in samples from 13 patients, including 5 samples from 4 patients yielding non-tuberculosis mycobacteria (2 samples for M. abscessus, 1 sample for mixed M. abscessus and M. chelonae, and 2 samples for unidentified non-tuberculosis mycobacteria).

Comparison of the diagnostic value of the two assays. For the 106 respiratory samples which were positive for MTB, based on clinical and microbiologic findings, the results of both amplification assays were concordantly positive in 58 samples and were both negative in 37 samples. In two samples, the results were DR. MTBC Screen positive and DTB negative; one of these samples was smear negative. In nine respiratory samples, the DR. MTBC Screen assay was negative and the DTB assay was positive. Five of these samples were smear negative.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major difference between MTB and NTM infections is that the former can spread via person-to-person contact. Therefore, delay in diagnosis and treatment can risk the dissemination of M. tuberculosis to others. In addition, recent studies found that early treatment improved survival in patients with tuberculosis (1, 14, 20). For these reasons, it is particularly important to diagnose tuberculosis as early as possible. Since conventional methods including acid-fast staining and culture are either insensitive or time consuming, new technological developments which facilitate rapid diagnosis are of great importance. From a clinical standpoint, the key aspect of any new rapid assay for detecting MTB is its negative predictive value. In the case of respiratory tract disease, it is critical to identify all cases of active tuberculosis and thereby interrupt the dissemination and transmission of the organism. The test should be sensitive, specific, and technically simple, as well as able to differentiate between live and dead mycobacteria.

The present study compared two semiautomatic assays, both of which can detect MTB in clinical samples within a few hours and are easy to perform. The negative predictive values of both assays approached 100%. The differences of the results from cutoff values, values in controls, and values in samples were broad enough to allow easy discrimination by both assays.

The DTB assay specificity of 98.4% and negative predictive value of 96.0% for respiratory specimens in this study are in agreement with previous reports (2, 10, 17, 21). However, the sensitivity of 63.21% and positive predictive value of 81.7% were lower than reported previously. This lower-than-expected sensitivity of the DTB assay found in both our previous report (21) and the current study probably resulted from the generally low bacterial load in the specimens. This speculation is supported by the lower percentage (33.3%) of smear-positive specimens in all culture-positive respiratory specimens than in these previous reports (64.7 to 83.5%) (2, 10, 17). The low positive predictive value of the DTB assay was due to the high false-positive rate. Of the 15 specimens from patients with a positive DTB result but having a negative result based on culture and clinical findings, 33.3% yielded NTM. This high rate of NTM isolation in the specimens may be responsible for the high false-positive rate for both of the assays in this study.

The lower rate of false-positive results for the DR. MTBC Screen assay compared with the DTB assay is likely due to the lower probability of a wrong locus being mistakenly amplified and subsequently nonspecifically hybridized with the hybridization probe. In clinical practice, a false-positive result can lead to unnecessary antituberculosis treatment and associated risk of subsequent drug-induced hepatitis. The colorimetric detection method used in the DR. MTBC Screen assay is less expensive, making it more cost effective for use in clinical practice. This study found a similar performance between the DR. MTBC Screen assay and the DTB assay with respiratory specimens. Further investigation is needed to assess the performance of the DR. MTBC Screen assay in nonrespiratory specimens.

In summary, the resurgence of tuberculosis has highlighted the urgent need for sensitive, correct, and fast methods for the laboratory detection of MTB. This study demonstrated that the diagnostic value of the PCR-based semiautomatic DR. MTBC Screen assay is similar to that of the DTB assay for respiratory specimens. The overall time for processing the DR. MTBC Screen assay is <5 h. In addition, the DR. MTBC Screen assay has a lower false-positive rate than the DTB assay.

 
* Corresponding author. Mailing address: Department of Laboratory Medicine, National Taiwan University Hospital, No. 7 Chun Shan South Road, Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 5363. Fax: 886-2-23224263. E-mail: hsporen{at}ha.mc.ntu.edu.tw.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, March 2006, p. 716-719, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.716-719.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, J.-Y. Articles by Luh, K.-T. Search for Related Content PubMed PubMed Citation Articles by Wang, J.-Y. Articles by Luh, K.-T.