Eleven-year experience on anti-TB drugs direct susceptibility testing from Siriraj Hospital, Thailand.

The early detection of drug-resistant tuberculosis (TB) strains is of utmost importance for patient management and effective TB control programs. This study aimed to demonstrate the performance of direct drug susceptibility testing (DST) performed in our laboratory in the past 11 years. The direct DST was performed on Middlebrook 7H10 medium using isoniazid (INH) and rifampicin (RIF), and the results were compared with those obtained from indirect DST (gold standard). The direct DST was performed with 15,598 smear-positive sputum samples, of which 11,284 (72%) yielded reportable results. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated and revealed 89%, 99%, 95%, and 99%, respectively, for RIF and 90%, 98%, 93%, and 97%, respectively, for INH. Direct DST results could be reported within 1 month after sample processing. This method was also shown to be suitable for use in resource-limited countries, particularly in settings with high numbers of TB cases.

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Distribution of hsp65 PCR-restriction enzyme analysis patterns among Mycobacterium avium complex isolates in Thailand.

A total of 227 clinical Mycobacterium avium complex isolates from Thailand were differentiated into species and types by using PCR-restriction enzyme analysis of hsp65. The distribution of types showed the predominance of M. avium I (77%) in blood specimens, whereas M. intracellulare I was more commonly found in pulmonary specimens (44.2%). In addition, infections with M. avium were more likely to be found in younger adults (20 to 39 years old), while infections with M. intracellulare were more likely to be found in older adults (> or =60 years old). Our results provide the useful epidemiological information that some particular types have more invasive and virulent characters than others.

Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compare to AFB staining in pathological sections.

BACKGROUND: Polymerase chain reaction (PCR) is a recent, rapid and reliable method in the detection of causative organism. The authors tried to determine the possibility of using PCR technique as an alternative way to detect mycobacterial DNA from paraffin-embedded tissue to avoid repeated biopsy from the patient. MATERIAL AND METHOD: Paraffin-embedded tissue blocks, the corresponding histopathologic slides, and cultural results were retrospectively searched for according to the patient's records, the granuloma clinic, Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand from 1994-2000. One hundred and thirty-one tissue blocks and slides were found but only 120 cultural results were retrieved Histologic sections were reviewed for AFB findings and PCR was done using 16S rRNA sequences to detect M. tuberculosis by one-tube nested technique and multiplex PCR for M. marinum and M. fortuitum complex. RESULTS: The causative organisms were identified by AFB staining in pathologic sections 31.29%, by PCR 35.87%, and by culture 30.00% of tested samples. The sensitivity of PCR when compared to AFB result was 29.26%, specificity 61.11% but when compared to cultural results, the sensitivity of PCR was 66.67% and AFB sensitivity was 41.66% with specificity 76.19% and 72.61% respectively. CONCLUSION: The low sensitivity of the PCR method may be due to formalin fixation, deparaffinization process, DNA extraction method, the use of 16S rRNA-based primers and the length of the expected product, and the tissue type that may have Taq polymerase inhibitor. Therefore, PCR should be used to augment the information of the conventional method in the diagnosis of mycobacterial infection.

Evaluation of polymerase chain reaction and restriction enzyme analysis for routine identification of mycobacteria: accuracy, rapidity, and cost analysis.

Polymerase chain reaction and restriction enzyme analysis (PCR-REA) of the hsp65 gene was evaluated for use as a routine identification method for identifying mycobacteria. The accuracy, rapidity, and cost were assessed compared with the conventional biochemical method. Five hundred and forty-one mycobacterial clinical isolates obtained from the Department of Microbiology, Faculty of Medicine at Siriraj Hospital, Mahidol University, were submitted for PCR-REA and biochemical identification. PCR-REA showed high concordant result with 100, 96.2, and 94.1% for identification of Mycobacterium tuberculosis, rapid- and slow-growing mycobacteria, respectively. Discordant results were obtained from 24 (4.4%) out of 541 isolates, consisting of 9 rapid growers (6 M. chelonae, 2 M. abscessus, and 1 M. fortuitum) and 15 slow growers (9 M. scrofulaceum, 2 M. gordonae, 1 M. avium, 1 M. kansasii, 1 M. malmoense, and 1 M. terrae complex). PCR-REA demonstrated not only accurate results but was also less expensive (2.1 US dollars/sample). This method was rapid with a turn-around time of 30 hours compared with 2-4 weeks for the conventional method.

Diagnostic value of gastric aspirate smear and polymerase chain reaction in smear-negative pulmonary tuberculosis.

OBJECTIVE: The aim of this study was to determine the validity of acid-fast bacilli (AFB) smear and polymerase chain reaction (PCR) from gastric aspirates for the diagnosis of smear-negative pulmonary tuberculosis. METHODOLOGY: A cross-sectional study was conducted in a university hospital. One hundred and nine patients with suspected pulmonary tuberculosis in whom either sputum smears were negative or who were not producing sputum were recruited to the study. All patients underwent gastric aspiration after an overnight fast followed by standard fibreoptic bronchoscopy. Specimens were subjected to AFB smear, culture, and pathological examination. PCR was performed on culture filtrate after 1 week of incubation. RESULTS: Eight patients did not complete the follow-up schedule. Of the 101 patients with final outcomes, a diagnosis of pulmonary tuberculosis from microbiological evidence was established in 54 patients. The gastric aspirate smear, PCR, or either one of them was positive in 34, 30, and 39 tuberculosis patients, respectively. There were 13 false positive smears from 47 non-tuberculosis patients, with five resulting from non-tuberculous mycobacteria (NTM). The PCR was falsely positive in eight patients, five of whom had previous histories of tuberculosis. The overall sensitivity, specificity, positive predictive value, and negative predictive value of gastric aspirate examination by combined smear and PCR were 72, 58, 66, and 64%, respectively. CONCLUSIONS: Gastric aspiration is a useful tool for the diagnosis of smear-negative pulmonary tuberculosis warranting institution of antituberculosis treatment. Interpretation of the results should be cautious in those who have had tuberculosis in the past or who have been at risk for acquisition of NTM.