Evaluation of a novel line-probe assay for genotyping-based diagnosis of Mycobacterium tuberculosis in Thailand.

SETTING: The Supranational Tuberculosis Reference Laboratory (NTRL), Bangkok, and Chiangrai Prachanukroh Hospital, Chiangrai, Thailand OBJECTIVE: To evaluate the diagnostic performance of newly developed line-probe assay (LiPA) kits in tuberculosis (TB) endemic settings. DESIGN: LiPA kits were used to evaluate 404 clinical isolates of Mycobacterium species and 163 sputum samples in Thailand. RESULTS: LiPA kits were able to identify M. tuberculosis, M. avium, M. intracellulare and M. kansasii with 100% sensitivity and specificity when compared with the commercially available AccuProbe assay. Testing of the LiPA kits for their ability to detect mutations in clinical isolates resistant to anti-tuberculosis drugs such as rifampicin, isoniazid, pyrazinamide and fluoroquinolones showed that the assay had very high sensitivity (65.9-100%) and specificity (98.2-100%) compared with drug susceptibility testing and DNA sequencing. LiPA had a sensitivity of 75.0-85.7% and a specificity of 96.4-100% in testing clinical sputum samples. CONCLUSION: The novel LiPA kits have high sensitivity and specificity, and may enhance the rapid detection of first- and second-line anti-tuberculosis drug resistance, improving the selection of suitable chemotherapy agents to treat multidrug-resistant and extensively drug-resistant TB.

Restriction fragment length polymorphism study of nationwide samples of Mycobacterium tuberculosis in Thailand, 1997-1998.

SETTING: During 1997-1998, a national anti-tuberculosis drug resistance survey was conducted in Thailand as a part of a global project. OBJECTIVE: To evaluate the IS6110 hybridisation patterns and the level of clustering, which was expected to be low due to the short duration of the sample collection. DESIGN: Eight hundred and twenty-eight bacterial isolates were available for fingerprinting by standard IS6110 hybridisation. RESULTS: The restriction fragment length polymorphism patterns varied with geographic locations, ages of the patients, and resistance to rifampicin and streptomycin. The Beijing strain was more common among younger patients, and their prevalence appeared to decrease with the distance from Bangkok, while the opposite was true for the single-banded isolates. Excluding isolates containing five or less copies of IS6110, 26.4% were clustered. Clustering was more common among females. The clustered isolates were sometimes from different provinces and, if resistant to drugs, usually possessed different resistance profiles. CONCLUSIONS: The results question the validity of inferring recent transmission from the clustering of IS6110 hybridisation patterns in some settings in Thailand. The level of recent transmission in a nationwide study in a country with a high incidence of tuberculosis should be evaluated with caution.

Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer.

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.

Differentiation of Mycobacterium species by restriction enzyme analysis of amplified 16S-23S ribosomal DNA spacer sequences.

SETTING: Mycobacteriology research and service laboratories in Thailand. OBJECTIVE: To evaluate the possibility of differentiating species of mycobacteria by amplifying 16S-23S ribosomal deoxyribonucleic acid (DNA) spacer and restriction enzyme analysis of the products. DESIGN: DNA of 113 strains of mycobacteria belonging to 18 species of the genus Mycobacterium were amplified by primers PL1 (5'-GAAGTCGTAACAAGG) and PL2 (5'-CAAGGCATCCACCAT). The amplified products as well as their HaeIII-, MspI- and BstXI-digested products were visualized after agarose gel electrophoresis. RESULTS: The amplified products of rapid-growing mycobacteria were different from the slow-growing mycobacteria. The restriction profiles of members of M. tuberculosis complex were the same as each other but different from other investigated species. The restriction profiles of some species, such as M. avium, M. intracellulare and M. gordonae, were unique, while those of the other species had more than one pattern. However, the restriction profiles of most investigated species were different from each other. CONCLUSION: This preliminary study suggested that the method might be useful for species differentiation of some commonly isolated pathogenic mycobacteria.

Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.