Evaluation of culture and PCR-based assay for detection of Mycobacterium tuberculosis from sputum collected and stored on filter paper.

We evaluated the use of culture and PCR-based assay for the direct detection of Mycobacterium tuberculosis (MTB) from sputum collected and stored on filter paper at room temperature for 5 days; the results were compared with those of staining and conventional culture of fresh sputum before storage (the 'gold standard'). Out of 231 sputum specimens examined, MTB was recovered from 124 samples by culture before storage. The culture positivity rate was significantly decreased to 70% after 5 days storage. For PCR assay, a fragment of 377 bp of the IS6110 sequence was amplified and detected using three methods: first PCR product combined with agarose gel electrophoresis (AGE); first PCR product with dot blot hybridization (DBH); nested PCR with AGE. Compared with culture, the sensitivity, specificity, and efficiency for first PCR with AGE were 71.8, 100 and 84.9% respectively; PCR with DBH gave results of 89.5, 96.3 and 92.6% respectively; the same values for nested PCR were 96.0, 97.2, and 96.5% respectively. Of these three methods, nested PCR gave excellent sensitivity and specificity with no significant difference (p = 0.727) from conventional culture. The storage of sputum on filter paper and storage at room temperature for 5 days had no apparent effect on the performance of nested PCR. We propose that this collection and storage method be considered for transporting sputum specimens from peripheral health centers or from the field; specimens may be sent by post to a central point for both culture and PCR analysis by trained technicians supervised in accordance with a well-established quality control system.

Comparison of the resistance ratio and proportion methods for antimicrobial susceptibility testing of Mycobacterium tuberculosis.

This study compared two in vitro antimicrobial susceptibility methods for determining drug susceptibilities of Mycobacterium tuberculosis isolated from newly diagnosed pulmonary tuberculosis patients to four front-line drugs. Of 250 strains of M. tuberculosis tested, 74.4 per cent were susceptible by the resistance ratio method, with 72.0 per cent by the proportion method. The results showed high agreement for both methods (P<0.0001) and agreement rates to streptomycin, isoniazid, rifampicin and ethambutol were 96.8, 98.0, 94.8 and 96.8 per cent, respectively. For drug resistance patterns, both methods showed the highest resistance to one drug, followed by two, three, and four drugs, respectively. Of the single drug resistance, both methods gave the highest resistance to streptomycin, followed by resistance to isoniazid, rifampicin and ethambutol, respectively. The correlation between both methods for determining susceptibility of M. tuberculosis to four drugs was not statistically significantly different by Mc Nemar chi2 (p>0.05). Thus, the resistance ratio method may be substituted. However, WHO recommended the use of the proportion method to be used for determining drug susceptibility of M. tuberculosis. The susceptibility testing result can be used as the guidance for proper treatment and is valuable for confirmation of drug resistance in patients showing unsatisfactory response to treatment, useful for identifying primary and acquired drug resistance trends in a community and for minimizing the spread of drug-resistant strains.

Evaluation of katG codon 315 mutations among isoniazid sensitive and resistant mycobacterium tuberculosis isolates from Thailand.

Nine isoniazid (INH)-susceptible and 11 INH-resistant Mycobacterium tuberculosis clinical isolates were analyzed for katG codon 315 mutations by polymerase chain reaction (PCR) assay using primers MYC-32 and MYC-33, followed by restriction fragment length polymorphism. After AciI digestion of PCR products, all 9 INH-susceptible isolates and 5 out of 11 (45%) INH-resistant isolates showed 0.12 kb band, which was previously reported to indicate wild type, whereas, 6 of 11 (55%) INH-resistant isolates lacked this band.

In vitro drug susceptibility of Mycobacterium other than tubercle bacilli.

MOTT were isolated from clinical specimens from the Division of Mycobacteriology, Department of Microbiology, Siriraj Hospital, Mahidol University, and also from natural sources, namely water and soil in the Bangkok area from January to December, 1987. The strains isolated were as follows: 30 M. fortuitum fortuitum, 5 M. fortuitum peregrinum 3 M. fortuitum 3 rd. biovariant complex, 3 M. chelonae chelonae, 7 M. chelonae abscessus. Of these, 5 strains were isolated from clinical specimens and the other 43 were from the environment. The pattern of drug susceptibility to antituberculous and antimicrobial agents revealed that they were resistant to antituberculous agents but susceptible to aminoglycosides (which contain a central ring of 2-deoxystreptamine) such as amikacin (83% of cultures), netilmicin (83%), gentamicin (77%) and kanamycin (75%). Some strains were sensitive to tetracyclines (35%), but all were resistant to penicillins and cephalosporins.