Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis.

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.

Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples.

Purpose: Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failure, thus necessitating the need to detect such resistance by a simple D test on routine basis. Materials and Method: 247 Staphylococcus aureus isolates were subjected to routine antibiotic susceptibility testing including oxacillin (1ìg) by Kirby Bauer disc diffusion method. Inducible clindamycin resistance was detected by D test, as per CLSI guidelines on erythromycin resistant isolates. Results: 36 (14.5%) isolates showed inducible clindamycin resistance, nine (3.6%) showed constitutive resistance while remaining 35 (14.1%) showed MS phenotype. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (27.6%, 24.3% and 1.6%, 4% respectively). Conclusion: Study showed that D test should be used as a mandatory method in routine disc diffusion testing to detect inducible clindamycin resistance.

Evaluation of rapid MTT tube method for detection of drug susceptibility of Mycobacterium tuberculosis to rifampicin and isoniazid.

PURPOSE: To evaluate MTT method for detection of drug resistance to rifampicin and isoniazid in M.tuberculosis . This method utilises the ability of viable mycobacterial cells to reduce MTT( 3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide). METHODS: The method was standardised with known resistant and sensitive strains of M.tuberculosis and was then extended to 50 clinical isolates. An inoculum of 10 7 cfu/mL was prepared in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose and catalase. For each drug three tubes were used, one with INH(0.2microg/mL) or RIF(1microg/mL), another as inoculum control and third as blank control. These were incubated at 37 degrees C for four and seven days respectively for RIF and INH after which MTT assay was performed. Results were read visually and by colorimeter at 570 nm. Relative optical density unit (RODU) of 0.2 was taken as cut off. Results were compared with drug sensitivity obtained by proportion method using LJ medium. RESULTS: For rifampicin, concordance with proportion method was 90% by visual and 94% by RODU. Sensitivity and specificity was 86.8% and 100% respectively by visual method and 95.2% and 87.5% respectively by RODU. For Isoniazid, concordance was 94% and sensitivity and specificity was 94.7 and 91.7% respectively by both visual and RODU. CONCLUSIONS: MTT assay proved to be rapid and cheap method for performing drug sensitivity of M.tuberculosis.