Drug resistance in mycobacterium tuberculosis

University dissertation from Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Abstract: Drug-resistant tuberculosis is a global public health problem. This investigation was performed to find ways of improving regimens that could be used for the treatment of drug- and multidrug-resistant TB and also to find a rapid method of diagnosis of drug resistant TB, particularly MDR-TB. Among 107 isolates of M. tuberculosis from re-treatment cases of pulmonary TB in Ethiopia (study 1), 48% were resistant at least to one of the four first-line drugs tested and 12 % were AMR. In this study, rifampicin resistance was a strong predictor of MDR-TB. The extended susceptibility of 35 polyresistant isolates including NMR isolates on ten other drugs showed that all were susceptible at least to five drugs (i.e., amikacin ciprofloxacin, clofazimine ethambutol, and ethionamide). The WHO re-treatment regimen would be effective in 86 % of cases. Moreover, the extended susceptibility pattern of MDR isolates indicated that it is possible to find a low cost treatment regimen for patients at risk of harbouring MDR-TB by replacing very expensive drugs like amikacin and ciprofloxacin. The studies on ß-lactams showed that cefepime (study II) and amoxicillin-clavulanate (study 111) are effective on M. tuberculosis isolates including MDR-isolates. Cefepime at a concentration of 32 mg/L was active on 54 % of M. tuberculosis isolates. The minimum inhibitory concentration (MIC) of amoxicillin-clavulanate was 2 or 4 mg/L for 90 %the isolates tested. The MIC was reduced to 5 0.5 mg/L for 97 %of isolates when subinhibitory concentration of ethambutol was added. The results suggested that ß-lactams, particularly in combination with ethambutol, could be important for the treatment of MDR-TB. An assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was standardised in both microtiter (study and tube formats (study V) for the detection of rifampicin resistance in clinical isolates of M. tuberculosis. MTT is converted to a blue formazan by live cells and the amount of formazan formation was proportional to the number of viable bacilli. In suspensions containing mixed bacterial population, the assay could detect a resistant subpopulation of I%. The evaluation of the assay with the BACTEC method using 92 clinical isolates of M. tuberculosis showed that the result obtained by the MIT assay (both on the third and sixth day) matched with the result obtained by the BACTEC method. Seventy-eight strains were identified as susceptible, 13 strains as resistant and one strain as borderline resistant. In the MIT assay, resistant and susceptible stains were identified based on the relative optical density (RODU) values and changes in OD from the third to the sixth day. AD results were interpretable by the naked eye. NM assay is rapid, reliable, safe and inexpensive assay for the detection of rifampicin resistance. Rifampicin resistance is a strong predictor of MDR-TB and the assay is appropriate for clinical application in low-income countries. Fifty isoniazid-resistant strains of M. tuberculosis (study VI) were characterised based on the catalase activity, MIC of isoniazid, growth pattern in the presence of different concentrations of isomazid, and mutations in katG gene (codons 315 and 463). The MIC of 72 % of the strains was < 4 mg/L. The results also showed that these stains could be classified into three groups based on their growth pattern. A majority (80%) of stains with high MIC (>= 16 mg/L) of isoiazid had no catalase activity. Mutations in codons 315 and 463 of katG gene were detected in 91 % of strains with MIC < 8 mg/L but only in 28 % of strains with higher MlC. This study showed that mutations in codons 315 and 463 of katG gene failed to detect 72 % of clinically most important group with high MlC of isoniazid (>= 16 mg/L). The sensitivity of molecular methods based on mutations in these codons could be increased by combining with the results from the catalase activity. The majority of strains with high MIC had no catalase activity and could be detected by a simple catalase test. The results also indicated that isoniazid-resistant strains of M. tuberculosis could be characterized based on their growth pattern.

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