Rifampicin resistance in Mycobacterium tuberculosis--rapid detection and implications in chemotherapy.
OBJECTIVE
Tuberculosis treatment and susceptibility testing are cumbersome, especially in the case of multidrug-resistant (MDR) Mycobacterium tuberculosis. It is known that mutations in the rpoB gene of M. tuberculosis lead to resistance to rifampicin (RMP). In this study, an attempt was made to apply molecular techniques for rapid detection of antibiotic resistance in clinical isolates of M. tuberculosis. DESIGN, SETTINGS AND SUBJECTS: RMP-resistant clinical isolates of M. tuberculosis from South Africa (N = 120) with unique resistant patterns were selected for calculation of resistance frequencies, and 74 MDR isolates of M. tuberculosis from different geographical origins were used for microbiological and molecular analysis. The polymerase chain reaction (PCR) technique was applied for amplification of a previously described region around a cluster of mutations in the rpoB gene, and single-stranded conformational polymorphism (SSCP) analysis was optimised to screen for mutations in the amplified region.


RESULTS
The results showed that an optimised PCR-SSCP procedure could detect a cluster of mutations in the rpoB gene (for RMP resistance) in 95% of RMP-resistant isolates. This procedure could therefore be used in the prediction of RMP resistance. Evidence was obtained that these mutations can be screened for directly from BACTEC cultures or even directly from Ziehl-Neelsen-positive sputum samples. Statistical analysis also showed that this locus can be used to predict the presence of an MDR isolate, which may have important implications in decisions concerning chemotherapy.


CONCLUSIONS
It is currently not feasible to test all tuberculosis cases, but application of the PCR-SSCP technology in the prediction of multidrug resistance in M. tuberculosis isolates may be important in patients, especially where frequencies are high for drug-resistant isolates This methodology could reduce the time required for sensitivity testing from approximately 6-12 weeks to a few days.
Pretorius
G S
GS
Department of Medical Biochemistry, University of Stellenbosch, Tygerberg, W. Cape.
Sirgel
F A
FA
Schaaf
H S
HS
van Helden
P D
PD
Victor
T C
TC
D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012293:Rifampin / Q000494:pharmacology*; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*
D000904:Antibiotics, Antitubercular; D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012293:Rifampin