Abstract
BackgroundThe importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined. This study aimed to assess the frequency and types of rpoB mutations in M. tuberculosis isolates from Cubal, Angola, a country with a high incidence of tuberculosis.MethodsAll isolates included (n = 308) were analyzed using phenotypic drug susceptibility testing and GenoType MTBDRplus assay. DNA sequencing of the rpoB gene and determination of rifampicin MIC by macrodilution method were additionally performed on isolates yielding discordant results (n = 12) and those in which the mutation detected was not characterized (n = 8).ResultsIn total, 85.1% (74/87) of rifampicin-resistant strains had undisputed rpoB mutations -S450L (49), D435V (15), H445D (3), H445Y (2), Q432ins (1), L449M plus S450F (1), S450F (1), S450W (1) and S450Y (1)-; 10.3% (9/87) had disputed rpoB mutations—L430P plus S493L (1), N437del (1), H445L (3), D435Y (2), L452P (2)-, 2.3% (2.3%) showed no rpoB mutations and 2.3% (2/87) showed heteroresistance—D435Y plus L452P and L430P plus S493L-.ConclusionDisputed rpoB mutations were common, occurring in 10.3% of rifampicin resistant isolates. Current phenotyping techniques may be unable to detect this resistance pattern. To increase their sensitivity, a lower concentration of RIF could be used in these tests or alternatively, rpoB mutations could be screened and characterized in all M. tuberculosis strains.
Highlights
The importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined
RIF resistance is mainly associated with single point mutations in a small 81 base pair hotspot region of the rpoB gene, referred to as the RIFresistance-determining region (RRDR), which can be amplified by PCR [4]
DNA sequencing of the rpoB gene and minimum inhibitory concentration (MIC) determination by the macrodilution method were performed on isolates that showed RIF resistance by culture-based drug susceptibility testing (DST), but were identified as susceptible by GenoType MTBDRplus Hain Lifescience or did not give hybridization results with the mutation-specific probes, and on those that showed RIF susceptibility by culturebased DST but were identified as resistant by molecular methods
Summary
The importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined. A study carried out by Supranational Reference Laboratories (SRL) showed that mutations in rpoB associated with low-level RIF resistance are missed by culturebased phenotypic DST methods, in particular automated broth-based Bactec Systems [2, 11]. What remains to be resolved is the importance of discordant strains with disputed rpoB mutations in terms of their relative frequency and impact on the outcome of RIF-based antituberculosis therapy [12]. In general they are considered rare, and their minimum inhibitory concentration (MIC) is usually below the critical concentration [12]. They are often considered RIFusceptible, there are reports of adverse treatment outcomes [12, 13]
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