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Abstract
Background
Despite the emerging danger of MDR-TB to human beings, there have only been a limited number of drugs developed to treat MDR-TB since 1970. This study investigated the cross-resistance rate between rifampicin (RFP) and rifabutin (RBU) in order to determine the efficacy of rifabutin in treating MDR-TB. In addition, the results of rifabutin were correlated with the rpoB mutations, which are believed to be markers for MDR-TB and RFP resistance.
Methods
The MICs of RBU were tested against 126 clinical isolates of MDR-TB submitted to the clinical laboratory of National Masan TB Hospital in 2004. Five different concentrations (10-160 µg/ml) were used for the MICs. The detection of the rpoB mutations was performed using a RFP resistance detection kit with a line probe assay(LiPA), which contains the oligonucleotide probes for 5 wide type and 3 specific mutations (513CCA, 516GTC, and 531TTG) The rpoB mutation was determined by direct sequencing.
Results
The rate of cross-resistance between RFP and RBU was 70.5%(74/105) at 20 µg/ml RBU(ed note: How much RFP?) Most mutations (86.3%) occurred in the 524~534 codons. The His526Gln, His526Leu, Leu533Pro, Gln513Glu, and Leu511Pro mutations(Ed note: Is this correct?) were associated with the susceptibilty to RBU.
Conclusion
Based on the cross-resistance rate between RFP and RBU, RBU may be used effectively in some MDR-TB patients. Therefore, a conventional drug susceptibility test for RBU and a determination of the critical concentration are needed. However, rpoB gene mutation test may be have limited clinical applications in detecting RBU resistance.
Figures and Tables
Figure 1
The representative figure of results of REBA MTB-Rifa®
No.1 shows no rpoB mutation. All of 5 sensitive bands are there.
No.2 shows TCG(Ser)531TTG(Leu) mutation. There is 531TTG band instead of wt5 band.
No.3 indicates the isolate has a rpoB mutation other than TCG(Ser)531TTG(Leu) on wt5(530-532) because of missing 531TTG band.
No.4 is GAC(Asp)516GTC(Val) mutation.
No.5 shows the isolate is not M. tuberculosis because of missing TB band.
Table 1
The number of MDR-TB isolates showing an initial inhibition at the following concentration of rifabutin.
Table 2
The number of MDR-TB isolates showing mutation at 5 different region or 3 specific mutations. The number is divided by resistance or susceptibility to rifabutin.
Table 3
Detection of rifabutin-susceptible isolates by LiPA (reverse blot hybridization method) compared to conventional method (drug susceptibility test).
While any mutation in wt2, wt5, Gln513CCA(Pro), and Asp516GTC(Val) were regarded as RBU susceptibility, any mutation in wt1, wt4, and Ser531TTG(Leu) were regarded as RBU resistance according to the majority of the DST results for each mutation of 5 wide type (wt1~wt5) and 3 specific mutation(513CCA, 516GTC, and 531TTG).
Table 4
Profile of rpoB mutations in 94 MDR-TB isolates and its relationship with rifabutin susceptibility by conventional drug susceptibility test
Table 5
Detection of rifabutin-susceptible isolates using direct sequencing of rpoB mutations
According to the majority of the DST results for each mutation, His526Gln, His526Leu, Leu533Pro, Gln513Glu, and Leu511Pro were regarded as RBU susceptibility, and Ser531Leu, Ser531Trp, Ser531Phe, His526Tyr, His526Arg, His526Gly, His526Asp, His526Pro, Gln513Pro, Gln513Lys, Gln513His, and Leu530Met as RBU resistance respectively. Asp516Tyr, Asp516Val, and Asp516Gly were regarded as RBU susceptibility based on the previous reports10,11
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