Comparison of PCR-heteroduplex characterization by automated DNA sequencing and line probe assay for the detection of rifampicin resistance in Mycobacterium tuberculosis isolates from KwaZulu-Natal, South Africa

Progress in understanding the basis of resistance to rifampicin (Rif(R)) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of Rif(R) by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RIP strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed, Most isolates (7/8) harboring Leu(533) --> Pro codon mutation required minimum inhibitory concentrations (MICs) of less than or equal to 8 mu g/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser(531) --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain Limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.