Background and objective: Tuberculosis (TB) is a devastating infectious disease causing high mortality and morbidity worldwide. The most serious threat related to tuberculosis control is the recent emergence of drug-resistant tuberculosis strains. The aim of the present study was to identify various types of mutations in the rpoB region in rifampicin-resistant strains isolated from sputum of tuberculosis patients.
Materials and methods: Drug susceptibility of 125 Mycobacterium tuberculosis isolates was determined using the CDC standard conventional proportional method. Target DNA of M. tuberculosis was amplified by polymerase chain reaction, hybridized, and scanned. We used the low cost density array (LCD-array) to detect mutations within the 90-bp rpoB region. Each array is a transparent, prestructured polymer support using a nonfluorescent detection principle based on the precipitation of a clearly visible dark substrate.
Results: Of the 125 M. tuberculosis isolates, 35 (28%) were found to be rifampicin-resistant and using the LCD array revealed point mutations at nine different codons as follows: S512T (AGC→ACC) (20%), D516V (GAC→GTC) (20%), H526D (CAC→GAC) (6%), H526R (CAC→CGC) (20%), H526Y (CAC→TAC) (23%), and S531W (TCG→TGG) (8%), and the most frequent site mutations were L511P (CCG→CTG) (46%), followed by S531l (TCG→TTG) (40%) and D516Y (GAC→TAC) (26%).
Conclusion: Our data significantly differs from previously reported mutation frequencies for codon 526 (CAC to GAC) among Italian isolates (40.1%) and Greek isolates (17.6%). Phenotypic testing is time-consuming and requires laboratory resources. Microarray rpoB is useful in detecting rifampicin resistance-determining region-associated site mutations of rifampicin-resistant M. tuberculosis isolates.