RESUMO
Aims: Detection of drug resistance M. tuberculosis isolates is one of the most important strategies to control the disease. Nowadays, with advances in molecular technology, various methods are available to detect drug resistant M. tuberculosis strains such as those based on capture specific probes. In this study, we aimed to investigate the frequency of mutation in the M. tuberculosis -rpoB gene by Polymerase Chain Reaction based on Enzyme Linked Immuno Sorbent Assay (PCR-ELISA) detection. Methodology: Thirty-three culture positive isolates were randomly selected for this study. All the isolates were subjected to a drug Susceptibility Test (DST) using the proportion method. Then the ability and the efficiency of Multiplex Allele Specific PCR (MAS-PCR) and PCR-ELISA to detect Rif resistant (Rifr) M. tuberculosis isolates was compared and evaluated. Results: Mutation of rpoB gene was detected in 19/33 isolates (57.6%) by PCR-ELISA. Hybridization with the specific mutant probes 516 and 526 codon occurred in 1/33 isolates each (3% respectively). Hybridization with the specific mutant probe 531 occurred in 13/33 isolates (39.4%). Three isolates (9.2%) showed simultaneous mutation in codons 516 and 531. The sensitivity and specificity of MAS-PCR in comparison to the Proportional Method was 100%. On the other hand, PCR-ELISA showed 75% sensitivity and 69.2% specificity. The positive predictive value for the PCR-ELISA method was 78.9% and the negative predictive value was 64.3%.The general efficacy of test was 72.7%. Conclusion: The study showed that the sensitivity and specificity of PCR-ELISA to detect mutations in the rpoB gene in Drug Resistant strains was low. Furthermore, this proved to be a complex, time consuming and expensive test. Therefore, this test is not recommended for determining Rifampicin resistance in M. tuberculosis strains.
RESUMO
The natural resistance-associated macrophage protein (NRAMP1), Vitamin-D receptor (VDR) and Tumor necrosis factor (TNF-¦Á) have been associated in susceptibility to tuberculosis, but the results have been inconsistent. This study aimed to determine the association of NRAMP1, VDR, and TNF-¨¢ variant with development of pulmonary tuberculosis (PTB) among Iranian patients. The single nucleotide polymorphisms (SNPs) at INT4, D543, 3'UTR of NRAMP1 gene, SNPs in restriction sites of BsmI, and FokI of the VDR gene and SNPs of TNF-¦Á at -238,-308, -244,857,-863 positions were analyzed by PCR-RFLP among two groups of individual; patients with PTB (n=117) and healthy controls (n=60). Thereafter, the frequencies of extended haplotypes and diplotypes were estimated. No statistically significant differences were observed in allele frequencies of INT4, D543, 3'UTR of NRAMPI, FokI of VDR and TNF-¦Á at -238, -244,-863 and -857 position. Although, the frequency of b allele of BsmI [ORs: 0.24 CI95 percent (0.07-0.67 (p=0.001)] and -308 A variant in TNF-¦Á promoter region [ORs:0.26 CI95 percent( 0.07-0.77) (p=0.006)] were significantly more in PTB patients than healthy controls. The frequency of extended diplotypes of NRAMP [GG TGTG++GA; 0.02(0.001-0.0035)], VDR [FFBB; 0.2(0.6-0.6] and TNF-¦Á [CCCCGGGGGG; 0.49(0.25-0.97)] were statistically different in patients and control subjects (p<0.05). This study confirmed the association of SNPs in BsmI (B/b + b/b) of VDR and SNPs in -308A (G/A +G/G) of TNF-¦Á genes with susceptibility to tuberculosis in Iranian PTB patients. Furthermore, the extended haplotypes and diplotypes analysis can be considered as an alternative way to determine the host susceptibility to TB.