Introduction: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. Methods: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. Results: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. Conclusions: The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.
Tuberculosis is an infectious disease with variable outcomes. This variability is due to host immune capacity in containing the infection process initiated by the Mycobacterium tuberculosis (MTB). Vitamin D is able to modulate a very specific immune response against MTB infection, and its action relies on vitamin D receptor (VDR) binding. Altered VDR forms may compromise vitamin D pathway and proper immune response after MTB infection. Herein we assessed the relationship of five potentially functional polymorphisms from VDR: rs2228570 FokI, rs11568820 Cdx-2, rs2248098, rs1540339 and rs4760648, with tuberculosis susceptibility. The SNP rs4760648 T/T was associated with differential susceptibility to tuberculosis (OR = 2.50, 95%CI = 1.20-5.36, p = 0.01). The SNP rs1540339 presented association to both T allele (OR = 0.55, 95%CI = 0.35-0.88, p = 0.01) and the T/T genotype (OR = 0.404, 95%CI = 0.20 -0.78, p = 0.005). The FokI T allele was identified as associated to diminished susceptibility (OR = 0.67, 95% CI = 0.45-0.99, p = 0.04) to active TB, as well as T/T genotype (OR = 0.15, 95%CI = 0.04-0.45, p = 9.58 × 10 -5 ). We also performed the expression analyses and observed a down-regulation of VDR in patients (-10.717 FC, p = 8.42e −12 ), and according to the presence of associated FokI SNP, we observed that the C/T and T/T genotypes presence increases VDR expression (+ 1.25 and + 2.35 FC, p = 0.425 and p = 0.506, respectively). This study shows that vitamin D receptor variants can influence upon pulmonary tuberculosis susceptibility and VDR mRNA levels are decreased in those patients.
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