Molecular characterisation of multidrug-resistantMycobacterium tuberculosisisolates from a high-burden tuberculosis state in Brazil

Abstract

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131 Mycobacterium tuberculosis (M.tb) DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of the katG, inhA and rpoB genes and RDRio sublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRio deletion was observed in 42 (32%) of the M.tb isolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%) M.tb isolates, we found mutations associated with rifampicin (RIF) resistance in rpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance in katG and inhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in the rpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil

“…The Euro-American lineage predominance in Santa Catarina State and in the Southern Brazil 17 , 34 , occurred due to migratory processes from Europe to South America that increased in the seventeenth century 35 . Therefore, LAM, T and Haarlem, the most common spoligotyping families identified in this study, were also found in other studies in Southern Brazil 17 , 20 , 34 , 36 , 37 . Our examination of the distribution of the fbpC 103 SNP, considered as a highly specific marker for the LAM family not only confirmed the identification of all spoligotyping-based identified LAM, but increased the frequency of this lineage to 69.5% (105/151).…”

A highly rifampicin resistant Mycobacterium tuberculosis strain emerging in Southern Brazil

“…WGS-based typing in concert to phylogenetic analysis corroborates with previous data[6] that associated the occurrence of the 12nt insertion with strains belonging to SIT 863 that presents an important and restricted distribution in Rio Grande do Sul State[9,42]. According to Dalla Costa et al[6], strains showing the SIT 863 pattern, and previously wrongly identified as Mycobacterium pinnipedii , are in fact M. tuberculosis Lineage 4, subfamily LAM strains.…”

“…[6] described six strains presenting the same insertion, all of them belonging to the SIT 863 and isolated in 2006 and 2010. Latest studies in the region have shown an increased frequency of strains carrying on this insertion[7,9,42], flagging its spreading and ongoing transmission. Currently, we are performing a population-based study on drug-resistant M. tuberculosis strains from Rio Grande do Sul, isolated between 2011 and 2014, and identified 16 MDR strains harboring the 12nt insertion.…”

“…These data are also confirmed by phylogenetic inference here conducted that showed the genomic closeness between 12nt insertion and SIT 863 without the insertion strains, as well as, with 4.3.3 sublineage strains. Although previous studies[5,42] related the presence of 12nt insertion strains classified as other sublineages, the genotyping techniques previously used, such as RFLP, MIRU-VNTR and spoligotyping, could produce some erroneous classification.…”

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

“…Upon analysis of the number of studies on MIRU-VNTR typing in the country, we observed that the Southeast and South regions present the highest number (82.4%; 14/17) and in general, these studies demonstrated that the MIRU-VNTR present a high discriminatory power. Two of these with the largest sampling 28 , 29 , 30 showed a low rate of clustering in the M. tuberculosis population (0.13 and 0.28, respectively). Additionally, two studies 32 , 33 investigated isolates from the same patient with up to three different loci and upon further characterisation, such closely related MIRU-VNTR types, demonstrated to belong to the same strain.…”