Multidrug Resistant <i>Mycobacterium tuberculosis</i>: A Retrospective <i>katG</i> and <i>rpoB</i> Mutation Profile Analysis in Isolates from a Reference Center in Brazil

<div><p>Background</p><p>Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in <i>Mycobacterium tuberculosis</i> isolates from a national reference center.</p><p>Methods and Findings</p><p>Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the <i>rpoB</i> and <i>katG</i> genes were screened for mutations. Genotypic diversity was investigated by <i>IS6110</i>-RFLP and Luminex 47 spoligotyping. Mutations in <i>rpoB</i> and <i>katG</i> were seen in 91% and 93% of the isolates, respectively. Codon 315 <i>katG</i> mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical <i>IS6110</i>-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 <i>rpoB</i> mutations while codon 531 mutations prevailed in the other isolates.</p><p>Conclusions</p><p>Our data suggest that there were no major multidrug resistant <i>M. tuberculosis</i> strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.</p></div

IS<i>6110</i>-RFLP fingerprint clusters and <i>rpoB</i> and <i>katG</i> mutations for clustered samples.

<p>NM- No mutations in the studied region.</p><p>*The Brazilian state from where the isolate was obtained is indicated as PA-Pará state; AM-Amazonas state; MA-Maranhão state. All the other isolates came from Rio de Janeiro state.</p><p>The susceptibility tests for pyrazinamide (PZA) were also conducted but these results were mostly inconclusive and therefore omitted.</p

Genotyping of <i>Mycobacterium leprae</i> for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil

<div><p>Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1–4 typing of <i>Mycobacterium leprae</i> in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from <i>M</i>. <i>leprae</i> in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique <i>M</i>. <i>leprae</i> genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.</p></div

The main spoligotyping lineages with all associated mutation profiles.

<p>NM - No mutation.</p><p>*<i>katG1</i> mutations and <i>katG2</i> mutations refer to mutations in the first and in the second regions of the gene analyzed, respectively.</p><p>*The numbers in brackets mean the number of times that each mutation appears.</p

Spoligotyping analysis of MDR-TB isolates.

<p>Eighty-two isolates were classified into 5 distinct lineages (Figure 1A). Isolates of LAM Family were distributed into 8 sublineages (Figure 1B) and Isolates of T Family were distributed into 5 sublineages (Figure 1C). U – Unknown, LAM - Latin-America and Mediterranean, T - T super-family, H - Haarlen, EAI - East African Indian.</p

Mutation patterns for the <i>rpoB</i> and <i>katG</i> genes found in 99 MDR isolates.

<p>*associated with another mutated codon.</p

Genotyping of <i>Mycobacterium leprae</i> for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil - Fig 3

<p>Two minimum spanning trees constructed on a UPGMA clustering on a similarity matrix that was calculated using categorical similarity index and allele copy numbers of all 17 microsatellites (A) and SNP-Type or leaving out the four with highest SI (B). The colors represent the SNP-Types as indicated in the indent; dark blue: no sequence available to differentiate type 1 and 2; light blue: no SNP type available. In Figure B, the node size represents the number of patients included.</p

Geographic position of the 19 and 23 patients from respectively cluster 12 and 14 in Fortaleza, State of Ceará.

<p>Map of Fortaleza (A), details from the region with two clustered pairs (B), the upper pair is localized in Bonsucesso and the lower in Vila Peri. Space/time distribution of clusters 12 (C) and 14 (D); the highlight in black refers to the first case for each cluster.</p

Allele distribution and simpson index among MLVA based genotypes and SNP-types.

<p>Allele distribution and simpson index among MLVA based genotypes and SNP-types.</p

Bivariate analysis of a selection of demographic, socioeconomic, behavioral, and environmental variables with leprosy genotype clustering.

<p>Bivariate analysis of a selection of demographic, socioeconomic, behavioral, and environmental variables with leprosy genotype clustering.</p