Emilyn Costa Conceição

Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Occurrence of Nontuberculous Mycobacterial Pulmonary Infection in an Endemic Area of Tuberculosis

The majority of investigations of the epidemiology of nontuberculous mycobacteria (NTM) have focused on highly developed nations with a low prevalence of tuberculosis. In contrast, the Para state of north Brazil represents an area of high tuberculosis prevalence and increasing NTM incidence. Toward the goal of understanding the dynamics of infection by all Mycobacterium species, we report patient characteristics and the identification of NTM strains isolated from sputum samples from patients that were residents of Para, a state in the Amazon region, Northern of Brazil, over the period January 2010 through December 2011 (2 years). The 29 NTM patients comprised 13.5% of positive mycobacterial cultures over the 2-year period. A major risk factor for NTM pulmonary disease was previous tuberculosis (76%). Further, the average age of NTM patients (52 years) was significantly higher than that of tuberculosis patients (39 years) and more were female (72.4% vs. 37.4%). Unlike other Brazilian states, NTM pulmonary patients in Para were infected with a different spectrum of mycobacteria; primarily the rapidly growing Mycobacterium massiliense and Mycobacterium simiae complex.

Genetic diversity of Mycobacterium tuberculosis from Pará, Brazil, reveals a higher frequency of ancestral strains than previously reported in South America

There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Pará, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-Indian (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4-9 and 23-24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Pará and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region.

Genotipagem por spoligotyping de Mycobacterium tuberculosis obtidos de lâminas de Ziehl-Neelsen em Belém, Estado do Pará, Brasil

Este trabalho foi realizado com apoio financeiro do Instituto Evandro Chagas/ SVS/ MS, Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior e Programa de Iniciacao Cientifica/ IEC/ CNPq.

Annual frequency and distribution of tuberculosis resistance in the public health laboratory network of Pará State, Brazil

This study was supported by the Instituto Evandro Chagas, Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) and the Programa de Pos-graduacao em Biologia Parasitaria/Universidade Estadual do Para.

Identificação genotípica de membros do complexo Mycobacterium avium isolados de infecções pulmonares no Estado do Pará, Brasil

INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.

Detailed analysis of potential household transmission events of tuberculosis in the city of Belem, Brazil

Tuberculosis (TB) is an infectious disease with a higher risk for infection and disease among household contacts (HHC). Here, we report a molecular epidemiology-based approach to study disease transmission and the genetic characteristics of Mycobacterium tuberculosis (Mtb) strains among HHC in the city of Belem, the capital of the state of Para in north Brazil. The study included 63 TB patients belonging to 26 HHC groups (HHC1 to HHC26). Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR) revealed indistinguishable bacterial genotypes among 26 patients in 14 (53.8%) HHC groups. Drug susceptibility testing (DST) revealed that 45 (71.4%) of the Mtb isolates were multidrug resistant. The major cluster composed of isolates from five HHCs and on three of these, whole genome sequencing (WGS) was performed confirming their high genetic similarity. These results pinpoint the need for improved vigilance for TB control in households in the city of Belém. When comparing WGS versus phenotypic resistance detection methods as DST and Minimum Inhibitory Concentration (MIC) our data suggest that depending on the colonies selection, results may present variation.

Genotypic identification of members of the Mycobacterium avium complex isolated from pulmonary infections in Pará State, Brazil

INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.

Identificación genotípica de miembros del complejo Mycobacterium avium aislados de infecciones pulmonares en el Estado de Pará, Brasil

INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.INTRODUCTION: The Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria naturally found in the environment capable of causing infections in a wide variety of living species, including birds, swines and humans. These infections can be asymptomatic, clinically significant and, in some cases, fatal. There is a demand for techniques that are capable of conclusively identifying closely related bacteria. Molecular biological techniques are promising tools for a more precise identification. MATERIAL AND METHODS: In this study, we evaluated the ability of 16S rRNA, hsp65 and rpoB molecular markers to distinguish between members of the MAC isolated in the Mycobacteria Laboratory at the Instituto Evandro Chagas. RESULTS: MAC samples collected from 15 patients were previously evaluated using an hsp65 gene restriction fragment length polymorphism analytical method (RFLP-hsp65), and they showed three different profiles: (I) BstEII: 235/115/100, HaeIII: 145/130/60; (II) BstEII: 235/210, HaeIII: 130/105; and (III) BstEII: 235/210, HaeIII: 145/130. We constructed phylogenetic trees using 16S rRNA analysis in which the samples were distributed into two groups, similarly to those found in the hsp65 analysis. However, the results from the rpoB analysis disagreed with those of the other trees due to changes in topology. CONCLUSION: The findings from this study warrant that various evolutionary forces may be acting on the rpoB gene. Thus, it is necessary to be cautious when using this target for taxonomic analyses. Additionally, we recommend that multiple markers (including16S rRNA) be evaluated when identifying mycobacteria.