RESUMO
To evaluate the genetic diversity and clustering rates of M. tuberculosis strains to better understand transmission among persons deprived of liberty (PDL) in Rio Grande do Sul (RS), southern Brazil. This is a cross-sectional study, including strains of M. tuberculosis isolated from PDL, stored at the Central Laboratory of RS, in the period from 2013 to 2018. The molecular characterization was performed using the MIRU-VNTR 15 loci method. A total of 598 M. tuberculosis strains were genotyped, and 37.5% were grouped into 53 clusters. Cluster sizes ranged from 2 to 34 strains. The largest cluster of the study had strains from 34 PDL, and 58.8% of the PDL of this cluster were in P01. Among the clusters formed, in 60.3%, there was at least one strain from P01. The most common strains in RS were LAM (53.2%) and Haarlem (31.1%). The LAM strain was the most likely to form clusters, and Haarlem was associated with anti-TB drug resistance. This was translational research, and the results can collaborate with the TB control programs, leading to improved strategies that allow the reduction of the TB burden in prisons.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Epidemiologia Molecular , Brasil/epidemiologia , Estudos Transversais , Genótipo , Repetições Minissatélites , Tuberculose/microbiologia , FilogeniaRESUMO
Treatment of drug-resistant tuberculosis requires extended use of more toxic and less effective drugs and may result in retreatment cases due to failure, abandonment or disease recurrence. It is therefore important to understand the evolutionary process of drug resistance in Mycobacterium tuberculosis. We here in describe the microevolution of drug resistance in serial isolates from six previously treated patients. Drug resistance was initially investigated through phenotypic methods, followed by genotypic approaches. The use of whole-genome sequencing allowed the identification of mutations in the katG, rpsL and rpoB genes associated with drug resistance, including the detection of rare mutations in katG and mixed populations of strains. Molecular docking simulation studies of the impact of observed mutations on isoniazid binding were also performed. Whole-genome sequencing detected 266 single nucleotide polymorphisms between two isolates obtained from one patient, suggesting a case of exogenous reinfection. In conclusion, sequencing technologies can detect rare mutations related to drug resistance, identify subpopulations of resistant strains, and identify diverse populations of strains due to exogenous reinfection, thus improving tuberculosis control by guiding early implementation of appropriate clinical and therapeutic interventions.
Assuntos
Resistência a Medicamentos/genética , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Mycobacterium tuberculosis/efeitos dos fármacos , Brasil , Resistência a Medicamentos/imunologia , Estudo de Associação Genômica Ampla/métodos , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.
Assuntos
Antibióticos Antituberculose/uso terapêutico , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/uso terapêutico , Brasil/epidemiologia , Perfilação da Expressão Gênica , Genoma Bacteriano/genética , Humanos , Isoniazida/uso terapêutico , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.
Assuntos
Mycobacterium tuberculosis , Filogenia , Tuberculose Resistente a Múltiplos Medicamentos , Adulto , Antituberculosos/farmacologia , Brasil/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Sequenciamento Completo do GenomaRESUMO
The Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.
Assuntos
DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/classificação , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Sequenciamento Completo do Genoma/métodos , Pequim , Brasil , Guiné-Bissau , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Filogeografia , PortugalRESUMO
Lack of routine surveillance in countries endemic for bovine tuberculosis (TB) and limited laboratory support contributes to the inability to differentiate the Mycobacterium tuberculosis Complex species, leading to an underestimated burden of the disease. Here, Whole-Genome Sequencing of Mycobacterium bovis isolated from tissues with TB-like lesions obtained from cattle and buffalos at Marajó Island, Brazil, demonstrates that recent transmission of M. bovis is ongoing at distinct sites. Moreover, the M. bovis epidemiology in this setting is herein found to be dominated by an endemic and unique clade composed of strains evolved from a common ancestor that are now genetically differentiated from other M. bovis clades. Additionally, envisioning a rapid strain differentiation and tracing across multiple settings, 28 globally validated strain-specific SNPs were identified, three of which considered as robust markers for the M. bovis Marajó strain. In conclusion, this study contributes with data regarding the identification of a novel M. bovis phylogenetic clade responsible for ongoing transmission events in both cattle and buffalo species in Brazil, provides a framework to investigate the dissemination of this highly prevalent strain and, holds the potential to inform TB control strategies that may help to prevent the spread of bovine and zoonotic TB.
Assuntos
Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Brasil , Búfalos , Bovinos , Mycobacterium tuberculosis/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Tuberculose/microbiologia , Sequenciamento Completo do Genoma/métodos , Zoonoses/microbiologiaRESUMO
Drug-resistant tuberculosis (DR-TB) is major problem in the fight against TB. Multidrug resistant (MDR) TB patients have a reduced treatment success rates and for, extensively drug-resistant (XDR) TB the cure rate does not exceed 25% in many countries. To evaluate the pre-XDR-TB and XDR-TB prevalence and transmission in Rio Grande do Sul State, in southern Brazil, we performed a retrospective WGS-based analysis of 87 MDR-TB cases, aiming to identify resistance-conferring mutations and its phylogenetic distinctiveness. Using a five SNP threshold for genomic clustering, 60 strains were genomically linked within 10 clusters, including 14 likely transmission events identified by retrospective conventional epidemiological investigation. Moreover, five likely transmission events involved 17 patients deprived of liberty in the same prison establishment. Mutations associated with isoniazid and rifampicin resistance were identified respectively in 97.70% and 98.85% of MDR M.tb strains, more frequently in katG and rpoB genes. In total, we identified eight (9.19%) pre-XDR and four (4.59%) XDR M.tb strains. Resistance to ofloxacin was observed in seven (8.04%) strains, all of them presenting resistance-conferring mutations. Phenotypic resistance from capreomycin and kanamycin was found in seven (8.04%) and four (4.59%) strains respectively, but no classic mutations associated with resistance to these drugs was identified. The results put in evidence a scenario involving multiple phylogenetically distinctive clades associated with pre-XDR and XDR-TB in the largest state of southern Brazil, while stressing the potential of using WGS to predict anti-TB drug resistance and need to halt MDR-TB transmission in the region.
Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto , Antituberculosos/farmacologia , Brasil/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Variação Genética , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Repetições Minissatélites , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Polimorfismo de Nucleotídeo Único , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Sequenciamento Completo do GenomaRESUMO
National border areas are special places for the spread of Mycobacterium tuberculosis (MTB). These regions concentrate vulnerable populations and constant population movements. Understanding the dynamics of the transmission of MTB is fundamental to propose control measures and to monitor drug resistance. We conducted a population-based prospective study of tuberculosis (TB) to evaluate molecular characteristics of MTB isolates circulating in Roraima, a state on the border of Venezuela and Guyana. Eighty isolates were genotyped by IS6110-RFLP (restriction fragment length polymorphism), spoligotyping, and 24-locus mycobacterial interspersed repetitive unit-variable number of repeats tandem (MIRU-VNTR). Drug susceptibility tests were performed by using the proportion method and GeneXpert® MTB/RIF (Cepheid, Sunnyvale, CA). Isolates showing a phenotypic resistance profile were submitted to polymerase chain reaction (PCR) and sequencing. Spoligotyping showed 40 distinct patterns with a high prevalence of Latin-American and Mediterranean (LAM), Haarlem (H), and the "ill-defined" T clades. Mycobacterial interspersed repetitive unit -VNTR and IS6110-RFLP showed clustering rates of 21.3% and 30%, respectively. Drug resistance was detected in 11 (15.1%) isolates, and all were found to have primary resistance; among these, six (8.2%) isolates were streptomycin mono-resistant, four (5.4%) isoniazid mono-resistant, and one (1.3%) multidrug resistant. This is the first study on the molecular epidemiology and drug resistance profile of MTB from Roraima. Herein, we describe high diversity of genetic profiles circulating in this region that may be driven by the introduction of new strain types because of large population flow in this region. In summary, our results showed that analyses of these circulating strains can contribute to a better understanding of TB epidemiology in the northern Brazilian border and be useful to establish public health policies on TB prevention.
Assuntos
Variação Genética , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Adolescente , Adulto , Brasil/epidemiologia , Análise por Conglomerados , Farmacorresistência Bacteriana , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites/genética , Epidemiologia Molecular , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Estudos Prospectivos , Tuberculose/microbiologia , Adulto JovemRESUMO
Tuberculosis (TB) in pinnipeds is typically caused by Mycobacterium pinnipedii, which has also been associated with infections in other species, such as cattle and humans. As a result, this pathogen has zoonotic potential and is a public health concern. In 2016, a female South American sea lion Otaria flavescens in southern Brazil presented with emaciation and severe dyspnea and died within 3 h of capture. Gross pathology identified pulmonary granulomas, and Ziehl-Neelsen stain identified acid-fast bacilli. M. tuberculosis complex bacteria were confirmed by a BD BACTEC™ MGIT™ 320 detection system using fibrinous exudate, lung granulomas and thoracic fluid. Molecular characterization by spoligotyping showed a hybridization pattern characteristic of M. pinnipedii (SIT593/PINI1). Currently, there is a paucity of data concerning the transmission and epidemiology of M. pinnipedii in pinniped populations in South America. The case report shows that the disease appeared in a free-ranging beached sea lion on the coast, and further surveillance is needed to determine the origin of this TB because of its potential impact on public health.