RESUMO
Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.
Assuntos
Hanseníase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , DNA Bacteriano/genética , Feminino , Humanos , Indicadores e Reagentes/normas , Lactente , Hanseníase/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Multiplex/normas , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Adulto JovemRESUMO
Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.
Assuntos
Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Brasil/epidemiologia , Genes Bacterianos , Genoma Bacteriano , Genótipo , Humanos , Epidemiologia Molecular , Tipagem Molecular , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Polimorfismo de Nucleotídeo Único , RNA Ribossômico 16S , Tuberculose Pulmonar/epidemiologiaRESUMO
Mycobacterium haemophilum is a nontuberculous mycobacterium that causes localized or disseminated disease, mainly in immunocompromised hosts. We report the case of a 35-year-old HIV-infected woman who presented with several enlarging cutaneous lesions over the arms and legs. Histopathological examination revealed the diagnosis of a cutaneous mycobacterial disease. Mycobacterial analyses unveiled M. haemophilum infection. Six months after completion of a successful antimycobacterial treatment, she developed an immune reconstitution inflammatory syndrome (IRIS). This paradoxical relapse presented as tenderness, redness and swelling at the precise sites of the healed lesions and took place in the setting of significant recovery of the CD4 cell count (from 05 to 318 cells/mm 3 ). Microbiological analyses of these worsening lesions were negative, and they spontaneously remitted without the initiation of a novel antimycobacterial treatment cycle. M. haemophilum infection should always be considered as a cause of skin lesions in immunocompromised subjects. Physicians should be aware of the possibility of IRIS as a complication of successful antiretroviral therapy in HIV-infected patients with M. haemophilum infection.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Antirretrovirais/efeitos adversos , Síndrome Inflamatória da Reconstituição Imune/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium haemophilum/isolamento & purificação , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Adulto , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Feminino , Humanos , Síndrome Inflamatória da Reconstituição Imune/imunologia , Síndrome Inflamatória da Reconstituição Imune/metabolismo , Hospedeiro Imunocomprometido , Masculino , Infecções por Mycobacterium/imunologiaRESUMO
BACKGROUND: In recent decades, Mycobacterium tuberculosis with the RDRio genotype, frequently isolated from tuberculosis patients in Rio de Janeiro, has become part of the Latin American - Mediterranean (LAM) family and has been associated with multidrug-resistant tuberculosis (MDR-TB). The aim of this study was to investigate the frequency of M. tuberculosis RDRio in the state of Minas Gerais, Brazil, and its relationship with MDR-TB. METHODS: For convenience, 172 susceptible and 63 MDR M. tuberculosis isolates were taken from pulmonary samples from patients diagnosed between January 2007 and December 2011. The DNA extracted from these isolates was analyzed by spoligotyping, PCR-RFLP to characterize fbpC103/Ag85C103, multiplex PCR to detect RDRio and RD174, and MIRU-VNTR 24 loci. RESULTS: Among the 235 isolates, the RDRio pattern was identified in 122 (51.9%) isolates (IC 0.45-0.58), with 100 (42.5%) wild-type and 13 (5.5%) mixed pattern isolates, whereas RD174 was identified in 93 of the 122 RDRio positive samples (76.3%). The LAM family and the LAM9 lineage were the most frequently identified among the isolates in this study. Among the 63 MDR isolates, 41 (65.1%) were RDRio and 28 (44.4%) RD174. CONCLUSION: The association of both deletions with MDR proved to be statistically significant, corroborating the few reports that have associated RDRio with MDR.
Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Frequência do Gene , Estudos de Associação Genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Repetições Minissatélites , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Tuberculose/epidemiologia , Tuberculose/microbiologiaRESUMO
Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.
Assuntos
Genoma Bacteriano , Genótipo , Pneumopatias/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium kansasii/genética , Brasil , Gráficos por Computador , DNA Bacteriano , Genômica , Humanos , Mycobacterium kansasii/isolamento & purificação , Polimorfismo de Fragmento de RestriçãoRESUMO
Tuberculosis (TB) remains a major public health problem in the world and Brazil is among the countries with the highest incidence and prevalence rates, and Rio Grande do Sul, a Brazilian state, occupy a prominent position. Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) further aggravates this scenario, making it more difficult to treat and control the disease. Isoniazid monoresistance (IMR) may increase the risk of progression to MDR-TB and treatment failure. However, most drug resistance molecular tests only focus on detecting rifampicin (RIF) resistance.In the present study, we characterized a total of 63 drug resistant isolates of M. tuberculosis (35 MDR, 26 IMR and two isolates monoresistant to rifampicin [RMR]) of the Rio Grande do Sul state by MIRU-VNTR (24 loci), spoligotyping, presence of RDRio, fbpC103, pks15/1 and sequencing of the katG, rpoB and inhA genes. We observed a higher proportion of the LAM family 30/63 (47.61%). In IMR, mutations were found in the katG gene (98% at codon 315) in 72.5%, and mutations in the promoter region of the inhA gene in 6.25% of the isolates. In MDR-TB and RMR-TB isolates, 92.1% had mutations in the rpoB gene (57% at codon 531). The presence of a 12 bp insertion between codons 516 and 517 of the rpoB gene in MDR-TB isolates was found in five isolates. In conclusion, we observed that the highest frequency of IMR-TB and MDR-TB strains belong to the LAM and Haarlem genotypes in Rio Grande do Sul state. A significant number of isolates previously characterized as Mycobacterium pinnipedi2 through spoligotyping were found to belong to the M. tuberculosis LAM family. This was responsible for a number of significant cases and the molecular profile of this strain and the pattern of mutations related to drug resistance were analyzed. These findings may contribute to a better understanding about the spread of M. tuberculosis resistant in southern of Brazil.
Assuntos
Antituberculosos/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos/genética , Variação Genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana/métodos , Repetições Minissatélites/genética , Mycobacterium tuberculosis/classificação , Fenótipo , Tuberculose/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.
Assuntos
DNA Bacteriano , Genoma Bacteriano/genética , Mycobacterium kansasii/genética , Gráficos por ComputadorRESUMO
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.
Assuntos
Variação Genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adolescente , Adulto , Distribuição por Idade , Brasil/epidemiologia , Criança , Pré-Escolar , DNA Bacteriano , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites , Tipagem Molecular , Filogenia , Filogeografia , Adulto JovemRESUMO
Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.
Assuntos
Mycobacterium bovis/genética , Animais , Bovinos , Análise por Conglomerados , Genes BacterianosRESUMO
Mycobacterium tuberculosis of the Bejing subtype (MtbB) is transmitted efficiently in high burden countries for this genotype. A higher virulence was associated with isolates of the "modern" Beijing genotype sub-lineages when compared to "ancient" ones. Here, we report the full genomes of the strain representing these two genotypes from Brazil, a country with a low incidence of MtbB.