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
This study analyzed the genetic diversity by MIRU-VNTR of Mycobacterium leprae isolates from nasal cavities and related to epidemiological and clinical data. The sample consisted of 48 newly diagnosed leprosy cases that tested positive for M. leprae PCR in nasal secretion (NS) attending to the National Reference Center of Dermatology Dona Libania (CDERM), Fortaleza, Brazil. Total DNA was extracted from NS of each patient and used for amplification of four M. leprae VNTR loci. Four clusters of M. leprae isolates were formed with identical genotypes. In the spatial analysis, 12 leprosy cases presented similar genotypes organized into 4 clusters. The most common genotypes in the current study was AC8b: 8, AC9: 7, AC8a: 8, GTA9: 10, which may represent a genotype of circulating strains most often in Ceará. A minimum set of four MIRU-VNTR loci was demonstrated to study the genetic diversity of M. leprae isolates from NS.
Assuntos
Variação Genética , Genótipo , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Repetições Minissatélites , Mycobacterium leprae/classificação , Cavidade Nasal/microbiologia , Adolescente , Adulto , Idoso , Líquidos Corporais/microbiologia , Brasil , Criança , Análise por Conglomerados , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Adulto JovemRESUMO
BACKGROUND: The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD: We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS: Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS: Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.
Assuntos
Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Brasil , Genótipo , Humanos , Técnicas de Diagnóstico Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
BACKGROUND The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.
Assuntos
Humanos , Técnicas Bacteriológicas/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Mycobacterium tuberculosis/genética , Brasil , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Patologia Molecular , GenótipoRESUMO
BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.
Assuntos
Variação Genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Alelos , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Brasil/epidemiologia , Análise por Conglomerados , DNA Bacteriano/análise , RNA Polimerases Dirigidas por DNA , Genótipo , Humanos , Isoniazida/uso terapêutico , Repetições Minissatélites/genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3%) isolates were resistant to at least one drug (R-TB) and 64 (55.7%) were sensitive to all the drugs tested (S-TB). A high frequency of resistance was found in previously treated cases (84%) and among new cases (16%; p < 0.001). a total of 74 (64%) isolates were grouped into 22 spoligotyped lineages, while 41 (36%) isolates were identified as new. among the predominant genotypes, 33% were latim american mediterranean (lam), 12% haarlem (h), and 5% u. there was no association of geographic distribution of rt-tb patients as compared to the controls and also the geographic location to the spoligotype patterns. the geospatial analysis revealed that 24 (23%) patients (hot spot zones) either shared the same residence or lived in a close neighborhood of a case. among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: it was observed that the spoligopatterns family distribution was similar to that reported for south america, prevailing the lam and h lineages. a high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Variação Genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/microbiologia , Antituberculosos/farmacologia , Brasil/epidemiologia , Estudos de Casos e Controles , DNA Bacteriano/genética , Genótipo , Sistemas de Informação Geográfica , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificação , Prevalência , Estudos Prospectivos , Tuberculose Pulmonar/epidemiologiaRESUMO
OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3%) isolates were resistant to at least one drug (R-TB) and 64 (55.7%) were sensitive to all the drugs tested (S-TB). A high frequency of resistance was found in previously treated cases (84%) and among new cases (16%; p<0.001). A total of 74 (64%) isolates were grouped into 22 spoligotyped lineages, while 41 (36%) isolates were identified as new. Among the predominant genotypes, 33% were Latim American Mediterranean (LAM), 12% Haarlem (H), and 5% U. There was no association of geographic distribution of RT-TB patients as compared to the controls and also the geographic location to the spoligotype patterns. The geospatial analysis revealed that 24 (23%) patients (hot spot zones) either shared the same residence or lived in a close neighborhood of a case. Among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: It was observed that the spoligopatterns family distribution was similar to that reported for South America, prevailing the LAM and H lineages. A high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.