RESUMEN
BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.
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Farmacorresistencia Bacteriana , Citometría de Flujo/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Costos y Análisis de Costo , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Citometría de Flujo/economía , Genotipo , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Regiones Promotoras Genéticas , Juego de Reactivos para Diagnóstico , Rifampin , Sensibilidad y Especificidad , Tuberculosis/economíaRESUMEN
INTRODUCTION: Mozambique is one of three countries with high prevalence of tuberculosis (TB), TB/human immunodeficiency virus coinfection, and multidrug-resistant TB. We aimed to describe Mycobacterium tuberculosis spoligotypes circulating among drug resistant (DR) strains from Beira, Mozambique comparing them with genotypes in the country. METHODS: We performed spoligotyping of 79 M. tuberculosis suspected of DR-TB compared all spoligotype patterns published on the international database and PubMed. RESULTS: Both in Beira and Mozambique (n=578), the main clades were Latin-American-Mediterranean, East-African-Indian, Beijing and T, with no extensively DR TB cases. CONCLUSIONS: Beira and Mozambique share the same population genetic structure of M. tuberculosis.
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Variación Genética/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Técnicas de Tipificación Bacteriana , Genotipo , Humanos , Mozambique , Mutación/genética , FilogeniaRESUMEN
Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sublineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48) and L1/EAI6-BGD1 (SIT591) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil.
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Variación Genética/genética , Mycobacterium tuberculosis/genética , Población Negra/genética , Brasil , Genotipo , Humanos , Madagascar , Mozambique , Países Bajos , Filogenia , Polimorfismo de Nucleótido Simple/genética , Tuberculosis/microbiologíaRESUMEN
We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/transmisión , Emigración e Inmigración , Europa (Continente)/epidemiología , Genoma Bacteriano , Genotipo , Humanos , Repeticiones de Minisatélite , Epidemiología Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Perú/epidemiología , Polimorfismo de Nucleótido Simple , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Secuenciación Completa del GenomaRESUMEN
Abstract INTRODUCTION Mozambique is one of three countries with high prevalence of tuberculosis (TB), TB/human immunodeficiency virus coinfection, and multidrug-resistant TB. We aimed to describe Mycobacterium tuberculosis spoligotypes circulating among drug resistant (DR) strains from Beira, Mozambique comparing them with genotypes in the country. METHODS: We performed spoligotyping of 79 M. tuberculosis suspected of DR-TB compared all spoligotype patterns published on the international database and PubMed. RESULTS: Both in Beira and Mozambique (n=578), the main clades were Latin-American-Mediterranean, East-African-Indian, Beijing and T, with no extensively DR TB cases. CONCLUSIONS: Beira and Mozambique share the same population genetic structure of M. tuberculosis.
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Humanos , Variación Genética/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mycobacterium tuberculosis/genética , Filogenia , Técnicas de Tipificación Bacteriana , Genotipo , Mozambique , Mutación/genéticaRESUMEN
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.
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Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Brasil , Genotipo , Humanos , Técnicas de Diagnóstico Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
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.
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Humanos , Técnicas Bacteriológicas/métodos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mycobacterium tuberculosis/genética , Brasil , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Patología Molecular , GenotipoRESUMEN
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.
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Variación Genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , Tuberculosis/microbiología , Adolescente , Adulto , Distribución por Edad , Brasil/epidemiología , Niño , Preescolar , ADN Bacteriano , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Repeticiones de Minisatélite , Tipificación Molecular , Filogenia , Filogeografía , Adulto JovenRESUMEN
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.
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Variación Genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Alelos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Brasil/epidemiología , Análisis por Conglomerados , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN , Genotipo , Humanos , Isoniazida/uso terapéutico , Repeticiones de Minisatélite/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
During the American colonization in the 18th and 19th century, Africans were captured and shipped to America. Harsh living and working conditions often led to chronic diseases and high mortality rates. Slaves in the Caribbean were forced to work mainly on sugar plantations. They were buried in cemeteries like Anse Sainte-Marguerite on the isle of Grande-Terre (Guadeloupe) which was examined by archaeologists and physical anthropologists. Morphological studies on osseous remains of 148 individuals revealed 15 cases with signs for bone tuberculosis and a high frequency of periosteal reactions which indicates early stages of the disease. 11 bone samples from these cemeteries were analysed for ancient DNA. The samples were extracted with established procedures and examined for the cytoplasmic multicopy ß-actin gene and Mycobacterium tuberculosis complex DNA (IS 6110) by PCR. An amplification product for M. tuberculosis with the size of 123 bp was obtained. Sequencing confirmed the result. This study shows evidence of M. tuberculosis complex DNA in a Caribbean slave population.
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Personas Esclavizadas/historia , Tuberculosis Osteoarticular/historia , Actinas/genética , Adolescente , Adulto , Población Negra/genética , Niño , ADN Bacteriano/genética , Femenino , Guadalupe , Historia del Siglo XVIII , Historia del Siglo XIX , Humanos , Masculino , Mycobacterium tuberculosis/genética , Técnicas de Amplificación de Ácido Nucleico , Paleopatología , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
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.
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Repeticiones de Minisatélite/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Tuberculosis/microbiología , Alelos , Proteínas Bacterianas/genética , Brasil , Frecuencia de los Genes , Genotipo , Humanos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , FilogeniaRESUMEN
BACKGROUND: 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 Mycobacterium tuberculosis isolates from a national reference center. METHODS AND FINDINGS: Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG 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 IS6110-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 rpoB mutations while codon 531 mutations prevailed in the other isolates. CONCLUSIONS: Our data suggest that there were no major multidrug resistant M. tuberculosis 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.
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Proteínas Bacterianas/genética , Catalasa/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mutación/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/genética , Adulto , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , Análisis por Conglomerados , ARN Polimerasas Dirigidas por ADN , Demografía , Femenino , Humanos , Incidencia , Masculino , Mycobacterium tuberculosis/clasificación , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Retrospectivos , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
Tuberculosis (TB) is still a serious public health problem, continuing to be an important threat for confined populations. We used spoligotyping to estimate the genotypic clades of Mycobacterium tuberculosis isolates from inmates in two blocks in a southeastern Brazilian prison unit, with TB incidence rate of 8185/100.000. The Latin American Mediterranean (LAM) clade is well represented in the country, and the LAM specific molecular markers, RD(Rio) large sequence polymorphism and the SNP on the Rv3062 [ligB(1212)], were used to characterize spoligotype signatures from prison isolates. Typing of RD(Rio) and ligB increase LAM clade from 66.7% (n=72/108) to 69.4% (n=75). The LAM2 SIT17 (n=23) and SIT179 (n=12) signatures comprised one third of all isolates, followed by Haarlem (11.5%, n=12), T (8.7%, n=9) and X (5.7%, n=6) clades. Strains with unknown signatures represented 5.5% (n=6), and four (3.7%) did not match any lineage. We observed RD(Rio) among 64 (59.2%) isolates, and 54 (50%) were of the LAM clade. In particular, the LAM2/RD(Rio) sub-lineage was significantly associated with clustering (p=0.02) and its frequency was higher (32%) when compared to that of the previous general TB cases in RJ (4.29%). Overall cluster frequency defined by spoligotyping/IS6110-RFLP was 62%. The two evolutionary markers helped to evaluate some LAM signature misconceptions and demonstrate that LAM2/RD(Rio) was found with high frequency, hitherto being unnoticed. All these data, allied to high clustering, imply that public health measures to minimize the escalation of TB in prison is essential, and both spoligotyping as well as RD(Rio) would be useful tools to monitor the effects of the measures with respect to M. tuberculosis lineage variation.
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Genotipo , Mycobacterium tuberculosis/genética , Polimorfismo Genético , Prisioneros , Prisiones , Tuberculosis/epidemiología , Tuberculosis/microbiología , Adulto , Alelos , Brasil/epidemiología , Frecuencia de los Genes , Humanos , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units-variable number of DNA tandem repeats (MIRU-VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU-VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a global vision of MTC genetic diversity worldwide based on 62,582 clinical isolates corresponding to 153 countries of patient origin (105 countries of isolation). We report a total of 7105 spoligotype patterns (corresponding to 58,180 clinical isolates) - grouped into 2740 shared-types or spoligotype international types (SIT) containing 53,816 clinical isolates and 4364 orphan patterns. Interestingly, only 7% of the MTC isolates worldwide were orphans whereas more than half of SITed isolates (n=27,059) were restricted to only 24 most prevalent SITs. The database also contains a total of 2379 MIRU patterns (from 8161 clinical isolates) from 87 countries of patient origin (35 countries of isolation); these were grouped in 847 shared-types or MIRU international types (MIT) containing 6626 isolates and 1533 orphan patterns. Lastly, data on 5-locus exact tandem repeats (ETRs) were available on 4626 isolates from 59 countries of patient origin (22 countries of isolation); a total of 458 different VNTR patterns were observed - split into 245 shared-types or VNTR International Types (VIT) containing 4413 isolates) and 213 orphan patterns. Datamining of SITVITWEB further allowed to update rules defining MTC genotypic lineages as well to have a new insight into MTC population structure and worldwide distribution at country, sub-regional and continental levels. At evolutionary level, the data compiled may be useful to distinguish the occasional convergent evolution of genotypes versus specific evolution of sublineages essentially influenced by adaptation to the host. This database is publicly available at: http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE.
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Bases de Datos de Ácidos Nucleicos , Variación Genética , Mycobacterium tuberculosis/genética , Técnicas de Tipificación Bacteriana , Biología Computacional/métodos , ADN Bacteriano , Genotipo , Humanos , Internet , Repeticiones de Minisatélite , Tipificación de Secuencias Multilocus , Mycobacterium tuberculosis/clasificación , Filogeografía , Programas Informáticos , Tuberculosis/epidemiologíaRESUMEN
One of the high tuberculosis (TB) incidence countries in the world, Brazil is characterized by considerable differences in TB incidence on regional and state level. In the present study, we describe Brazilian spoligotypes of 1991 Mycobacterium tuberculosis complex (MTC) clinical isolates from patients residents of 11 states from different regions of the country, diagnosed between 1996 and 2005. By performing spoligotyping on a large number of M. tuberculosis clinical isolates, one of the main objectives of this study was to determine the major genotype families causing TB in Brazil and to verify the region-associated genotype distribution. We observed a total of 577 distinct spoligopatterns, 12.6% of these corresponded to orphan patterns while 87.4% belonged to 326 shared-types (SITs). Among the latter, 86 SITs (isolated from 178 patients) had been observed for the first time in this study, the most frequent being SIT2517 which belonged to the T3-ETH lineage and was exclusively found among patients residents of Belém, the capital of the state of Pará (n=8 isolates). Irrespective of shared-type labeling, a total of 19.5% strains were unique (unclustered) in our study as opposed to 80.5% clustered isolates (189 clusters, size range from 2 to 205 isolates). The three largest clusters were SIT42 of the Latin-America & Mediterranean (LAM) 9 clade (10.3%), SIT53 of the T clade (7.6%), and SIT50 of the Haarlem clade (5.4%). The predominant MTC lineages in Brazil in decreasing order belonged to the LAM (46%); the ill-defined T (18.6%); the Haarlem (12.2%), the X (4.7%), the S (1.9%), and the East African Indian (EAI) (0.85%) families. The rest of clades grouped together as Mycobacterium africanum, Mycobacterium bovis, Beijing, Central Asian (CAS), and the Manu types, represented less than 1% of the strains. Finally, about 15% of the isolates showed spoligotype signatures that were not yet classified among well-defined lineages. In conclusion, we provide hereby a first insight into the population structure of MTC isolates in Brazil, showing the predominance of both LAM and T family and the existence of region-associated genotypes.
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Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , Análisis por Conglomerados , Genotipo , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Tuberculosis/epidemiologíaRESUMEN
BACKGROUND: French Guiana has the highest tuberculosis (TB) burden among all French departments, with a strong increase in the TB incidence over the last few years. It is now uncertain how best to explain this incidence. The objective of this study was to compare three different methods evaluating the extent of recent TB transmission in French Guiana. METHODS: We conducted a population-based molecular epidemiology study of tuberculosis in French Guiana based on culture-positive TB strains (1996 to 2003, n = 344) to define molecular relatedness between isolates, i.e. potential transmission events. Phylogenetic relationships were inferred by comparing two methods: a "cluster-graph" method based on spoligotyping results, and a minimum spanning tree method based on both spoligotyping and variable number of tandem DNA repeats (VNTR). Furthermore, three indices attempting to reflect the extent of recent TB transmission (RTIn, RTIn-1 and TMI) were compared. RESULTS: Molecular analyses showed a total amount of 120 different spoligotyping patterns and 273 clinical isolates (79.4%) that were grouped in 49 clusters. The comparison of spoligotypes from French Guiana with an international spoligotype database (SpolDB4) showed that the majority of isolates belonged to major clades of M. tuberculosis (Haarlem, 22.6%; Latin American-Mediterranean, 23.3%; and T, 32.6%). Indices designed to quantify transmission of tuberculosis gave the following values: RTIn = 0.794, RTIn-1 = 0.651, and TMI = 0.146. CONCLUSION: Our data showed a high number of Mycobacterium tuberculosis clusters, suggesting a high level of recent TB transmission, nonetheless an estimation of transmission rate taking into account cluster size and mutation rate of genetic markers showed a low ongoing transmission rate (14.6%). Our results indicate an endemic mode of TB transmission in French Guiana, with both resurgence of old spatially restricted genotypes, and a significant importation of new TB genotypes by migration of TB infected persons from neighgouring high-incidence countries.
Asunto(s)
Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Tuberculosis/transmisión , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , Guyana Francesa/epidemiología , Genotipo , Humanos , Repeticiones de Minisatélite , Epidemiología Molecular , Mycobacterium tuberculosis/genética , Filogenia , Tuberculosis/epidemiologíaRESUMEN
The present investigation focused on genetic diversity and drug resistance of 101 Mycobacterium tuberculosis strains isolated between July 2003 and February 2005 in the Okinawa prefecture, Ryukyu Islands, Japan. A high rate of clustering (87%, eight clusters, 2 to 69 strains/cluster) was observed upon spoligotyping; most of it was due to the lower discriminatory power of this method for the Beijing lineage (n = 72; 71.3% of the isolates). The remaining diversity was limited to seven clusters (two to five isolates/cluster), with the following distribution of major lineages: ill-defined T (n = 13; 12.8%), ancestral East African-Indian (n = 6; 5.9%), Haarlem (n = 4; 4%), Latin American-Mediterranean (n = 2; 2%), X1 (n = 1; 1%), and a total absence of the central Asian clade. Three remaining strains could not be classified on the basis of their spoligotype pattern and were labeled "unknown." Subtyping with mycobacterial interspersed repetitive units (MIRUs) in association with additional QUB minisatellites was performed to discriminate among the Beijing strains. Based on an "in-house" spoligotyping/MIRU database (n = 694 Beijing strains), eight highly discriminative MIRU loci for Beijing strains were selected (loci numbered 10, 16, 23, 26, 27, 31, 39, and 40). The highest discriminatory power (h) observed in our sample (n = 72; M-26, 0.385; M-10, 0.38; M-31, 0.255; M-16, 0.238) was too low, and 73.6% of the Beijing strains from Okinawa remained clustered. Typing of Beijing strains with additional QUB loci (with the exception of "one-copy" QUB-1451) resulted in higher discriminatory powers: QUB-11b, 0.68; QUB-11a, 0.656; QUB-26, 0.644; QUB-18, 0.553; QUB-4156, 0.5; and QUB-1895, 0.453. A definitive algorithm on the use of QUB markers to subtype Beijing isolates in expanded studies would shed light on their hypervariability, which may sometimes blur recognition between epidemiologically linked Beijing isolates. The total absence of multiple drug resistance among Beijing isolates from Okinawa, as well as the relatively older ages of the patients (majority above 60 years), shows that tuberculosis (TB) is a declining disease in Okinawa, and an adequate TB control program has successfully avoided both the emergence and the spread of multidrug-resistant TB in this insular setting.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Repeticiones de Minisatélite , Mycobacterium tuberculosis/clasificación , Anciano , Anciano de 80 o más Años , Farmacorresistencia Bacteriana , Femenino , Variación Genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
BACKGROUND: Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD). RESULTS: Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. CONCLUSION: This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Recolección de Datos , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Tuberculosis/tratamiento farmacológico , Tuberculosis/epidemiología , Venezuela/epidemiologíaRESUMEN
The present investigation evaluated the PCR-restriction fragment length polymorphism (RFLP) analysis of hsp65 and gyrB targets for differentiation of the species within the Mycobacterium tuberculosis complex (MTC) both by including new restriction enzymes and previously unstudied species. The hsp65 restriction analysis using HhaI resulted in a characteristic 'Mycobacterium canettii' pattern. A study of the gyrB gene polymorphism using TaqIalpha and HinfI allowed the initial division of MTC into two major groups, one consisting of M. tuberculosis and 'M. canettii' as opposed to another single group with other species. Three different patterns were observed with RsaI, the first characteristic of Mycobacterium microti, the second with Mycobacterium bovis, M. bovis BCG and Mycobacterium caprae (M. caprae was easily separated from M. bovis, and M. bovis BCG by SacII digestion), and the third with M. tuberculosis, 'M. canettii', Mycobacterium africanum, Mycobacterium pinnipedii, and the dassie bacillus. Although further discrimination within the last group was not obtained using additional restriction enzymes, the HaeIII and RsaI digestions highlighted an important gyrB polymorphism among 'M. canettii' strains. A study of the single nucleotide polymorphisms (SNP) within the gyrB by sequence analysis not only confirmed the results of the restriction analysis, but showed further differences among 'M. canettii' isolates that were not picked up using the existing battery of restriction enzymes. As many as 11 different SNPs were identified in the collection of eight 'M. canettii' isolates studied. Considering that gyrB variability among MTC member species other than 'M. canettii' is as restricted as hsp65 variability among MTC, our data corroborate a recent proposition that the 'M. canettii' group is evolutionary much older than the other MTC members. In conclusion, gyrB PCR-RFLP is a simple and rapid low-cost method that combined with phenotypic characteristics, may be helpful to differentiate most of the subspecies within the MTC.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Girasa de ADN/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Datos de Secuencia Molecular , Mycobacterium tuberculosis/clasificación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los ResultadosRESUMEN
The three French overseas departments of the Americas are characterized both by insular (Guadeloupe and Martinique) and continental (French Guiana) settings with a tuberculosis case detection rate that varies from less than 10 per 100,000 per year in insular areas to an estimated incidence of more than 55 per 100,000 in French Guiana. Under a long-term genotyping program, more than three-fourths of all the Mycobacterium tuberculosis isolates (n = 744) received from the three settings were fingerprinted over a 10-year period (1994 to 2003) by spoligotyping and variable number of tandem DNA repeats (VNTRs) in order to understand the current trends in their detection rates, drug resistance, and groups and subpopulations at risk of contracting the disease and to pinpoint the circulating phylogeographical clades of the bacilli. The major difference in the study populations was the nationality of the patients, with a high percentage of immigrants from high-incidence neighboring countries in French Guiana and a low but increasing percentage in the French Caribbean. The rate of recent transmission was calculated to be 49.3% in French Guiana, compared to 27.2% and 16.9% in Guadeloupe and Martinique, respectively. At the phylogeographic level, 77.9% of the isolates studied belonged to four major clades (Haarlem, Latin-American and Mediterranean, T, and X) which are already reported from neighboring Caribbean islands in an international database and may underline potential interregional transmission events.