RESUMO
Buruli ulcer is an emerging and neglected tropical disease caused by Mycobacterium ulcerans. Few cases have been reported so far in the Americas. With 250 cases reported since 1969, French Guiana is the only Buruli ulcer endemic area in the continent. Thus far, no genetic diversity studies of strains of M. ulcerans from French Guiana have been reported. Our goal in the present study was to examine the genetic diversity of M. ulcerans strains in this region by using the Multilocus Variable Number Tandem Repeat Analysis (MLVA) approach. A total of 23 DNA samples were purified from ulcer biopsies or derived from pure cultures. MVLA was used in the study of six previously-described Variable Number of Tandem Repeat (VNTR) markers. A total of three allelic combinations were characterized in our study: genotype I which has been described previously, genotype III which is very similar to genotype I, and genotype II which has distinctly different characteristics in comparison with the other two genotypes. This high degree of genetic diversity appears to be uncommon for M. ulcerans. Further research based on complete genome sequencing of strains belonging to genotypes I and II is in progress and should lead soon to a better understanding of genetic specificities of M. ulcerans strains from French Guiana.
Assuntos
Heterogeneidade Genética , Repetições Minissatélites , Mycobacterium ulcerans/genética , Sequência de Bases , DNA , Guiana Francesa , Humanos , Dados de Sequência Molecular , Mycobacterium ulcerans/classificação , Filogenia , Homologia de Sequência do Ácido NucleicoRESUMO
In this study we used spoligotyping and 15-loci MIRU-VNTRs for a finer characterization of Mycobacterium tuberculosis strains isolated from patients residing in Guyana (n=74) and Suriname (n=80). The mean age of the patients was 38.5years (36.5 and 40.2years for Guyana vs. Suriname), with a male-to-female sex-ratio of 2.25 for Guyana vs. 4.27 in Suriname. Spoligotyping and 15-loci MIRU-VNTRs led to a total of 41 and 65 different patterns respectively, with an overall clustering rate of 83.8% vs. 68.8%. Combined spoligotyping and VNTR analysis led to the detection of 18 clusters of 2-41 isolates, with an overall clustering of 67.5% and a recent "n-1" transmission rate of 55.8%. Importantly, Guyana was characterized by a significantly higher percentage of clustered isolates than Suriname (79.7% vs. 56.3%; p=0.0019). Three big spoligo/MIRU (SIT/MIT) clusters containing >10 isolates each were shared between the 2 countries, and concerned: T1 sublineage cluster 53/861 (n=41, 37 in Guyana vs. 4 in Suriname); EAI6-BGD1 sublineage cluster 1340/860 (n=16, 3 in Guyana vs. 13 in Suriname); and T1 sublineage cluster 131/146 (n=11, 6 in Guyana vs. 5 in Suriname); as well as 2 smaller clusters of 2 and 3 isolates respectively. However, the relative phylogeographical specificities of strains in circulation as well as a lack of drug-resistance observed among strains from Suriname suggested that trans-border transmission of drug-resistant isolates occurred less frequently than thought. Tracing and interrupting transmission channels of a specific clone (SIT53/15-MIT861) should become a priority in Guyana, not only because it is by far most abundant but also because it accounts for almost half of the drug resistant isolates (n=8/17, 47.1%) in our study, and clustered 5/12 (41.7%) MDR isolates.
Assuntos
Sequências Repetitivas Dispersas/genética , Repetições Minissatélites/genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Análise por Conglomerados , Feminino , Guiana/epidemiologia , Humanos , Masculino , Tipagem Molecular , Filogeografia , Suriname/epidemiologia , Tuberculose/epidemiologiaRESUMO
INTRODUCTION: The advent of molecular typing using MIRU-VNTR mini-satellites has largely facilitated tuberculosis (TB) molecular epidemiological studies. Apart from detecting the chains of transmission and risk factors, these markers have also allowed to study the phenomena of mixed strain infections versus microevolutionary events. METHODS: An initial set of Mycobacterium tuberculosis strains (n=161) genotyped using spoligotyping and MIRU-VNTRs in Guyana and Suriname was evaluated for indications mixed strain infections (characterized by the detection of double alleles in 2 or more MIRU loci) versus "in-patient" microevolutionary events (characterized by the detection of double alleles in a single locus). RESULTS: The present study hereby reports evidence of microevolution in 3.7% (n=6/161) of the studied population, vs. 0.6% (n=1/161) for mixed infection. The strains belonged to three different spoligotyping-based lineages, namely the T (SITs 44, 53, and 1081), Haarlem (SIT47), and EAI (SITs 72 and 349) lineages, while 1 isolate (SIT237) could not be assigned to any lineage. DISCUSSION: By comparing these results on microevolutionary cases (n=6) to 112,000 strains present in the SITVIT2 database, evidence is presented that in 2/6 cases (each case corresponding to 2 patterns due to MIRU double bands), one of the patterns corresponded to a shared type found exclusively in Suriname or Guyana. Phylogenetic analysis showed that no spoligotyping lineage in particular was more prone to microevolutionary events in this study's sample. Overall, the observations fortify the awareness regarding the existence of microevolution and polyclonal TB infections which has important implications for patient care.
Assuntos
Técnicas de Tipagem Bacteriana , Evolução Molecular , Repetições Minissatélites , Epidemiologia Molecular , Tipagem Molecular , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Feminino , Guiana/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Suriname/epidemiologia , Tuberculose/microbiologia , Adulto JovemRESUMO
BACKGROUND: The present study aimed to characterize Mycobacterium tuberculosis population structure and to identify transmission chains and risk factors by prospective molecular typing in conjunction with conventional epidemiological investigations in the French overseas department of Guadeloupe. METHODS: The study included all the culture-positive TB cases (1 clinical isolate per patient; n = 129) diagnosed between a seven year period (April 4th, 1999 to December 31st, 2005). Prospective molecular typing was performed using spoligotyping and VNTRs, and a subset of 44 M. tuberculosis isolates found to be clustered was retrospectively typed using 12-loci MIRUs. Data were compared using the SITVIT2 database, followed by analysis of risk factors in function of clustering of the isolates and available demographic and socioeconomic data. RESULTS: The study sample was characterized by a majority of new cases (87.4%); a moderate proportion of drug-resistance (7.8%); a high level of immigration (51.2% foreign-born) originating from high TB/HIV incidence neighboring islands such as Haiti or Dominican Republic; lower socioeconomic conditions (70.7% of jobless, average income 824 EUR/month); and a significantly higher proportion of TB/HIV co-infected cases (38.2% vs. 8.5%; p < 0.001), and extrapulmonary disease (18.2% vs. 4.8%; p < 0.02) among migrants as compared to French patients. The study revealed an important delay in access to healthcare with a median delay of 74.5 days between the 1st symptoms and clinical suspicion of TB. Prospective molecular typing based on spoligotyping and 5-loci VNTRs showed that evolutionary recent Euro-American lineages predominated in Guadeloupe (91.5% of isolates). In conjunction with epidemiological data, it allowed to estimate a recent transmission rate of 18.6%, which was close to the rate of 16.7% estimated using retrospective 12-loci MIRU typing. Although a higher proportion of cases in older age-group were apparently linked to reactivation; univariate analysis of risk factors did not allow pinpointing specific risk factors for a patient to belong to a TB transmission group. CONCLUSIONS: Ongoing TB transmission in the insular, low TB-incidence setting of Guadeloupe can be defined as follows: (i) a significant proportion of imported cases of the disease from neighboring islands; (ii) significantly higher TB/HIV coinfection among foreign-born cases; and, (iii) a higher proportion of cases affecting older age-group among French patients due to reactivation. This study emphasizes the need for universal typing using spoligotyping and 15-loci MIRUs in prospective studies.
Assuntos
Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Adolescente , Adulto , Criança , Pré-Escolar , Análise por Conglomerados , Farmacorresistência Bacteriana , Feminino , Genótipo , Guadalupe/epidemiologia , Acessibilidade aos Serviços de Saúde , Humanos , Incidência , Lactente , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites , Tipagem Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Estudos Prospectivos , Estudos Retrospectivos , Fatores de Risco , Tuberculose/epidemiologia , Adulto JovemRESUMO
Nontuberculous mycobacteria (NTM) are ubiquitous environmental organisms able to cause severe opportunistic human infections. Their distribution patterns are subject to geographical variations. This study describes their isolation frequencies from clinical specimen in the three French overseas departments of the Americas, namely, Guadeloupe, Martinique, and French Guiana during 1994-2012. A total of 651 strains from as many patients (one isolate per species per patient) were analysed regarding regional isolation patterns and potential pattern changes over time. The Mycobacterium avium complex was the most common group of NTM in Guadeloupe and French Guiana. In Martinique it was the second most common after the rapidly growing mycobacteria. M. fortuitum was the most commonly isolated species in all three departments. Some species (M. kansasii, M. xenopi, and M. terrae complex) displayed a clear regional preference. Furthermore a change in isolation frequency was observed for M. intracellulare (increase) and M. kansasii (decrease) in Guadeloupe. In conclusion, marked regional differences in isolation frequencies of NTM species were observed in the study area. Results are discussed in context of variables such as study populations, risk factors, methodology employed, isolation from pulmonary versus sterile isolation sites (blood, urine, and CSF), and in vitro drug-susceptibility patterns.
RESUMO
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.
Assuntos
Bases de Dados de Ácidos Nucleicos , Variação Genética , Mycobacterium tuberculosis/genética , Técnicas de Tipagem Bacteriana , Biologia Computacional/métodos , DNA Bacteriano , Genótipo , Humanos , Internet , Repetições Minissatélites , Tipagem de Sequências Multilocus , Mycobacterium tuberculosis/classificação , Filogeografia , Software , Tuberculose/epidemiologiaRESUMO
Mycobacterium tuberculosis Beijing lineage is highly prevalent in Japan. The aim of the present study was to describe the population structure of the Beijing lineage in this country based on 12-, 15-, and 21-loci MIRU-VNTR genotyping schemes. The MIRU-VNTR patterns of Beijing strains from Okinawa, Ryukyu Islands were compared to those recently published from the Osaka-Kobe megalopolis of the main island of Japan, Honshu (Wada et al., 2009). We also compared proportions of "modern/typical" vs. "ancient/atypical" Beijing strains as defined by structure of the NTF locus. Contrarily to the 12-loci Minimum Spanning Tree (MST), the 15- and 21-loci trees allowed the distinction of two groups of strains in Okinawa. A 12-loci MIRU-VNTR pattern (223325173533) corresponding to MIRU international type MIT17 was identified as the most prevalent Beijing genotype in Japan. In the SITVIT2 database, this pattern was found to be disseminated worldwide and corresponded to the most widely distributed Beijing profile in East Asia and former USSR countries. A comparison of 15- and 21-loci MIRU-VNTR patterns showed that two loci (QUB-4156 and Mtub21) were most polymorphic in our study, and could be potential candidates to distinguish between NTF locus based subclassification of Beijing strains. High-resolution VNTR typing using 15- and 21-loci underlined an evolutionarily distinct "ancient/atypical" subpopulation of the Beijing genotype in Okinawa as well as a subgroup of strains closely related to "modern/typical" Beijing strains observed in Osaka/Kobe.
Assuntos
Técnicas de Tipagem Bacteriana , Genótipo , Tipagem de Sequências Multilocus , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , DNA Bacteriano , Humanos , Japão , Repetições Minissatélites , Mycobacterium tuberculosis/isolamento & purificação , FilogeniaRESUMO
This study shows the benefit of spoligotyping coupled to mycobacterial interspersed repetitive-unit (MIRU) typing to pinpoint circulating Mycobacterium tuberculosis genotypes in Guadeloupe, Martinique, and French Guiana. We hereby propose reduced 4-locus and 6-locus subsets for LAM and Haarlem lineage strains that predominate in South America and the Caribbean, retaining 99.35% and 99.64% of the total discriminatory power of the 12-locus scheme, respectively.
Assuntos
Tipagem Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Bacteriano/genética , Guiana Francesa/epidemiologia , Genótipo , Guadalupe/epidemiologia , Humanos , Masculino , Martinica/epidemiologia , Pessoa de Meia-Idade , Epidemiologia Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Adulto JovemRESUMO
We report on a fine molecular and phylogenetical characterization of circulating Mycobacterium tuberculosis strains isolated from patients during a 1-year period in Trinidad and Tobago (T&T). The spoligotyping data coupled to minisatellite typing and available epidemiological data showed that a single major clone of "evolutionary modern" tubercle bacilli (SIT566) was responsible for more than half of the tuberculosis (TB) cases. It preferentially infected younger age groups (mean 39.1 years versus 47.7 years for other genotypes, p<0.0005), and was overrepresented in Port-of-Spain (1 out of 3 patients). A comparison of genotyping results to data gathered for 6 Caribbean countries (n=2653 clinical isolates) showed that the overall lineage distribution in T&T was completely different from its neighbors, e.g., T&T was the only country harboring a unique sublineage of the Latin American & Mediterranean (LAM) family, designated LAM-10CAM with phylogeographical specificity for Cameroon and neighboring countries in West Africa; interestingly 3/4 of the patients within this group in T&T were African descendants. Similarly, strains belonging to East African Indian (EAI) lineage with phylogeographical specificity for the Indian subcontinent, were found in T&T (13% of all strains), but were absent among the neighboring countries. Although the predominant SIT566 was not yet detected elsewhere in the Caribbean, available information underlined that this genotype was already present in the United States as imported cases of disease among T&T-born patients. Characterization of SIT566 strains using 12-, 15- and 24-loci MIRU typing, and comparison of results to international databases showed that these isolates were characterized by a common 12-loci MIRU pattern 224315153324 corresponding to MIRU International Type-MIT633 in 21/25 strains tested, as well as its 4 variants; an orphan pattern , MIT27-, MIT117-, and MIT1158-. Extended 24-loci MIRU typing led to a predominant pattern 224315153324323483334323 in a total of 16/21 MIT633 isolates, as well as identification of 3 supplemental patterns. Comparison of 24-loci MIRU data with the international database MIRU-VNTRplus showed the unique nature of the patterns obtained in T&T. Further analysis using the Levenshtein algorithm showed that the first 2 closest matches with the SIT566/MIT633 clone belonged to the X lineage strains in MIRU-VNTRplus. This observation corroborates our preliminary spoligotyping-based analysis using minimum spanning and neighbor-joining trees, which suggested a phylogenetical relatedness of the SIT566 clone with SIT119, which represents X1 lineage prototype in SpolDB4 database. We hypothesize that the predominant SIT566 clone might have evolved from a pool of X lineage M. tuberculosis strains with phylogeographical affinity for Anglo-Saxon descendants.
Assuntos
Geografia , Mycobacterium tuberculosis/genética , Filogenia , Tuberculose Pulmonar/epidemiologia , Adulto , Fatores Etários , Idoso , DNA Bacteriano/análise , DNA Bacteriano/genética , Evolução Molecular , Variação Genética , Genoma Bacteriano , Infecções por HIV/complicações , Interações Hospedeiro-Patógeno , Humanos , Incidência , Sequências Repetitivas Dispersas , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Fatores Sexuais , Trinidad e Tobago/epidemiologia , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/microbiologiaRESUMO
This report is based on a 1-year recruitment of all of the culture-positive Mycobacterium tuberculosis cases in Trinidad and Tobago (n = 132). The study population was characterized by a high male-to-female sex ratio of 4 and a human immunodeficiency virus-tuberculosis (TB) coinfection rate of 30 per cent. It mainly occurred among African descendants, who represent 37.5 per cent of the total population but 69.7 per cent of all TB cases (P < 0.001). Spoligotyping resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster), with the predominance of a highly conserved spoligotype international type clone, SIT566.
Assuntos
Humanos , Mycobacterium tuberculosis , Epidemiologia , Variação Genética , Trinidad e TobagoRESUMO
This report is based on a 1-year recruitment of all of the culture-positive Mycobacterium tuberculosis cases in Trinidad and Tobago (n = 132). The study population was characterized by a high male-to-female sex ratio of 4 and a human immunodeficiency virus-tuberculosis (TB) coinfection rate of 30%. It mainly occurred among African descendants, who represent 37.5% of the total population but 69.7% of all TB cases (P < 0.001). Spoligotyping resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster), with the predominance of a highly conserved spoligotype international type clone, SIT566.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/epidemiologia , Adolescente , Adulto , Idoso , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , Etnicidade , Feminino , Variação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Fatores Sexuais , Trinidad e Tobago/epidemiologia , Adulto JovemRESUMO
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.