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1.
J Infect Dev Ctries ; 14(8): 878-885, 2020 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-32903232

RESUMEN

INTRODUCTION: Data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in Lebanon are scarce. This study was undertaken to reveal the spoligotypes of MTB isolates recovered from patients in Lebanon. METHODOLOGY: MTB isolates from 49 patients living in Lebanon were recovered and identified. The samples were heat killed and subjected to DNA extraction. Spoligotyping was performed using microbeads from TB-SPOL Kit and the fluorescence intensity was measured using Luminex 200®. Generated patterns were assigned to families using the SITVIT2 international database of the Pasteur Institute of Guadeloupe and compared. RESULTS: The spoligotyping of the 49 MTB isolates revealed that 31 isolates belonged to Lineage 4 (Euro-American, 63.3%), 12 to Lineage 3 (East- African Indian, 24.5%), 3 to Lineage 2 (East Asian, 6%) and 2 were unknown. Over half of the genotypes (16 of 30) harbored SIT127 supposed to belong to the L4.5 sublineage. One isolate belonging to the rare Manu-Ancestor SIT523 was recovered for the first time in Lebanon, being associated with highly virulent extensively drug-resistant (XDR) MTB phenotype. CONCLUSION: The application of the Spoligotyping Multiplex Luminex® method is an efficient, discriminatory and rapid method to use for first-lane genotyping of MTB isolates. Though humble numbers were tested, this study is one of the first to describe the genomic diversity and epidemiology of MTB isolates of Lebanon, and suggests an increasing prevalence of SIT127 in the country.

2.
Int J Infect Dis ; 95: 22-27, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32251801

RESUMEN

BACKGROUND: Patients with mixed-strain Mycobacterium tuberculosis infections may be at a high risk of poor treatment outcomes. However, the mechanisms through which mixed infections affect the clinical manifestations are not well recognized. Evidence suggests that failure to detect the pathogen diversity within the host can influence the clinical results. We aimed to investigate the effects of different genotypes in mixed infections and determine their relationship with heteroresistance in the treatment of Iranian tuberculosis patients. METHODS: One of the genotypes was identified in the culture and another genotype pattern in the mixed infection was predicted by comparing the pattern of MIRU-VNTR between the clinical specimens and their respective cultures in each patient. For all patients, the drug susceptibility testing was carried out on three single colonies from each clinical sample. The follow-up of patients was carried out during six months of treatment. RESULTS: Based on MIRU-VNTR profiles of clinical samples, we showed that 55.6% (25/45) of the Iranian patients included in the study had mixed infections. Patients with mixed infections had a higher rate of treatment failure, compared to others (P=0.03). By comparing clinical sample profiles to profiles obtained after culture, we were able to distinguish between major and hidden genotypes. Among hidden genotypes, Haarlem (L4.1.2) and Beijing (L2) were associated to treatment failure (6/8 patients). CONCLUSIONS: To conclude, we propose a procedure using the MIRU-VNTR method to identify the different genotypes in mixed infections. The present findings suggest that genotypes with potentially higher pathogenicity may not be detected when performing experimental culture in patients with mixed infections.


Asunto(s)
Coinfección/microbiología , Genotipo , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Adulto , Farmacorresistencia Bacteriana , Femenino , Técnicas de Genotipaje , Humanos , Irán , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Resultado del Tratamiento
3.
Tuberculosis (Edinb) ; 120: 101894, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32090855

RESUMEN

The most widely discussed antibiotic-resistant tuberculosis strains ("W" and "B0/W148", "CAO") belong to L2/Beijing Lineage and are characterized by IS6110 insertion sequences at the NTF locus. We present a high-throughput, microbead-based method, called NTF-RINT for detection of IS in NTF and Rifampicin and Isoniazid Typing. This method provides tuberculosis diagnostic confirmation, screens for the so-called modern L2/Beijing sublineage and detects mutations involved in resistance to Rifampicin (RIF) and Isoniazid (INH).

4.
Sci Rep ; 9(1): 15549, 2019 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-31664101

RESUMEN

Tuberculosis remains the world's leading cause of death from an infectious agent, and is a serious health problem in Papua New Guinea (PNG) with an estimated 36,000 new cases each year. This study describes the genetic diversity of Mycobacterium tuberculosis among tuberculosis patients in the Balimo/Bamu region in the Middle Fly District of Western Province in PNG, and investigates rifampicin resistance-associated mutations. Archived Ziehl-Neelsen-stained sputum smears were used to conduct microbead-based spoligotyping and assess genotypic resistance. Among the 162 samples included, 80 (49.4%) generated spoligotyping patterns (n = 23), belonging predominantly to the L2 Lineage (44%) and the L4 Lineage (30%). This is consistent with what has been found in other PNG regions geographically distant from Middle Fly District of Western Province, but is different from neighbouring South-East Asian countries. Rifampicin resistance was identified in 7.8% of the successfully sequenced samples, with all resistant samples belonging to the L2/Beijing Lineage. A high prevalence of mixed L2/L4 profiles was suggestive of polyclonal infection in the region, although this would need to be confirmed. The method described here could be a game-changer in resource-limited countries where large numbers of archived smear slides could be used for retrospective (and prospective) studies of M. tuberculosis genetic epidemiology.

5.
Infect Genet Evol ; 73: 337-341, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31170529

RESUMEN

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.


Asunto(s)
Variación Genética/genética , Mycobacterium tuberculosis/genética , Grupo de Ascendencia Continental Africana/genética , Brasil , Genotipo , Humanos , Madagascar , Mozambique , Países Bajos , Filogenia , Polimorfismo de Nucleótido Simple/genética , Tuberculosis/microbiología
6.
J Microbiol Methods ; 152: 10-17, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29913189

RESUMEN

Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus. Basic analysis can be performed manually. An upgraded interpretation is made available in Excel 2016®. Using a reference set of 61 DNA extracts, we show that TB-EFI provides perfect concordance with pyrosequencing. Concordance between genotypic resistance and phenotypic DST was relatively good (72 to 98% concordance), with lower efficiency for fluoroquinolones and ethambutol due to some untargeted mutations. When compared to phenotypical resistance, performances were similar to those obtained with Hain MTBDRsl assay, possibly thanks to the use of automatized processing of data although some mutations involved in fluoroquinolone resistance could not be included. When applied on three uncharacterized sets, phenotype could be predicted for 51% to 98% depending on the setting and the drug investigated, detecting one extensively drug-resistant isolate in each of a Pakistan and a Brazilian set of 91 samples, and 9 XDR among 43 multi-resistant Kazakhstan samples. By allowing high-throughput detection of second-line drugs resistance and of resistance to ethambutol that is often combined to second-line treatments, TB-EFI is a cost-effective assay for large-scale worldwide surveillance of resistant tuberculosis and XDR-TB control.


Asunto(s)
Antituberculosos/farmacología , Pruebas Diagnósticas de Rutina/métodos , Etambutol/farmacología , Microesferas , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Alelos , Antituberculosos/uso terapéutico , ADN Bacteriano/genética , Fluoroquinolonas/farmacología , Genotipo , Técnicas de Genotipaje , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Microfluídica/métodos , Mutación , Mycobacterium tuberculosis/genética , Pentosiltransferasa , Sensibilidad y Especificidad
7.
Tuberculosis (Edinb) ; 110: 52-55, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29779773

RESUMEN

More and more Single Nucleotide Polymosrphisms of interest among pathogenic organisms are described with the advent of Whole Genome Sequencing but WGS approach is still too expensive, time consuming, and relying on bioinformatical means that are not available in many developing countries. This study presents a low-cost reverse hybridization line probe technique for detecting SNPs in Mycobacterium tuberculosis. The proposed test is able to detect mutations in the RRDR of rpoB gene in M. tuberculosis with specificity and sensitivity of 98% and 100%, respectively and for an average cost of less than €3 per sample. The technique proved efficient not only on pure DNA samples extracted from culture isolates but also on crude extracts from clinical samples. The flexibility of the platform allows to get it transformed to any kind of test detection, hence, building a bridge between rich countries performing SNP discovery and countries with high burden that can target these SNPs on the collected samples.


Asunto(s)
Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Hibridación de Ácido Nucleico/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN
8.
Mem Inst Oswaldo Cruz ; 112(11): 769-774, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29091137

RESUMEN

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.


Asunto(s)
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/microbiología
9.
Mem. Inst. Oswaldo Cruz ; 112(11): 769-774, Nov. 2017. tab
Artículo en Inglés | LILACS | ID: biblio-894852

RESUMEN

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.


Asunto(s)
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 , Genotipo
10.
BMC Infect Dis ; 17(1): 562, 2017 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-28806916

RESUMEN

BACKGROUND: Tuberculosis (TB) remains a public health problem in Madagascar. A crucial element of TB control is the development of an easy and rapid method for the orientation of TB control strategies in the country. Our main objective was to develop a TB spatial hotspot identification method by combining spatial analysis and TB genotyping method in Antananarivo. METHODS: Sputa of new pulmonary TB cases from 20 TB diagnosis and treatment centers (DTCs) in Antananarivo were collected from August 2013 to May 2014 for culture. Mycobacterium tuberculosis complex (MTBC) clinical isolates were typed by spoligotyping on a Luminex® 200 platform. All TB patients were respectively localized according to their neighborhood residence and the spatial distribution of all pulmonary TB patients and patients with genotypic clustered isolates were scanned respectively by the Kulldorff spatial scanning method for identification of significant spatial clustering. Areas exhibiting spatial clustering of patients with genotypic clustered isolates were considered as hotspot TB areas for transmission. RESULTS: Overall, 467 new cases were included in the study, and 394 spoligotypes were obtained (84.4%). New TB cases were distributed in 133 of the 192 Fokontany (administrative neighborhoods) of Antananarivo (1 to 15 clinical patients per Fokontany) and patients with genotypic clustered isolates were distributed in 127 of the 192 Fokontany (1 to 13 per Fokontany). A single spatial focal point of epidemics was detected when ignoring genotypic data (p = 0.039). One Fokontany of this focal point and three additional ones were detected to be spatially clustered when taking genotypes into account (p < 0.05). These four areas were declared potential TB transmission hotspots in Antananarivo and will be considered as priority targets for surveillance in the future. CONCLUSION: This method, combining spatial analysis and TB genotyping will now be used for further focused clinical and epidemiological studies in Madagascar and will allow better TB control strategies by public health authorities.


Asunto(s)
Mycobacterium tuberculosis/genética , Tuberculosis Pulmonar/epidemiología , Variación Genética , Genotipo , Humanos , Madagascar/epidemiología , Mycobacterium tuberculosis/aislamiento & purificación , Análisis Espacial , Esputo/microbiología
11.
Am J Trop Med Hyg ; 97(3): 806-809, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28722603

RESUMEN

We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.


Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Genotipo , Pruebas de Sensibilidad Microbiana/métodos , Tipificación Molecular/métodos , Sensibilidad y Especificidad
12.
Tuberculosis (Edinb) ; 101: 160-163, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27865388

RESUMEN

While minisatellites are usually typed using capillary sequencers or qiaplex systems in developed countries, many low-resource regions cannot afford it. We propose an optimized agarose gel electrophoresis method to genotype Mycobacterium tuberculosis species complex minisatellites in their standardized format (24 MIRU-VNTR). It is based on duplex PCRs combining VNTR loci harboring distinct amplicon sizes whatever the repetition number of each locus. This method performs well both on DNA extracts of good quality and on thermolysates while reducing workload and reagents costs.


Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , ADN Bacteriano/genética , Electroforesis en Gel de Agar/métodos , Técnicas de Genotipaje/métodos , Humanos , Repeticiones de Minisatélite/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Tuberculosis/microbiología
13.
Infect Genet Evol ; 45: 461-473, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27746295

RESUMEN

Two geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny.


Asunto(s)
Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , Tuberculosis/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Epidemias/historia , Epidemias/estadística & datos numéricos , Evolución Molecular , Genotipo , Historia del Siglo XVI , Historia del Siglo XVII , Historia del Siglo XVIII , Historia Antigua , Historia Medieval , Humanos , Japón , Repeticiones de Minisatélite/genética , Epidemiología Molecular , Tipificación Molecular , Filogenia , Polimorfismo de Nucleótido Simple/genética , Tuberculosis/historia , Turquia
14.
BMC Infect Dis ; 15: 306, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26231661

RESUMEN

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.


Asunto(s)
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/microbiología
15.
PLoS One ; 10(7): e0130912, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26154264

RESUMEN

Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.


Asunto(s)
Genómica , Aprendizaje Automático , Mycobacterium tuberculosis/clasificación , Tuberculosis/microbiología , Algoritmos , Técnicas de Tipificación Bacteriana/métodos , Análisis por Conglomerados , Biología Computacional , ADN Bacteriano/genética , Bases de Datos Genéticas , Genotipo , Internet , Epidemiología Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Prevalencia
16.
BMC Infect Dis ; 14: 602, 2014 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-25407589

RESUMEN

BACKGROUND: We aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) isolates in the province of Palermo, Sicily, Italy, by characterizing 183 isolates identified in the years 2004-2012. A comparison with 104 MTBC strains identified in the same geographic area in the years 1994-2000 was also carried out. METHODS: One hundred eighty-three MTBC isolates identified in Palermo, Italy, in the years 2004-2012 were analyzed by spoligotyping and the 24 mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem-repeat (VNTR) method typing. Susceptibility testing to streptomycin, isoniazid, rifampin and ethambutol was also performed. Furthermore, the spoligotyping dataset obtained from 104 MTBC isolates identified from 1994 to 2000 was reanalyzed. Distribution into lineages and clustering of isolates in the two periods was compared. RESULTS: One hundred seventy-seven out of the 183 isolates of MTBC submitted to molecular typing were fully characterized. Of these, 108 were from Italian-born and 69 from foreign-born individuals. Eleven different lineages and 35 families-subfamilies were identified with the most represented lineages being Haarlem (26.5%), T (19.2%), LAM (13.6%) and S (8.5%). Except for the Haarlem lineage, where isolates from foreign-born patients were overrepresented, the distribution of isolates in the families belonging to the Euro-American clone reflected the proportions of the two subpopulations. A total of 27 (15.2%) strains were clustered and three clusters were mixed. Approximately 25% of the 183 MTBC isolates under study proved to be resistant to at least one antiTB drug, with only three isolates categorized as multidrug resistant (MDR). When MTBC isolates identified in the years 1994-2000 were reanalyzed, lineages T (30.8%), LAM (29.8%), Haarlem (16.3%) and S (13.5%) proved to be predominant. No MTBC isolates belonging to CAM, U, CAS, Turkish and Ural lineages were identified. CONCLUSIONS: A wide heterogeneity was detected among the MTBC strains isolated in the years 2004-2012. Six lineages were not present among the isolates of the period 1994-2000. Comparison between distribution of lineages in the two consecutive periods depicts rapid and deep changes in the TB epidemiology in Palermo, Italy. An universal and continued laboratory-based surveillance of TB in Sicily is required.


Asunto(s)
Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Adulto , Antibióticos Antituberculosos/farmacología , Farmacorresistencia Bacteriana/fisiología , Etambutol/farmacología , Femenino , Genotipo , Humanos , Isoniazida/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Repeticiones de Minisatélite , Epidemiología Molecular , Tipificación Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Rifampin/farmacología , Sicilia/epidemiología , Tuberculosis/epidemiología
17.
Infect Genet Evol ; 27: 6-14, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24981519

RESUMEN

Multi-Drug Resistant Tuberculosis (MDR-TB), i.e. bacilli resistant to rifampicin (RIF) and isoniazid (INH), is a major Public Health concern in Pakistan according to WHO estimates (3.5% and 32% of new and retreated cases, respectively). Previous Pakistanis reports identified a correlation between being MDR and belonging to Beijing or EAI lineages in one study, and belonging to "H4"-Ural Euro-American sublineage in another study. In addition, MDR-TB transmission was suspected in Karachi. We tested MDR characteristics on a Punjab sample of 278 clinical isolates (without selection for Multi-Drug Resistance) including new and retreated cases collected from 2008 to 2012. All samples were characterized by a new, microbead-based method named "TB-SPRINT" (molecular diagnostic including spoligotype identification, and genetic resistance determinants to first-line anti-TB drugs RIF and INH). Isolates from 2011 to 2012 (n=100) were further analyzed using 24-loci MIRU-VNTR. We detected 8.7% MDR isolates (CI95%=[5.0; 12.5]), mainly among CAS lineage that predominates in this central-East region of Pakistan. Out of 20 MDR-TB cases, 12 different TB-SPRINT profiles were identified, limiting the suspicion of MDR-TB transmission. 24 MIRU-VNTR confirmed the unrelatedness of isolates with different TB-SPRINT profiles and discriminated 3 isolates with identical TB-SPRINT profiles. In conclusion, our study did not confirm any of the correlations between Multi-Drug Resistance and lineage or sublineage in Punjab, Pakistan. MDR-TB isolates were diverse indicating that transmission is not pervasive. TB-SPRINT proved useful as a first step for detecting MDR-TB likely transmission events, before more extensive genotyping such as 15 or 24 MIRU-VNTR and thorough epidemiological investigation.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Variación Genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/transmisión , Antituberculosos/farmacología , Análisis por Conglomerados , Genotipo , Geografía , Humanos , Pruebas de Sensibilidad Microbiana , Repeticiones de Minisatélite , Tipificación de Secuencias Multilocus , Mutación , Mycobacterium tuberculosis/clasificación , Pakistán/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología
18.
J Clin Microbiol ; 52(7): 2410-5, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24759720

RESUMEN

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.


Asunto(s)
Legionella pneumophila/clasificación , Legionella pneumophila/genética , Microesferas , Tipificación Molecular/métodos , Automatización de Laboratorios , Francia , Ensayos Analíticos de Alto Rendimiento , Humanos , Epidemiología Molecular/métodos
19.
J Clin Microbiol ; 51(11): 3527-34, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23966495

RESUMEN

As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Tipificación Molecular/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Monitoreo Epidemiológico , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Epidemiología Molecular/métodos , Mutación Missense , Mycobacterium tuberculosis/genética , Medicina de Precisión/métodos , Rifampin/farmacología , Sensibilidad y Especificidad
20.
J Clin Microbiol ; 50(10): 3172-9, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22814456

RESUMEN

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.


Asunto(s)
Tipificación Molecular/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico/métodos , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/farmacología , Bulgaria , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana , Genotipo , Alemania , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Microesferas , Mycobacterium tuberculosis/efectos de los fármacos , Nigeria , Oligonucleótidos/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico
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