Genetic characterization of Mycobacterium tuberculosis complex isolates circulating in Abuja, Nigeria

WOS:000446783800001Objective: Nigeria ranks fourth among the high tuberculosis (TB) burden countries. This study describes the prevalence of drug resistance and the genetic diversity of Mycobacterium tuberculosis in Abuja's Federal Capital Territory. Materials and methods: Two hundred and seventy-eight consecutive sputum samples were collected from adults with presumptive TB during 2013-2014. DNA was extracted from Lowenstein-Jensen cultures and analyzed for the identification of nontuberculous mycobacteria species, detection of drug resistance with line probe assays, and high-throughput spacer oligonucleotide typing (spoligotyping) using microbead-based hybridization. Results: Two hundred and two cultures were positive for M. tuberculosis complex, 24 negative, 38 contaminated, and 15 positive for nontuberculous mycobacteria. Five (2.5%)M. tuberculosis complex isolates were resistant to rifampicin (RIF) and isoniazid (multidrug resistant), nine (4.5%) to RIF alone, and 15 (7.4%) to isoniazid alone; two RIF-resistant isolates were also resistant to fluoroquinolones and ethambutol, and one multidrug resistant isolate was also resistant to ethambutol. Among the 180 isolates with spoligotyping results, 164 (91.1%) were classified as lineage 4 (Euro-American), 13 (7.2%) as lineage 5 (West African 1), two (1.1%) as lineage 2 (East Asia), and one (0.6%) as lineage 6 (West African 2). One hundred and fifty-six (86.7%) isolates were grouped in 17 clusters (2-108 isolates/cluster), of which 108 (60.0%) were grouped as L4.6.2/Cameroon (spoligotype international type 61). Conclusion: The description of drug resistance prevalence and genetic diversity of M tuberculosis in this study may be useful for improving TB control in Nigeria

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Fil: Abadia, Edgar. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Zhang, Jian. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Kremer, Kristin. National Institute for Public Health and the Environment; Paises Bajos.Fil: Ruimy, Raymond. Université Paris- Diderot & Microbiology Laboratory; Francia.Fil: Rigouts, Leen. Prince Leopold Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Gomes, Harrison Magdinier. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Elias, Atina Ribeiro. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Fauville-Dufaux, Maryse. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; Bélgica.Fil: Stoffels, Karolien. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; Bélgica.Fil: Rasolofo-Razanamparany, Voahangy. Institut Pasteur de Madagascar. Unité des Mycobactéries; Madagascar.Fil: Garcia de Viedma, Dario. Hospital Gregorio Marañón. Servicio de Microbiología Clínica y Enfermedades Infecciosas; España.Fil: Herranz, Marta. Hospital Gregorio Marañón. Servicio de Microbiología Clínica y Enfermedades Infecciosas; España.Fil: Al-Hajoj, Sahal. King Faisal Specialist Hospital and Research Center. Department of Comparative Medicine; Arabia Saudita.Fil: Rastogi, Nalin. Institut Pasteur de Guadeloupe. Unité de la Tuberculose et des Mycobactéries - WHO Supranational TB Reference Laboratory; Guadalupe.Fil: Garzelli, Carlo. Università di Pisa. Dipartimento di Patologia Sperimentale Biotecnologie Mediche Infettivologia ed Epidemiologia; Italia.Fil: Tortoli, Enrico. Careggi Hospital. Regional Reference Center for Mycobacteria; ItaliaFil: Suffys, Philip N. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: van Soolingen, Dick. National Institute for Public Health and the Environment; Paises Bajos.Fil: Refregier, Guislaine. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Sola, Christophe. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Contains fulltext : 124321.pdf (publisher's version ) (Open Access)BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping

Identificação molecular de cepas do complexo Mycobacterium tuberculosis e detecção de resistência a antibióticos

Submitted by Repositório Arca ([email protected]) on 2019-07-02T18:45:11Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) harrison_gomes_ioc_mest_2006.pdf: 1819113 bytes, checksum: 6d2f4ef165bf7e152724d58df79c3647 (MD5)Approved for entry into archive by Raquel Dinelis ([email protected]) on 2019-09-12T18:37:29Z (GMT) No. of bitstreams: 2 harrison_gomes_ioc_mest_2006.pdf: 1819113 bytes, checksum: 6d2f4ef165bf7e152724d58df79c3647 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-09-12T18:37:29Z (GMT). No. of bitstreams: 2 harrison_gomes_ioc_mest_2006.pdf: 1819113 bytes, checksum: 6d2f4ef165bf7e152724d58df79c3647 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A tuberculose mata aproximadamente 3 milhões de pessoas no mundo a cada ano. A pobreza, a falta de informação, a demora no diagnóstico de cepas resistentes e multi-resistentes e fatores de risco associados à infecção por Mycobacterium tuberculosis são os principais responsáveis pela manutenção de sua transmissão. Em nosso estudo, utilizamos duas abordagens moleculares, baseadas na reação de PCR, \201CSingle Strand Conformational Polymorphism-SSCP\201D e \201CReverse Line Blot Hybridization\201D-RLBH, para verificarmos genótipos mutantes e a freqüência de mutações no genoma de M. tuberculosis associadas à resistência a drogas, assim como avaliarmos a RLBH para a detecção rápida de resistência. Utilizando o SSCP, verificamos que a versão radioativa apresentou uma melhor resolução que a não radioativa. Na versão não-radioativa uma discordância foi observada (9,1%) ao comparar os resultados com a análise por sequenciamento. Na RLBH, uma membrana contendo sondas para detecção de resistência a: rifampicina, isoniazida, estreptomicina e etambutol foi avaliada em um estudo multi-cêntrico e 50% das cepas resistentes a rifampicina apresentavam mutações em rpoB nos códons 531-TTG (43%) e 526-GTC (7%) enquanto que 49% das cepas resistentes a etambutol apresentavam mutações no códon 306-GTG (34%) e ATA (14%) Uma segunda versão da RLBH, para detecção de resistência a rifampicina com sondas específicas para o gene rpoB foi avaliada. Analisamos 50 cepas de Mycobacterium Bovis, sensíveis e 157 cepas de M. tuberculosis sensíveis e resistentes a rifampicina. Todas as cepas de M. bovis apresentaram o genótipo de sensível e entre as cepas resistentes de M. tuberculosis, 93% apresentaram mutações na região de 157pb do gene rpoB. O Inno-LiPA (teste comercial), baseado em RLBH, para detecção de cepas de M. tuberculosis resistente a rifampicina, também foi analisado, apresentando uma sensibilidade de 97,6%.Concluímos que a hibridação reversa tem a sensibilidade adequada para detectar cepas de M. tuberculosis resistentes a rifampicina e que os mecanismos relacionados à resistência a estreptomicina e isoniazida necessitam de uma melhor compreensão. Na parte deste estudo referente à variabilidade genética de cepas brasileiras de M. tuberculosis, avaliamos a composição de uma região especifica do Complexo M. tuberculosis, a \201CDirect repeat\201D (DR), pela técnica de \201Cspoligotyping\201D Através desta técnica determinamos os genótipos de cepas de M. tuberculosis, de pacientes, provenientes de 11 estados brasileiros e os comparamos com \201Cspoligotypes\201D de 122 países depositados no Banco de Dados Internacional de \201Cspoligotypes\201D. 80% das cepas brasileiras pertencem às famílias dos \201Cspoligotypes\201D \201CLatin-American and Mediterranean\201D (LAM; 46,5%), T (19,7%) and Haarlem (13,3 %). 302 \201Cspoligotypes\201D novos estão distribuídos entre todas as famílias. Dentro da família LAM, uma deleção denominada de RDRio de 26 kB foi detectada e esta deleção está relacionada a 30% dos casos de TB na cidade do Rio de Janeiro. A distribuição de cepas de M. tuberculosis no Brasil parece estar relacionada à colonização brasileira e ao moderno fluxo migratório.Each year, tuberculosis kills, approximately, 3 million people worldwide. Poverty, lack of knowledge (information), late diagnosis of resistant and multi-resistant strains and risk factors associated to Mycobacterium tuberculosis infection are the main reasons (principally responsible) for the maintenance of the (ongoing) transmission. In our study, two molecular approaches, based on PCR amplification, have been performed, “Single Strand Conformational Polymorphism - SSCP” and “Reverse Line Blot Hybridization - RLBH”, for the identification of mutant genotypes and the frequency of mutations present in M. tuberculosis genome associated to drug resistance, as well as the evaluation of RLBH for the rapid detection of resistance. The radioactive version of SSCP presented a better resolution than the non radioactive version. In the non-radioactive version, discordance of 9.1% was observed when the results were compared to those obtained by sequencing. Concerning the RLBH, a membrane containing probes designed to detect resistance to: rifampin, isoniazid, streptomycin and ethambutol was evaluated in a multi-center study and 50% of the rifampin resistant strains showed mutations in rpoB in codons 531-TTG (43%) and 526-GTC (7%), while 49% of the resistant strains to ethambutol presented mutations in codons 306-GTG (34%) and ATA (14%) from embB gene. A second version of RLBH, designed for the detection of rifampin resistance by specific probes for the rpoB gene, was evaluated. Fifty sensitive strains of Mycobacterium bovis and 157 sensitive and rifampin-resistant strains of M. tuberculosis were analyzed. All M. bovis strains presented a sensitive genotype, while 93% of the M. tuberculosis resistant strains presented mutations in the 157 bp region of the rpoB gene. The Inno-LiPA (commercial test), based in the RLBH for the detection of strains resistant to rifampin, was also analyzed presenting a sensitivity of 97.6%. We have concluded that the RLBH has an adequate sensitivity to detect rifampin-resistant strains and that the mechanisms involved in resistance to streptomycin and isoniazid need a better understanding. Concerning the part of this study related to the genotypic variability of the Brazilian strains of M. tuberculosis, we have evaluated the composition of the M. tuberculosis complex specific region, the “Direct repeat” (DR) locus, by the spoligotyping technique. Through this technique, we have determined the genotypes of M. tuberculosis strains from patients attended in 11 Brazilian states, which were compared to spoligotypes from 122 countries deposited in the International Database of Spoligotypes. Eighty percent of the Brazilian strains belong to the spoligotyping families “Latin-American and Mediterranean” (LAM; 46.5%), T (19.7%) and Haarlem (13.3%). Three-hundred-and-two new spoligotypes are distributed in all families. Within the LAM family, a deletion called RDRio was detected and it is related to 30% of TB cases in the city of Rio de Janeiro. The distribution of strains of M. tuberculosis in Brazil seems to be related to the Brazilian colonization and the modern migratory flux

Método para identificação de cepas resistentes de Mycobacterium tuberculosis e kit de diagnóstico/prognóstico de tuberculose

Universidade Federal do Rio Grande do SulFundação Estadual de Produção e Pesquisa em Saúde (RS)Ciências BiológicasDepositad

Método para identificação de cepas resistentes de Mycobacterium tuberculosis e kit de diagnóstico/prognóstico de tuberculose

Universidade Federal do Rio Grande do SulFundação Estadual de Produção e Pesquisa em Saúde (RS)Ciências BiológicasDepositad

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis