Pulmonary Nontuberculous Mycobacterial Infections in the State of Para, an Endemic Region for Tuberculosis in North of Brazil

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Federal University of Para. Tropical Medicine Nucleus. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Para. Tropical Medicine Nucleus. Belém, PA, Brazil.Oswaldo Cruz Institute. Oswaldo Cruz Foundation. Rio de Janeiro, RJ, Brazil.Federal University of Para. Department of Integrative Medicine. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil

Método para a predição do desfecho do tratamento de doenças humanas utilizando fármacos metabolizados pela N-acetiltransferase 2 humana (NAT2) com base em polimorfismos genéticos

Em 04/01/2016: Anuidade de pedido de patente de invenção no prazo ordinário.DepositadaMétodo para a predição do desfecho do tratamento de doenças humanas utilizando fármacos metabolizados pela N- acetiltransferase 2 humana (NAT2) com base em polimorfismos genéticos. A presente invenção está relacionada ao campo da Biologia Molecular e Genâmica, especialmente a Farmacogenâmica. A invenção descreve a presença de novos polimorfismos no gene que codifica para a enzima Arilamina N-acetiltransferase 2 humana (NAT2), a qual é responsável pela metabolização de fármacos importantes na terapêutica de várias doenças de etiologias diversas, bem como de inúmeras toxinas e carcinógenos presentes em alimentos, cigarro e no ambiente. A presente invenção inclui metodologia passível de utilização na terapêutica utilizando os polimorfismos descritos

Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

International audienceRio 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

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America

Background Mutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains. Results We, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH ≥2 μg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families. Conclusion Our data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.Fil: Dalla Costa, Elis R. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Ribeiro, Marta O. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Silva, Márcia S. N. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Arnold, Liane S. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Rostirolla, Diana C. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Cafrune, Patricia I. State Foundation for Production and Research in Health (FEPPS); Brasil.Fil: Espinoza, Roger C. Blufstein Clinic Laboratory; Perú.Fil: Palaci, Moises. Federal University of Espírito Santo; Brasil.Fil: Telles, Maria A. Adolfo Lutz Institute; Brasil.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Micobacterias; Argentina.Fil: Suffys, Philip N. Oswaldo Cruz Institute; Brasil.Fil: Lopes, Maria L. Evandro Chagas Institute; Brasil.Fil: Campelo, Creuza L. LACEN Ceará; BrasilFil: Miranda, Silvana S. Federal University of Minas Gerais; Brasil.Fil: Kremer, Kristin. National Institute for Public Healthand the Environment (RIVM). Mycobacteria Reference Unit (CIb-LIS); Países Bajos.Fil: Almeida da Silva, Pedro E. Federal Foundation of Rio Grande; Brasil.Fil: de Souza Fonseca, Leila. Federal University of Rio de Janeiro. Tuberculosis Academic Program; Brasil.Fil: Ho, John L. Cornell University; Estados Unidos.Fil: Kritski, Afrânio L. Federal University of Rio de Janeiro. Tuberculosis Academic Program; Brasil.Fil: Rossetti, María L. R. State Foundation for Production and Research in Health (FEPPS); Brasil

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

Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation

Human N-acetyltransferase 2 (NAT2) gene variability in Brazilian populations from different geographical areas

Introduction: Several polymorphisms altering the NAT2 activity have already been identified. The geographical distribution of NAT2 variants has been extensively studied and has been demonstrated to vary significantly among different ethnic population. Here, we describe the genetic variability of human N-acetyltransferase 2 (NAT2) gene and the predominant genotype-deduced acetylation profiles of Brazilians.Methods: A total of 964 individuals, from five geographical different regions, were genotyped for NAT2 by sequencing the entire coding exon.Results: Twenty-three previously described NAT2 single nucleotide polymorphisms (SNPs) were identified, including the seven most common ones globally (c.191G>A, c.282C>T, c.341T>C, c.481C>T, c.590G>A, c.803A>G and c.857G>A). The main allelic groups were NAT2*5 (36%) and NAT2*6 (18.2%), followed to the reference allele NAT2*4 (20.4%). Combined into genotypes, the most prevalent allelic groups were NAT2*5/*5 (14.6%), NAT2*5/*6 (11.9%) and NAT2*6/*6 (6.2%). The genotype deduced NAT2 slow acetylation phenotype was predominant but showed significant variability between geographical regions. The prevalence of slow acetylation phenotype was higher in the Northeast, North and Midwest (51.3%, 45.5% and 41.5%, respectively) of the country. In the Southeast, the intermediate acetylation phenotype was the most prevalent (40.3%) and, in the South, the prevalence of rapid acetylation phenotype was significantly higher (36.7%), when compared to other Brazilian states (p < 0.0001). Comparison of the predicted acetylation profile among regions showed homogeneity among the North and Northeast but was significantly different when compared to the Southeast (p = 0.0396). The Southern region was significantly different from all other regions (p < 0.0001).Discussion: This study contributes not only to current knowledge of the NAT2 population genetic diversity in different geographical regions of Brazil, but also to the reconstruction of a more accurate phenotypic picture of NAT2 acetylator profiles in those regions