Homozygotes NAT2*5B slow acetylators are highly associated with hepatotoxicity induced by anti-tuberculosis drugs.

BACKGROUND: Distinct N-acetyltransferase 2 (NAT2) slow acetylators genotypes have been associated with a higher risk to develop anti-tuberculosis drug-induced hepatotoxicity (DIH). However, studies have not pointed the relevance of different acetylation phenotypes presented by homozygotes and compound heterozygotes slow acetylators on a clinical basis. OBJECTIVES: This study aimed to investigate the association between NAT2 genotypes and the risk of developing DIH in Brazilian patients undergoing tuberculosis treatment, focusing on the discrimination of homozygotes and compound heterozygotes slow acetylators. METHODS/FINDINGS: The frequency of NAT2 genotypes was analysed by DNA sequencing in 162 patients undergoing tuberculosis therapy. The mutation analyses revealed 15 variants, plus two new NAT2 mutations, that computational simulations predicted to cause structural perturbations in the protein. The multivariate statistical analysis revealed that carriers of NAT2*5/*5 slow acetylator genotype presented a higher risk of developing anti-tuberculosis DIH, on a clinical basis, when compared to the compound heterozygotes presenting NAT2*5 and any other slow acetylator haplotype [aOR 4.97, 95% confidence interval (CI) 1.47-16.82, p = 0.01]. CONCLUSION: These findings suggest that patients with TB diagnosis who present the NAT2*5B/*5B genotype should be properly identified and more carefully monitored until treatment outcome in order to prevent the occurrence of anti-tuberculosis DIH.

Homozygotes NAT2*5B slow acetylators are highly associated with hepatotoxicity induced by anti-tuberculosis drugs

BACKGROUND Distinct N-acetyltransferase 2 (NAT2) slow acetylators genotypes have been associated with a higher risk to develop anti-tuberculosis drug-induced hepatotoxicity (DIH). However, studies have not pointed the relevance of different acetylation phenotypes presented by homozygotes and compound heterozygotes slow acetylators on a clinical basis. OBJECTIVES This study aimed to investigate the association between NAT2 genotypes and the risk of developing DIH in Brazilian patients undergoing tuberculosis treatment, focusing on the discrimination of homozygotes and compound heterozygotes slow acetylators. METHODS/FINDINGS The frequency of NAT2 genotypes was analysed by DNA sequencing in 162 patients undergoing tuberculosis therapy. The mutation analyses revealed 15 variants, plus two new NAT2 mutations, that computational simulations predicted to cause structural perturbations in the protein. The multivariate statistical analysis revealed that carriers of NAT2*5/*5 slow acetylator genotype presented a higher risk of developing anti-tuberculosis DIH, on a clinical basis, when compared to the compound heterozygotes presenting NAT2*5 and any other slow acetylator haplotype [aOR 4.97, 95% confidence interval (CI) 1.47-16.82, p = 0.01]. CONCLUSION These findings suggest that patients with TB diagnosis who present the NAT2*5B/*5B genotype should be properly identified and more carefully monitored until treatment outcome in order to prevent the occurrence of anti-tuberculosis DIH.

Exploring the parasite load and molecular diversity of Trypanosoma cruzi in patients with chronic Chagas disease from different regions of Brazil.

Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and parasite load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The parasite load median for all positive patients was 2.54 [1.43-11.14] parasite equivalents/mL (par. Eq./mL), with the load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the parasite load was significantly higher in patients over 70 years old (median 20.05 [18.29-86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII in general (parasite load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher parasitemia levels.

ACTN3 gene variants as potential phenotype and performance biomarkers in Brazilian sport horses training for eventing in a tropical climate.

The aim of this study was to look for mutations in the equine ACTN3 gene and to identify sequence variants that might be associated with the phenotype and performance of Brazilian sport horses training for events in a tropical climate. Among 17 such horses direct DNA sequencing and mutation analysis of the exon 15 and the intron-exon boundaries of ACTN3 revealed 2 new sequence variants in the ACTN3 intron 14-15, designated c.1681-86G > A and c.1681-129delA. Wild-type/deletion heterozygotes (A/del) had a lower mean subcutaneous fat layer in the region of the gluteus medius, as measured by ultrasonography, than the del/del homozygotes; the correlation was significant (P = 0.017). This single base-pair deletion in ACTN3 intron 14-15 may have resulted in metabolic changes that led to increased deposition of body fat in the homozygous state. However, neither sequence variant was correlated with the time to fatigue in a test on a high-speed treadmill with an incremental-speed protocol.

Le but de la présente étude était de vérifier pour la présence de mutations dans le gène ACTN3 équin et d'identifier des variants de séquence qui pourraient être associés avec le phénotype et la performance de chevaux de sport brésiliens qui s'entraînent pour des concours dans un climat tropical. Parmi 17 chevaux qui correspondent à ces critères, le séquençage direct de l'ADN et l'analyse de mutation de l'exon 15 et des frontières de l'intron-exon d'ACTN3 a révélé deux nouveaux variants de séquence dans l'intron 14­15 d'ACTN3, désigné c.1681­86G > A et c.1681­129delA. Chez les hétérozygotes type-sauvage/délétion (A/del) la moyenne de l'épaisseur de la couche de gras sous-cutané dans la région du gluteus medius était plus petite, telle que mesurée par échographie, que celle des homozygotes del/del; la corrélation était significative (P = 0,017). Cette délétion unique de paire de bases dans l'intron 14­15 d'ACTN3 pourrait avoir résulté dans des changements métaboliques qui auraient mené à une augmentation du dépôt de gras chez les homozygotes. Toutefois, aucun des variants de séquence n'était corrélé avec le temps de fatigue dans un test sur un tapis-roulant à haute vitesse avec un protocole d'augmentation de vitesse.(Traduit par Docteur Serge Messier).

Effect of selection for eventing on the MSTN gene in Brazilian sport horses.

Polymorphisms in MSTN have previously been associated with equine performance. Therefore, the aim of this study was to identify variants in MSTN intron 1 in 16 Brazilian Sport Horses selected for competition in eventing and their possible effects of selection on performance. Among the nine variants identified, eight had already been reported in previous studies or genomic databases, although they showed differences in frequencies when compared with other horse breeds. Moreover, a new mutation was identified in two horses, both in heterozygous form. Considering the absence of molecular studies in this valuable Brazilian breed, these findings represent an important contribution to the characterization of its genetic profile and may possibly aid in further genotype-phenotype association studies.

ABCB1 gene polymorphism associated with clinical factors can predict drug-resistant tuberculosis.

Polymorphism in the ABCB1 gene encoding P-glycoprotein, a transmembrane drug efflux pump, contributes to drug resistance and has been widely studied. However, their association with rifampicin and ethambutol resistance in tuberculosis (TB) patients is still unclear. Genotype/allele/haplotype frequencies in c.1236C > T (rs1128503), c.2677G > T/A (rs2032582), and c.3435C > T (rs1045642) were obtained from 218 patients. Of these, 80 patients with rifampicin and/or ethambutol resistance were selected as the case group and 138 patients were selected for the control group through the results of their culture and drug-sensitive tests. Patients aged <18 years and HIV-positive serologic tests were excluded. ABCB1 polymorphisms were determined using a PCR direct-sequencing approach, and restriction fragment length polymorphism (RFLP). A nomogram was constructed to simulate a combined prediction of the probability of anti-TB drug resistance, with factors including genotype c.1236C > T (rs1128503) (P=0.02), clinical form (P=0.03), previous treatment (P=0.01), and skin color (P=0.03), contributing up to 90% chance of developing anti-TB drug resistance. Considering genotype analyses, CT (rs1128503) demonstrated an increased chance of anti-TB drug resistance (odds ratio (OR): 2.34, P=0.02), while the analyses for ethambutol resistance revealed an association with a rare A allele (rs2032582) (OR: 12.91, P=0.01), the haplotype TTC (OR: 5.83, P=0.05), and any haplotype containing the rare A allele (OR: 7.17, P=0.04). ABCB1 gene polymorphisms in association with others risk factors contribute to anti-TB drug resistance, mainly ethambutol. The use of the nomogram described in the present study could contribute to clinical decision-making prior to starting TB treatment.

FIRST REPORT OF ACUTE CHAGAS DISEASE BY VECTOR TRANSMISSION IN RIO DE JANEIRO STATE, BRAZIL.

Chagas disease (CD) is an endemic anthropozoonosis from Latin America of which the main means of transmission is the contact of skin lesions or mucosa with the feces of triatomine bugs infected by Trypanosoma cruzi. In this article, we describe the first acute CD case acquired by vector transmission in the Rio de Janeiro State and confirmed by parasitological, serological and PCR tests. The patient presented acute cardiomyopathy and pericardial effusion without cardiac tamponade. Together with fever and malaise, a 3 cm wide erythematous, non-pruritic, papule compatible with a "chagoma" was found on his left wrist. This case report draws attention to the possible transmission of CD by non-domiciled native vectors in non-endemic areas. Therefore, acute CD should be included in the diagnostic workout of febrile diseases and acute myopericarditis in Rio de Janeiro.

First report of acute Chagas disease by vector transmission in Rio de Janeiro State, Brazil / Primeiro relato de doença de Chagas aguda por transmissão vetorial no Estado do Rio de Janeiro, Brasil

SUMMARYChagas disease (CD) is an endemic anthropozoonosis from Latin America of which the main means of transmission is the contact of skin lesions or mucosa with the feces of triatomine bugs infected by Trypanosoma cruzi. In this article, we describe the first acute CD case acquired by vector transmission in the Rio de Janeiro State and confirmed by parasitological, serological and PCR tests. The patient presented acute cardiomyopathy and pericardial effusion without cardiac tamponade. Together with fever and malaise, a 3 cm wide erythematous, non-pruritic, papule compatible with a "chagoma" was found on his left wrist. This case report draws attention to the possible transmission of CD by non-domiciled native vectors in non-endemic areas. Therefore, acute CD should be included in the diagnostic workout of febrile diseases and acute myopericarditis in Rio de Janeiro.

RESUMOA doença de Chagas é antropozoonose endêmica na América Latina que tem como principal mecanismo de transmissão humana o contato da pele lesada ou da mucosa com as fezes de triatomíneos infectados por Trypanosoma cruzi. Neste artigo descrevemos o primeiro caso de doença de Chagas aguda adquirida no Estado do Rio de Janeiro por transmissão vetorial com confirmação parasitológica, sorológica e pela PCR. O paciente apresentou miocardite aguda e derrame pericárdico de evolução benigna. Juntamente com as manifestações sistêmicas da fase aguda, foi notada pápula eritematosa de três cm de diâmetro compatível com chagoma em punho esquerdo. Este relato de caso chama a atenção para a possibilidade de transmissão da doença de Chagas por vetores nativos não domiciliados e em áreas consideradas indenes. Portanto, a doença de Chagas aguda deve ser incluída entre os diagnósticos diferenciais de doenças febris e miopericardites agudas no Rio de Janeiro.

Autochthonous transmission of Chagas disease in Rio de Janeiro State, Brazil: a clinical and eco-epidemiological study.

BACKGROUND: After the control of the main modes of Chagas disease (CD) transmission in most endemic countries, it is important to identify the participation of native sylvatic vectors in CD transmission. Although CD is not considered endemic in Rio de Janeiro State (RJ), Brazil, we identified patients with CD born in RJ and investigated the possible autochthonous transmission in the state. METHODS: Patients born in RJ and followed in our institution between 1986 and 2011 were retrospectively analyzed. The cases identified as autochthonous transmission were submitted to epidemiological, clinical, serological, parasitological and molecular studies. Sectional field study with serological survey, research of sylvatic reservoirs and vectors was conducted in rural areas where patients were born. RESULTS: Among 1963 patients, 69 (3.5%) were born in RJ. From these, 15 (21.7%) were considered to have acquired the infection by autochthonous transmission. Cardiac form was the commonest form of presentation (60%). In rural areas in RJ northern region, sylvatic cycles of Trypanosoma cruzi and domestic invasion by Triatoma vitticeps were identified, and CD prevalence among inhabitants was 0.74%.TcI genotype was identified in sylvatic reservoirs and vectors. The genotype (mixed infection TcI/TcVI) could be identified in one of the autochthonous cases. CONCLUSIONS: The autochthonous vectorial transmission of CD occurs in RJ, probably due to wild cycles of T. cruzi and sylvatic vectors, such as T. vitticeps. Therefore, the health authorities should evaluate if RJ should be included in the original endemic area of CD and CD should be included in the diagnostic work out of cardiomyopathy of patients born in RJ. Moreover, control and educational measures should be put into place in the risk areas.

The role of cigarette smoking and liver enzymes polymorphisms in anti-tuberculosis drug-induced hepatotoxicity in Brazilian patients.

Tuberculosis (TB) is still a major health concern and side-effects related to the treatment, especially drug-induced hepatotoxicity (DIH), should be better investigated. In the present study, a possible association between anti-TB DIH and cigarette smoking, N-acetyltransferase 2 (NAT2), Cytochrome P450 2E1 (CYP2E1) and Cytochrome P450 3A4 (CYP3A4) genotypes was studied in 131 TB Brazilian patients. The NAT2 and CYP3A4 genetic polymorphisms were determined using a polymerase chain reaction (PCR) direct sequencing approach and genetic polymorphisms of CYP2E1 gene were determined by restriction fragment length polymorphism (RFLP). The risk of anti-TB DIH was lower in rapid/intermediate acetylators when compared to slow acetylators (OR: 0.34, CI 95: 0.16-0.71; p < 0.01). A decreased risk of developing anti-TB DIH was also observed in active smokers when compared to non-smokers (OR: 0.28, 95 CI: 0.11-0.64; p < 0.01). Significant association between CYP3A4 genotypes and hepatotoxicity was not observed, as well as between CYP2E1 genotype and hepatotoxicity, whose frequency of patients with wild homozygous was more prevalent. The anti-TB drugs interactions with smoking on hepatotoxicity, as well as the NAT2 phenotype, may require to adjust therapeutic regimen dosages or alarm in case of adverse event developments.

HLA-A*01 allele: a risk factor for dengue haemorrhagic fever in Brazil's population.

Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients.

Unique CYP3A4 genetic variant in Brazilian tuberculosis patients with/without HIV.

CYP3A4 is involved in tuberculosis (TB) and human immunodeficiency virus (HIV) drug metabolism. Transcriptional activation by rifampicin involves the CYP3A4 gene 5'-upstream region. Consequently, variation may interfere with transcription and enzymatic activity and even drug response. However, genetic polymorphisms and distribution of CYP3A4 allelic frequencies in individuals from Rio de Janeiro remain unknown. The aim of this study was to conduct research into sequencing the CYP3A4 5'-upstream region in Brazilian patients with and without HIV. This follow-up study involved 106 individuals undergoing treatment for TB and/or HIV. The CYP3A4 5'-upstream region was analyzed using PCR, sequencing and clinical data. Male patients revealed a higher HIV frequency (p=0.021). The TB forms observed were pulmonary (48.1%), extrapulmonary (22.64%) and disseminated (27.36%). Lymph node form was the most frequent (70.83%) extrapulmonary form of TB. The only single nucleotide polymorphism detected in the population was c.-392A>G. Genotypes observed were CYP3A4*1A/CYP3A4*1A (45.3%), CYP3A4*1A/CYP3A4*1B (40.6%) and CYP3A4*1B/CYP3A4*1B (14.2%), revealing a different distribution with extrapulmonary TB cases (17.6% CYP3A4*1A/CYP3A4*1B and 23.5% CYP3A4*1B/CYP3A4*1B). The CYP3A4*1A allele was found to be associated with tobacco use. The CYP3A4*1B mutant allele occurred in 34% of patients. This study revealed that the CYP3A4 5'-upstream regulatory region was highly conserved with the exception of the -392 position. Genotype association with tobacco suggests that CYP3A4 may participate in tobacco metabolism. Genotype distribution inversion in extrapulmonary TB cases suggests that CYP3A4 may be involved in TB prognosis.

ELISA versus PCR for diagnosis of chronic Chagas disease: systematic review and meta-analysis.

BACKGROUND: Most current guidelines recommend two serological tests to diagnose chronic Chagas disease. When serological tests are persistently inconclusive, some guidelines recommend molecular tests. The aim of this investigation was to review chronic Chagas disease diagnosis literature and to summarize results of ELISA and PCR performance. METHODS: A systematic review was conducted searching remote databases (MEDLINE, LILACS, EMBASE, SCOPUS and ISIWeb) and full texts bibliography for relevant abstracts. In addition, manufacturers of commercial tests were contacted. Original investigations were eligible if they estimated sensitivity and specificity, or reliability -or if their calculation was possible - of ELISA or PCR tests, for chronic Chagas disease. RESULTS: Heterogeneity was high within each test (ELISA and PCR) and threshold effect was detected only in a particular subgroup. Reference standard blinding partially explained heterogeneity in ELISA studies, and pooled sensitivity and specificity were 97.7% [96.7%-98.5%] and 96.3% [94.6%-97.6%] respectively. Commercial ELISA with recombinant antigens studied in phase three investigations partially explained heterogeneity, and pooled sensitivity and specificity were 99.3% [97.9%-99.9%] and 97.5% [88.5%-99.5%] respectively. ELISA's reliability was seldom studied but was considered acceptable. PCR heterogeneity was not explained, but a threshold effect was detected in three groups created by using guanidine and boiling the sample before DNA extraction. PCR sensitivity is likely to be between 50% and 90%, while its specificity is close to 100%. PCR reliability was never studied. CONCLUSIONS: Both conventional and recombinant based ELISA give useful information, however there are commercial tests without technical reports and therefore were not included in this review. Physicians need to have access to technical reports to understand if these serological tests are similar to those included in this review and therefore correctly order and interpret test results. Currently, PCR should not be used in clinical practice for chronic Chagas disease diagnosis and there is no PCR test commercially available for this purpose. Tests limitations and directions for future research are discussed.