High Prevalence of ITPA Alleles Associated with Ribavirin-Induced Hemolytic Anemia Among Mexican Population

ABSTRACT Background. The prevalence of two functional polymorphisms (rs1127354 and rs7270101) of the inosine triphosphatase (ITPA) gene associated with ribavirin-induced hemolytic anemia (RIHA) during antiviral therapy for hepatitis C virus (HCV) infection varies by ethnicity. In Mexico, the distribution of these polymorphisms among Native Amerindians (NA) and admixed population (Mestizos) is unknown. This study aimed to determine the prevalence of the ITPA polymorphisms among healthy NA and Mestizos, as well as in HCV patients from West Mexico. Material and methods. In a cross-sectional study, 600 unrelated subjects (322 Mestizos, 100 NA, and 178 treatment-naïve, HCV-infected Mestizos patients) were enrolled. A medical history was registered. ITPA genotype was determined by Real-Time PCR. Fst-values and genetic relatedness between study and reference populations were assessed. Results. The frequency of the risk genotypes rs1127354CC and rs7270101AA was higher among NA (98-100%) than in Mestizos (87-92.9%), (p < 0.05). The NA presented the highest prevalence of the rs1127354CC genotype reported worldwide. The Fst-values revealed a genetic relatedness among Mexican NA, South Americans and African populations (p > 0.05). The frequency of the predicted risk for RIHA was higher among NA (98%) than in Mestizos (80.5%) and HCV-infected patients (81.5%) (p < 0 .01). The CC/AA alleles were associated with lower values of total bilirubin, aspartate/alanine aminotransferases, and aspartate-to-platelet-ratio-index score among HCV-patients. Conclusion. A high prevalence of the ITPA polymorphisms associated with RIHA was found in Mexican NA. These polymorphisms could be a useful tool for evaluating potential adverse effects and the risk or benefit of antiviral therapy in Mexicans and other admixed populations.

HBV carrying drug-resistance mutations in chronically infected treatment-naive patients.

BACKGROUND: Nucleoside/nucleotide analogue (NA) treatment causes selection pressure for HBV strains carrying mutations conferring NA resistance. Drug-resistance mutations occur in the reverse transcriptase (RT) region of the HBV polymerase gene and spontaneously arise during viral replication. These mutations can also alter the hepatitis B surface (HBs) protein and in some cases reduce binding to HBs antibodies. The spread of NA-resistant HBV may impact the efficacy of antiviral treatment and hepatitis B immunization programmes. In this study, we used direct sequencing to assess the occurrence of HBV carrying known mutations that confer NA resistance in the largest cohort of treatment-naive patients with chronic hepatitis B (CHB) to date. METHODS: HBV DNA samples isolated from 702 patients were sequenced and the RT region subjected to mutational analysis. RESULTS: There was high genetic variability among the HBV samples analysed: A1 (63.7%), D3 (14.5%), A2 (3.3%), A3 (0.1%), B1 (0.1%), B2 (0.1%), C2 (0.9%), D1 (0.9%), D2 (4.6%), D4 (5.1%), D unclassified subgenotype (0.7%), E (0.6%), F2a (4.6%), F4 (0.4%) and G (0.4%). HBV strains harbouring mutations conferring NA resistance alone or combined with compensatory mutations were identified in 1.6% (11/702) of the patients. CONCLUSIONS: HBV strains harbouring resistance mutations can comprise the major population of HBV quasispecies in treatment-naive patients. In Brazil, there is a very low frequency of untreated patients who are infected with these strains. These findings suggest that the spread and natural selection of drug-resistant HBV is an uncommon event and/or most of these strains remain unstable in the absence of NA selective pressure.

Resistance mutations are rare among protease inhibitor treatment-naive hepatitis C genotype-1 patients with or without HIV coinfection.

BACKGROUND: HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. This diversity may lead to emergence of resistant strains in direct-acting antiviral therapy. The frequency of naturally occurring HCV protease inhibitor (PI) mutations has been addressed in many countries, but there are few data on the prevalence of these mutations in Brazilian patients. METHODS: We evaluated the sequence of HCV NS3 protease gene in 247 patients (135 HCV-monoinfected and 112 HIV-HCV-coinfected patients). HCV RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. RESULTS: HIV-HCV-coinfected patients were more likely to be older than 40 years and have an HCV subtype-1a infection. Overall, 21.9% of patients had at least one amino acid substitution in the NS3 region; 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R) and the Q80K mutation was not found in our case series. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. CONCLUSIONS: Baseline HCV NS3 amino acid substitutions identified herein are considered mostly natural polymorphisms with no clinical impact on PI-based therapy. The identified resistance mutations may be associated with low-level resistance to PIs in vitro. Q80K substitution seems to be a rare event in Brazil. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample.

Epidemiological update of hepatitis B, C and delta in Latin America.

Viral hepatitis B, C and delta still remain a serious problem in Latin America. Data from the 1980s indicated that HBV and HDV infection are the main causes of chronic hepatitis. However, the spread of HBV infection could be controlled through the implementation of immunization programmes. Different countries from Mexico to Argentina display marked differences in terms of HBV genotype distribution. HBV genotype F has been identified as the most frequent in most Latin America countries, except for Mexico and Brazil, where genotypes H and A are the most frequent, respectively. In Latin America, the overall prevalence of HCV antibody is estimated to be 1.5%. Latin American countries have been very proactive in screening their blood supplies, thus minimizing risk of HCV transmission through transfusion. The number of diagnosed and treated patients is still low, thereby increasing the burden of complications such as liver cirrhosis or hepatocellular carcinoma. The most prevalent HCV genotype is 1, which is the genotype with the greatest worldwide spread, but it is a different genotype from other regions like Africa and Asia. HDV is present worldwide but its distribution pattern is not uniform. HDV was recently detected in novel geographic regions, reinforcing that it is a very serious health threat in under-developed countries. The main prevalence areas are the Mediterranean basin, the Middle East, central and northern Asia, western and central Africa, the Amazonian basin (Brazil, Peru, Venezuela and Colombia) and the Pacific islands. Novel strategies to increase HBV immunization in the Latin American population are needed to warrant thorough coverage in the rural areas.

An update on HDV: virology, pathogenesis and treatment.

Hepatitis delta is an inflammatory liver disease caused by infection with HDV. HDV is a single-stranded circular RNA pathogen with a diameter of 36 nm. HDV is classified in the genus Deltavirus and is still awaiting a final taxonomic classification up to the family level. HDV shares similarities with satellite RNA and viroids including a small circular single-stranded RNA with secondary structure that replicates through the 'double rolling circle' mechanism. The HDV RNA genome is capable of self-cleavage through a ribozyme and encodes only one structural protein, the hepatitis delta antigen (HDAg), from the antigenomic RNA. There are two forms of HDAg, a shorter (S; 22 kDa) and a longer (L; 24 kDa) form, the latter generated from an RNA editing mechanism. The S form is essential for viral genomic replication. The L form participates in the assembly and formation of HDV. For complete replication and transmission, HDV requires the hepatitis B surface antigen (HBsAg). Thus, HDV infection only occurs in HBsAg-positive individuals, either as acute coinfection in treatment-naive HBV-infected persons, or as superinfection in patients with pre-existing chronic hepatitis B (CHB). HDV is found throughout the world, but its prevalence, incidence, clinical features and epidemiological characteristics vary by geographic region. There are eight genotypes (1 to 8) distributed over different geographic areas: HDV-1 is distributed worldwide, whereas HDV-2 to 8 are seen more regionally. Levels of HDV viraemia change over the course of HDV infection, being significantly higher in patients with early chronic hepatitis than in cirrhosis. Chronic HDV infection leads to more severe liver disease than chronic HBV monoinfection with an accelerated course of fibrosis progression, an increased risk of hepatocellular carcinoma and early decompensation in the setting of established cirrhosis. Current treatments include pegylated interferon-α and liver transplantation; the latter of which can be curative. Further studies are needed to develop better treatment strategies for this challenging disease.

Genetic diversity of HCV in Brazil.

BACKGROUND: Many studies have documented the molecular epidemiological scenario of HCV within individual Brazilian states, but we still have an incomplete understanding of the dispersion dynamics of the virus in different regions throughout the country. METHODS: A total of 676 HCV NS5B gene sequences of subtypes 1a (n=321), 1b (n=170) and 3a (n=185), isolated from seven different Brazilian states covering four out of five regions were analysed in the present study. We also analysed 22 HCV NS5B gene sequences of minor genetic variants including genotype 2 (n=13), genotype 4 (n=6) and subtype 5a (n=3). Brazilian HCV sequences were aligned with sequences of non-Brazilian origin and subjected to maximum likelihood phylogenetic analyses. RESULTS: These analyses revealed that the Brazilian HCV epidemic resulted from multiple introductions and autochthonous transmission of subtypes 1a, 1b, 3a and genotypes 2, 4 and 5. Brazilian HCV subtype 1a epidemic is dominated by the dissemination of one major clade; while Brazilian HCV subtypes 1b and 3a epidemics are characterized by concurrent dissemination of several independent HCV lineages. Some HCV Brazilian lineages of subtypes 1a, 1b, 2b and 3a were successful in becoming established and disseminated through several regions in the country. Despite significant phylogenetic intermixing of Brazilian sequences, the distribution of HCV strains from different states across lineages was not completely homogeneous. CONCLUSIONS: These results demonstrate the existence of multiple introductions and local propagation of both prevalent and uncommon HCV genetic variants in Brazil and identify some major Brazilian HCV clades with nationwide dissemination. This study also suggests that the observed HCV diversity in Brazil has been shaped by both frequent viral migration among regions and in situ viral dissemination.

Evolutionary rates and HBV: issues of rate estimation with Bayesian molecular methods.

BACKGROUND: HBV infection is a public health problem affecting approximately 2 billion people and leading to >350 million chronic carriers of the virus worldwide. Phylogenetic analysis can give valuable insight to help in clarifying the history of viral infections around the world and in elucidating routes of transmission of the different viral strains present in the infected host population. These analyses rely on an accurate estimate of the rate of mutations. METHODS: In this study, we investigated the robustness of rate estimations based on Bayesian analysis obtained so far and examined, in particular, the choice of prior for the substitution rate. RESULTS: Most previous studies have concentrated on estimating the parameters of simple demographic models for HBV, such as exponential growth and constant population size. Here, we introduce a method that automatically partitions the genome in components that show a different rate of mutation and fit different substitution models. CONCLUSIONS: In conclusion, we find that, due to inaccuracy in the sampling dates from the samples where viral sequences were obtained, lack of a sufficiently large geographical and time spread of available and trustworthy sample dates, sensitivity to priors and model misspecification and rate estimation based on molecular methods, are not reliable. We suggest that rate estimates taking into account calibration points based on relevant historical events are more robust due to the lack of trustworthy sampling dates. For example, the known history of colonization of the Americas should be used to accurately study the current diversity of genotype F, which is the most frequent genotype in almost all Spanish speaking countries in South America.

Distribution of HBV genotypes in Latin America.

Approximately 2 billion people worldwide are infected with HBV, and 350 million people are chronic carriers. HBV is classified into nine genotypes (A to I). Genotype F is the most prevalent in the Spanish-speaking countries and in the Amerindian population in South America. HBV genotype F was primarily found in indigenous populations from South America and is divided into four subgenotypes (F1 to F4). Subgenotype F1 is further divided into F1a (found in Costa Rica and El Salvador) and F1b (found in in Alaska, Argentina and Chile). Subgenotypes F2 and F3 cocirculate in the north of South America: F2a is found in Brazil and Venezuela, F2b is described only in Venezuela, F3 is frequent in Colombia, Venezuela and Panama, and F4 is reported from the central and south areas of South America, including Bolivia, Argentina and southern Brazil. HBV genotypes and subgenotypes have distinct geographical distributions. It is currently under discussion whether they are associated with different prognoses, considering the patterns of severity of liver diseases in various populations. Furthermore, global human migrations affect the pattern of genotype distribution, introducing genotypes differing from those found in the original inhabitants.

Origin of HBV and its arrival in the Americas--the importance of natural selection on time estimates.

BACKGROUND: The strong geographic structure shown by the global pattern of HBV lineages suggests an ancient origin for this virus; however, estimates based on the molecular clock suggest a very recent origin for the Native American genotypes F and H. In this study, we contribute to this debate by estimating the divergence times of genotypes F and H and by discussing how evolutionary rates estimated from recent samples may underestimate the divergence time of more ancient nodes in HBV phylogenies. METHODS: A total of 108 complete HBV genotype F and H genomes were compared to 44 reference genomes from other genotypes. Time estimates were based on a Bayesian method with evolutionary rates taken from the literature. To assess the pattern of substitutions in recent versus old branches we mapped the phylogenetic distribution of all mutations occurring in genotypes F and H using a maximum likelihood approach and compared the number of synonymous and non-synonymous mutations in young and old branches of HBV genotype F and H phylogeny using a χ² test. RESULTS: Estimated divergence times between genotypes F and H depend heavily on the evolutionary rate. While fast rates suggest a recent separation of these genotypes (approximately 800 years ago), slow rates suggest an earlier divergence (up to approximately 13,000 years ago). There is a clear excess of non-synonymous substitutions in the most recent branches of HBV phylogeny (P=4.87×10⁻¹5), most likely suggesting the action of purifying selection. CONCLUSIONS: These results suggest that rates estimated based on recent samples will overestimate the evolutionary rate and underestimate the coalescence times for ancient nodes in HBV phylogeny.

Analysis of hepatitis C virus intrahost diversity across the coding region by ultradeep pyrosequencing.

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naïve patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Characterization of hepatitis B virus (HBV) genotypes in patients from Rondônia, Brazil.

BACKGROUND: Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondônia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM® 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. RESULTS: The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). CONCLUSIONS: These results for the first HBV genotypic characterization in Rondônia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.