Incursion of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus, Brazil, 2023.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 Replication by Hypertonic Saline Solution in Lung and Kidney Epithelial Cells.

An unprecedented global health crisis has been caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed experiments to test if a hypertonic saline solution was capable of inhibiting virus replication. Our data show that 1.2% NaCl inhibited virus replication by 90%, achieving 100% of inhibition at 1.5% in the nonhuman primate kidney cell line Vero, and 1.1% of NaCl was sufficient to inhibit the virus replication by 88% in human epithelial lung cell line Calu-3. Furthermore, our results indicate that the inhibition is due to an intracellular mechanism and not to the dissociation of the spike SARS-CoV-2 protein and its human receptor. NaCl depolarizes the plasma membrane causing a low energy state (high ADP/ATP concentration ratio) without impairing mitochondrial function, supposedly associated with the inhibition of the SARS-CoV-2 life cycle. Membrane depolarization and intracellular energy deprivation are possible mechanisms by which the hypertonic saline solution efficiently prevents virus replication in vitro assays.

Detection and Genetic Characterization of Hepatitis B and D Viruses: A Multi-Site Cross-Sectional Study of People Who Use Illicit Drugs in the Amazon Region.

Hepatitis B (HBV) and delta (HDV) viruses are endemic in the Amazon region, but vaccine coverage against HBV is still limited. People who use illicit drugs (PWUDs) represent a high-risk group due to common risk behavior and socioeconomic factors that facilitate the acquisition and transmission of pathogens. The present study assessed the presence of HBV and HBV-HDV co-infection, identified viral sub-genotypes, and verified the occurrence of mutations in coding regions for HBsAg and part of the polymerase in HBV-infected PWUDs in municipalities of the Brazilian states of Amapá and Pará, in the Amazon region. In total, 1074 PWUDs provided blood samples and personal data in 30 municipalities of the Brazilian Amazon. HBV and HDV were detected by enzyme-linked immunosorbent assay and polymerase chain reaction. Viral genotypes were identified by nucleotide sequencing followed by phylogenetic analysis, whereas viral mutations were analyzed by specialized software. High rates of serological (32.2%) and molecular (7.2%) markers for HBV were detected, including cases of occult HBV infection (2.5%). Sub-genotypes A1, A2, D4, and F2a were most frequently found. Escape mutations due to vaccine and antiviral resistance were identified. Among PWUDs with HBV DNA, serological (19.5%) and molecular (11.7%) HDV markers were detected, such as HDV genotypes 1 and 3. These are worrying findings, presenting clear implications for urgent prevention and treatment needs for the carriers of these viruses.

The role of PNPLA3 and TM6SF2 polymorphisms on liver fibrosis and metabolic abnormalities in Brazilian patients with chronic hepatitis C.

BACKGROUND: Despite the growing body of knowledge about TM6SF2 and PNPLA3 polymorphisms in non-alcoholic fatty liver disease, their influence in the spectrum of HCV liver disease is not yet fully defined. Besides that, admixed populations, such as Brazilians, were not included in most of the studies. METHODS: This cross-sectional study enrolled 365 treatment-naïve patients with HCV and 134 healthy individuals. TM6SF2 (rs58542926 c.499C > T) and PNPLA3 (rs738409 c.444C > G) polymorphisms were evaluated regarding their association with clinical and laboratory data, histological liver steatosis and fibrosis, and with components of the metabolic syndrome. RESULTS: In HCV subjects, the frequencies of TM6SF2 CC and CT + TT were 89% and 11%, while PNPLA3 frequencies of CC and CG + GG were 51.4% and 48.6%. In the univariate logistic regression analysis, the TM6SF2 CT + TT genotype in HCV was associated with significant liver fibrosis (p = 0.047; OR 1.953; 95% CI 1.009-3.788). In comparison to the CT + TT genotype, the TM6SF2 CC genotype in HCV was associated with older age (p = 0.002), higher frequency of arterial hypertension (p = 0.032), obesity (p = 0.030), metabolic syndrome (p = 0.014) and lower total cholesterol levels (p = 0.036). The PNPLA3 GG subjects had lower body mass index than CG/ CC individuals (p = 0.047). None of the polymorphisms, or their combinations, was independently associated with hepatic steatosis or fibrosis. On the other hand, older age, lower serum levels of total cholesterol, and higher serum levels of alanine aminotransferase and alkaline phosphatase were associated with liver fibrosis in the multivariate logistic regression analysis. CONCLUSION: In this evaluation of an admixed HCV population, neither TM6SF2 nor PNPLA3 polymorphisms were independently associated with hepatic steatosis or fibrosis. Other factors seem more influential than these specific polymorphisms in isolation. More studies are warranted to clarify the role of the TM6SF2 and PNPLA3 polymorphisms in Brazilians with HCV.

Effectiveness of direct-acting antivirals for hepatitis C virus infection in hepatitis C/HIV coinfected individuals: A multicenter study.

In a hepatitis C virus (HCV)/HIV-positive Brazilian cohort, evaluate the safety and efficacy of HCV DAAs, the frequency of resistance substitutions in the HCV NS5A and NS5B genes and identify predictors of treatment failure.Retrospective multicenter study of HCV/HIV patients treated with sofosbuvir (SOF)-based regimens at 10 reference centers in Brazil.Clinical and virological data were collected. Genetic diversity in the NS5A and NS5B genes was assessed by direct nucleotide sequencing. The primary outcome was sustained virological response (SVR) 12 weeks after DAA completion.Of 643 HCV/HIV patients analyzed, 74.7% were male, median CD4+ T cell count was 617 cells/mm, 90% had an undetectable HIV viral load. HCV genotype 1 was detected in 80.2%, and 60% were taking at least 1 medication other than antiretroviral drugs during their DAA therapy. Cirrhosis was present in 42%. An SOF/daclatasvir (DCV) regimen was used in most patients (98%). The frequency of NS5A polymorphisms associated with clinically relevant resistance to DCV was 2%; no relevant NS5B variants were identified. The SVR12 rate was 92.8% in an intention to treat (ITT) analysis and 96% in a modified ITT (m-ITT) analysis. AE occurred in 1.6% of patients. By multivariate analysis, therapeutic failure was associated, in the m-ITT analysis, with concomitant use of anticonvulsant drugs (P = .001), age (P = .04), and female gender (P = .04).SOF/DCV regimens were associated with a high SVR rate in an HCV/HIV population. The use of concurrent anticonvulsant drugs and DAAs decreases the chances of achieving an SVR.

Hepatitis B Virus Infection among People Who Use Illicit Drugs: Prevalence, Genotypes and Risk Factors in the State of Amapá, Northern Brazil.

Background: Several records of high prevalence of HBV have been made in northern Brazil. Among risk groups for viral infection, people who use illicit drugs (PWUDs) stand out, especially if they inject drugs and engage in risky sexual behavior. Objectives: In this study, the prevalence, genotype distribution and factors associated with hepatitis B virus (HBV) exposure in PWUDs were estimated. Methods: This cross-sectional study used snowball sampling from nine different sites. Socio-demographic, economic, drug use and health-related information were collected of 308 PWUDs from nine municipalities in the state of Amapá, northern Brazil. Blood samples were tested for the presence of HBV using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Logistic regressions were run to identify factors independently associated with HBV exposure. Results: In total, seventy (22.7%) PWUDs were exposed to HBV, and 12 (3.9%) had DNA HBV. Genotypes A (58.4%), D (33.3%) and F (8.3%) were identified. Age ≥35 years, use of injectable drug, daily use of drugs, use of drugs over 12 years, unprotected sex, exchange sex for money/drugs, and >12 sexual partners in the last 12 months were associated with exposure to HBV. Conclusions: This study identified important information on the epidemiological scenario of HBV infection in PWUDs, highlighting the high prevalence of HBV exposure and the urgent need for measures for control and prevention, especially vaccination against this hepatotropic virus.

Hepatitis B Virus: Alternative phylogenetic hypotheses and its impact on molecular evolution inferences.

Characterizing molecular evolution patterns of the Hepatitis B Virus (HBV) is important for a better understanding of the natural history of this infection. However, several molecular evolution estimates are conditioned on tree topology. There is no consensus about the phylogenetic relationships of HBV genotypes, and different studies often find alternative topologies. While most studies consider HBV genotypes F and H as sister to all other human genotypes, a recent study suggested an alternative HBV phylogeny that indicates an accelerated substitution rate for HBV-F/H partially driven by positive selection. In this study, we evaluate the impact of alternative HBV topologies on inferences of HBV phylogeny, rate acceleration, and positive selection on the HBV-F/H branch. Our results indicate that under certain methodological approaches alternative HBV topologies are equally likely. Considering phylogenetic uncertainty, there is no evidence that HBV-F/H had an accelerated substitution rate, even though inferences of positive selection are robust to alternative background topologies. Our results further suggest that, under reasonable assumptions, HBV-F/H most likely represents the sister lineage to all other human/ape HBV genotypes. Understanding the full range of likely topologies will be crucial for elaborating, testing, and refining hypothesis about the evolutionary HBV origins in our species.

Hepatitis C Virus among Female Sex Workers: A Cross-Sectional Study Conducted along Rivers and Highways in the Amazon Region.

BACKGROUND: Previous studies found a high prevalence of pathogens among female sex workers (FSWs) in the Amazon region, and established their parenteral and sexual transmission. This study estimated the prevalence of hepatitis C virus (HCV) infection and associated risk factors, and the frequency of HCV genotypes and resistance-associated substitutions (RASs) in this vulnerable group. METHODS: Distinct sampling methods were used to access 412 FSWs in cities and riverside communities in the Amazon region from 2015 to 2018. Three methods for HCV diagnosis were used to determine infection status. HCV genotypes and RASs were identified by sequencing and nucleotide fragment analysis. An association between HCV infection and exposure factors was determined by bivariate and multivariate analysis. RESULTS: In total, 44 (10.7%) FSWs were exposed to HCV, and 32 (7.8%) of them had active infection. Nine socioeconomic characteristics and risky sexual behaviors were associated with HCV exposure, particularly unprotected sex and condom exemption for the clients who paid extra money. Genotype 1 (81.3%) and 3 (18.7%) were detected. The frequency of FSWs with RASs was 23.1% (6/26) for grazoprevir related to the occurrence of substitutions Y56F and S122G. CONCLUSIONS: HCV infection among FSWs is highly prevalent and dominated by genotype I. Urgent preventive and treatment measures are required to reduce HCV infection in FSWs and the general population.

Hepatitis C virus infection status and associated factors among a multi-site sample of people who used illicit drugs in the Amazon region.

BACKGROUND: Elevated rates of Hepatitis C Virus (HCV) infection have been reported in epidemiological studies with people who used illicit drugs (PWUIDs) in different Brazilian regions. In Brazil's Amazon region, studies have already identified the common use of illicit drugs among adolescents and the high prevalence of HCV infections among PWUIDs. However, all studies done with PWUIDs were conducted with small samples and within limited geographic coverage. This study determined the prevalence and risk factors for HCV infection in PWUIDs in the Amazon region, northern Brazil, as well as estimating the prevalence and factors associated with the HCV spontaneous clearance (HSC). METHODS: This cross-sectional study accessed 1666 PWUIDs from multiple municipalities of the Amazon region. Socio-demographic, economic, drug use and health-related information were collected through interviews. Blood samples collected were tested for the presence of anti-HCV antibodies and RNA-HCV. HCV genotypes were identified by real-time polymerase chain reaction (PCR). Logistic regressions were run to identify factors independently associated with HCV infection status and HSC. RESULTS: In total, 577 (34.6%) featured HCV antibodies, of which 384 (23.1%) had active HCV infection and 193 (11.6%) indicated HSC. Genotypes 1 (80.2%) and 3 (18.8%) were detected. HCV infection status was associated with the length of illicit drug use history, factors related to parenteral and sexual transmission, and factors of socio-economic marginalization leading to potential risk activities for HCV. HSC was associated with the ethnic (including indigenous) background of participants. CONCLUSIONS: High levels of HCV infection were detected in PWUIDs. Genotype 1 was predominant. Intense use of illicit drugs, unprotected sexual intercourse, high number of sexual partners and social marginalization were associated with all HCV infection. HSC was associated with origin (Amazonian-born) and non-white (e.g., Black or Indigenous) of PWUIDs. These findings emphasize the need for improve HCV prevention and control services and care for PWUIDs in the Brazilian Amazon region.

Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population.

INTRODUCTION AND AIM: Studies have shown that two polymorphisms were associated with steatosis and progression of non-alcoholic fatty liver disease (NAFLD) in different populations: the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) and Transmembrane 6 Superfamily Member 2 (TM6SF2). However, the frequency and significance of these polymorphisms in an admixed population, i.e., Brazilian, is unknown. Therefore, we aimed to evaluate them in healthy subjects in comparison to patients with NAFLD. MATERIAL AND METHODS: This was a multicenter cross-sectional study in 248 patients with biopsy-proven NAFLD and in 134 healthy controls from two tertiary centers in Brazil. PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) polymorphisms were evaluated. RESULTS: In controls, the frequencies of PNPLA3 CC and CG+GG were 49.25% and 50.74%, respectively; in NAFLD patients, this was 31.05% and 68.88% (p=0.0044, 95% CI 1.037-2.977). PNPLA3 GG subjects had an increased risk (3.29-fold) of having NAFLD when compared to CC subjects (p=0.0044, 95% CI 1.504-7.225). In patients with nonalcoholic steatohepatitis (NASH), PNPLA3 GG compared to CC was associated with higher AST levels [38.4±25.3 versus 36.7±40.1IU/L, p=0.0395)] and with the presence of liver fibrosis (≥F2 fibrosis, p=0.0272). TM6SF2 polymorphisms were not in Hardy-Weinberg equilibrium in our NAFLD group precluding further analysis. CONCLUSION: We demonstrated for the first time that PNPLA3 CG+GG increase the risk of NAFLD among Brazilian subjects. Moreover, PNPLA3 GG was associated with liver enzyme elevation and fibrosis in NASH patients.

Hepatitis C virus infection among illicit drug users in an archipelago of the Amazon.

Hepatitis C virus (HCV) infection causes acute and chronic liver disease and may lead to cirrhosis, liver failure or hepatocellular carcinoma. The behavior of illicit drug users (DUs) typically exposes them to risks of viral infection. In the Brazilian Amazon region, a number of studies have identified high rates of drug use among adolescents, and a high prevalence of HBV infection in DUs, disseminated by sexual and parenteral activities. However, the epidemiological scenario of HCV infection in the region is still poorly understood. This study determined the prevalence, genotypes, and risk factors for HCV infection among DUs of the Marajó Archipelago. This cross-sectional study collected samples and epidemiological information from DUs in 11 municipalities. The diagnosis was established by EIA and real-time PCR, and the samples were genotyped by multiplex real time PCR. The data were analyzed by simple and multiple logistical regression. In 466 DUs, 28.3% had anti-HCV antibodies, and 25.5% had HCV-RNA. In 92 injecting drug users, 88.0% had anti-HCV antibodies, and 80.4% had HCV-RNA. Genotypes 1 and 3 were detected, with three cases of mixed infections. The multivariate analysis indicated associations of HCV infection with age (≥ 35 years), tattoos, intravenous drug use, shared use of injection equipment, and the daily and long-term (> 3 years) use of illicit drugs. These findings will contribute to the development of effective measures for the prevention of HCV infection among Brazilian DUs, as well as its general population.

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients.

BACKGROUND: About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. FINDINGS: Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. CONCLUSIONS: Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.

Genetic diversity of NS5A protein from hepatitis C virus genotype 3a and its relationship to therapy response.

BACKGROUND: The quasispecies nature of HCV may have important implications for viral persistence, pathogenicity and resistance to antiviral agents. The variability of one of the viral proteins, NS5A, is believed to be related to the response to IFN therapy, the standard treatment for infection. In this study we analyzed the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a, before IFN therapy. METHODS: Viral RNA was isolated from samples of 12 patients: four sustained virological responders (SVR), four non-responders (NR), and four end-of-treatment responders (ETR). cDNA was synthesized, the NS5A region was amplified and the fragments obtained were cloned. Fifteen clones from each patient were sequenced with eight primers, generating 179 contigs. RESULTS: Higher values for substitution (either synonymous or non-synonymous) and for distance were found in the SVR group. However, the NR group showed relatively more non-synonymous mutations than the other groups, owing to the higher values of dN/dS in complete NS5A and most specific regions. Overall, NS5A protein is undergoing purifying selection, since all dN/dS ratios values are below 0.5. CONCLUSIONS: Our study provides an overview of the genetic variability of complete NS5A protein in HCV genotype 3a.

Soroprevalência da hepatite B e avaliação da resposta imunológica à vacinação contra a hepatite B por via intramuscular e intradérmica em profissionais de um laboratório de saúde pública / Hepatitis B seroprevalence and evaluation of immune response to hepatitis B vaccination using intramuscular and intradermal routes in public health laboratory employees

OBJETIVOS: Determinar a prevalência dos marcadores da hepatite B (HBsAg e anti-HBs) e avaliar a resposta à vacinação contra hepatite B por via intradérmica (ID) em profissionais de saúde que não responderam à vacinação por via intramuscular (IM). MÉTODO: Todos os funcionários do Instituto Adolfo Lutz (IAL) foram convidados a participar do estudo. Amostras de soro foram colhidas no momento da administração da primeira dose de vacina (Engerix® B) e o HBsAg e o anti-HBs foram pesquisados, utilizando-se kits comerciais (Laboratórios Abbott®). Aos funcionários que não responderam à vacinação convencional (três doses por via IM) foram oferecidas doses de 5µg da mesma vacina por via ID. RESULTADOS: Foram envolvidos nesse estudo 404 funcionários do IAL, dos quais dois (0,5 por cento) eram HBsAg e 42 (10,5 por cento), anti-HBs reagentes. Dos 360 voluntários com sorologia negativa, 316 (87,8 por cento) receberam três doses de vacina (IM) e, desses, 259 colheram soros para avaliação pós-vacinal. Do total, 242 (93,4 por cento) apresentaram anticorpos acima de 10 UI/L após completarem o esquema inicial. Foram administradas duas doses de reforço, porém sete funcionários permaneceram sem resposta imunológica. A vacinação intradérmica foi realizada em cinco voluntários, e todos produziram anticorpos após a utilização dessa via de administração. CONCLUSÕES: A prevalência da hepatite B não foi maior nessa população do que na população geral. A vacinação por via intradérmica pode ser uma boa alternativa na imunização de pessoas que não respondem ao esquema convencional.

OBJECTIVES: To determine the prevalence of HBsAg and anti-HBs and to evaluate the response of intradermal hepatitis vaccination in healthcare workers non-responsive to previous repeated intramuscular vaccination. MATERIAL AND METHOD: All of the employees from Instituto Adolfo Lutz were invited to participate on this study. Serum samples were obtained and HBsAg and anti-HBs were detected using commercial kits (Abbott® Laboratories). Employees were submitted to the conventional three-dose vaccination by intramuscular route. To those employees who did not respond to intramuscular vaccination, 5 µg doses of Engerix® B were then administered by intradermal route up to nine doses. RESULTS: Four hundred and four healthcare workers were enrolled in this study. Initially, two (0.5 percent) and 42 (10.4 percent) were HBsAg and anti-HBs reagent, respectively. Among the 360 negative volunteers, 316 (87.8 percent) received three vaccine doses and in 259 of them, serum samples were collected to evaluate vaccine efficacy. Among them, 242 (93.4 percent) showed antibodies titer higher than 10 UI/l. Intradermal vaccination was carried out in five volunteers and all of them responded to this vaccine administration route. CONCLUSION: The prevalence of hepatitis B was not higher than in general population. Intradermal vaccine administration could be a good alternative in people that did not respond to previous intramuscular route.

The precore mutation is associated with severity of liver damage in Brazilian patients with chronic hepatitis B.

BACKGROUND: The clinical relevance of the G1896A precore mutation in chronic hepatitis B is still poorly understood. OBJECTIVES: To assess the frequency of G1896A precore mutation in Brazilian patients with chronic hepatitis B, as well as its relation to the viral genotype, serum HBV-DNA levels and liver damage. STUDY DESIGN: Fifty chronic hepatitis B patients (29 HBeAg-negative and 21 HBeAg-positive) were studied. HBV-DNA was quantified by the Amplicor HBV Monitor test and precore region and S gene were amplified and submitted to automatic sequencing. The histological activity index (HAI), degrees of hepatic fibrosis and distribution of core antigen (HBcAg) in hepatocytes were determined. RESULTS: Precore mutation occurred in 1/21 (4.8%) HBeAg-positive patients and in 17/29 (58.6%) HBeAg-negative (p < 0.0001). Genotype D was identified in 56.5%, genotype A in 41.3%, and genotype F in 2.2%. The frequency of genotypes D and A, as well as serum levels of ALT and HBV-DNA were similar in patients infected with wild type and with precore mutant. Patients infected with precore mutant presented a higher frequency of moderate/severe HAI (p: 0.03) and moderate/severe fibrosis and cirrhosis (p: 0.03) than those infected with wild type. There was no association between G1896A mutation and cytoplasmic expression of HBcAg. CONCLUSIONS: Precore mutation was frequent among Brazilian subjects with chronic hepatitis B and its presence was associated with greater severity of liver disease.