Applicability of oral fluid samples for tracking hepatitis B virus mutations, genotyping, and phylogenetic analysis.

Little is known about the usefulness of saliva samples for hepatitis B virus (HBV) genotyping and mutation analysis. The aim of this study was to evaluate the usefulness of oral fluid samples to determine HBV genotype distribution, S/polymerase mutations, and HBV subpopulation diversity among chronically HBV-infected individuals. Serum and oral fluid samples were obtained from 18 individuals for PCR and nucleotide sequencing of the HBV surface antigen gene. Biochemical analysis of liver enzymes (ALT, AST, GGT) and HBV, HCV, and HIV serological tests were also performed. All serum samples were HBsAg (+), anti-HBc (+), and anti-HBs (-); 55.6% were HBeAg (+)/anti-HBe (-), and 11.1% were anti-HIV (+). The mean HBV DNA viral load was 6.1 ± 2.3 log IU/mL. The HBV genotype distribution was as follows: A, 72.2%; D, 11.1%; E, 5.6%; F, 11.1%. A concordance of 100% in genotype classification and 99.8% in sequence similarity between paired oral fluid and serum samples was observed. HBsAg mutations were detected in all samples, but no resistance mutations were found in the polymerase gene. This study demonstrates that oral fluid samples can be used reliably for tracking HBV mutations, genotyping, and phylogenetic analysis. This could be important for molecular epidemiology studies with hard-to-reach populations.

Epidemiology of hepatitis B and C virus infection in Central West Argentina.

Little information is available regarding the prevalence of viral hepatitis in Central West Argentina. This study aims to give new information regarding HBV and HCV prevalence, genotypes, and risk factors in Central West Argentina and the suitability of dried blood spot (DBS) sampling for HBV and HCV screening. METHODS: A total of 622 individuals were included; the mean age was 36.6 ± 14.3 years and 55.4% were females. HBV and HCV markers were detected using serological and molecular analysis, and risk factors were evaluated using statistical analysis. RESULTS: Using serum samples, the HBsAg prevalence was 1.8%, the rate of HBV exposure (anti-HBc positivity) was 5.3%, and the rate of HBV immunity was 34.9%. HBV DNA was found in four out of 11 HBsAg+ samples, and the viruses in three of these samples were classified as genotypes A1, A2 and F2a. Multivariate analysis showed that anti-HBs positivity was associated with the level of schooling and history of HBV vaccination. The anti-HCV prevalence was 2.6%, and HCV RNA was found in 11 samples, seven of which contained viruses of genotypes 1a (n = 2), 1b (n = 3) and 2 (n = 2). The sensitivity of the DBS assay for HBsAg, anti-HBc, and anti-HCV was 100%, 66.6%, and 75%, respectively, and the specificity was above 98% for all markers when compared to serum. CONCLUSION: A low rate of HBV immunity was observed, demonstrating the importance of HBV vaccination. High HCV prevalence was found, and HCV 1b was closely related to other Argentinian isolates. Finally, the performance of DBS testing in this population needs more optimization to increase its sensitivity and specificity.

Molecular and serological characterization of occult hepatitis B among blood donors in Maputo, Mozambique.

BACKGROUND: Occult hepatitis B virus (HBV) - characterized by the absence of detectable HBsAg in the presence of HBV DNA - represents a potential threat for blood safety. OBJECTIVES: This study was conducted with the aim to investigate the serological and molecular characterization of occult HBV infection (OBI) among blood donors in Mozambique. METHODS: 1,502 blood donors were tested for HBsAg. All HBsAg-negative individuals were tested for HBV DNA. Antibodies against HBV core, surface and HBe antigen (anti-HBc, anti-HBs, HBeAg) were measured in HBV DNA positive individuals. FINDINGS: 1435 serum samples were HBsAg negative and 16 positive for HBV DNA, 14 confirmed to have OBI, corresponding to a frequency of 0.98%. Of the 14 OBI infections identified, 13/14 (92.8%) were positive for anti-HBc, 4/14 (28.5%) for anti-HBs, and no samples were reactive for HBeAg. Of the 14 OBI cases, nine samples (64.2%) were sequenced for the S/P region. Eight samples (88.9%) belonged to genotype A1 and one (11.1%) to genotype E. One escape mutation (T123A) associated with OBI and various amino acid substitutions for genotype A1 and E were observed. MAIN CONCLUSIONS: Our results show the importance of using nucleic acid amplification test to detect occult hepatitis B infection in blood donors in Mozambique.

Hepatitis D infection in Brazil: Prevalence and geographical distribution of anti-Delta antibody.

In Brazil, the Amazon Basin is endemic for hepatitis D virus (HDV) infection; however, studies in other regions of the country are scarce. This study aims to map the seroepidemiological situation of anti-Delta antibodies in chronic hepatitis B carriers in all five Brazilian geographic regions. Serum samples from 1240 HBsAg positive individuals (55.4% men; mean age 43.1 ± 13.4 years) from 24 of 26 Brazilian states were tested for the presence of anti-Delta antibodies using a commercial immunoassay. Anti-Delta antibodies were detected in 40 samples (3.2%; 52.5% female; mean age of 38.1 ± 13.8 years). Age less than 20 years was significantly associated with anti-HDV positivity (P < 0.001). The distribution of anti-Delta differed markedly in the diverse regions of the country. The highest prevalence of anti-HDV was found in the North (8.5%; P < 0.001), followed by Central West (2.5%), Southeast (1.7%), Northeast (0.8%), and South (0.0%). Anti-Delta antibodies were detected in 12 states, but more than 60% of the positive cases were observed in two states, Amazonas and Acre, located in the western portion of the Amazon region. The overall HDV prevalence of 3.2% emphasizes that HDV is far from being a disease under control in Brazil. Despite the low HDV prevalence in non-endemic regions, this infection persists as a major concern in two states (Acre and Amazonas) in the north of the country, indicating that a continuous epidemiological surveillance program should be implemented in all Brazilian regions.

HTLV-1 intrafamilial transmission among Japanese immigrants in Brazil.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The aim of this study was to investigate the intrafamilial transmission of HTLV-1 among Japanese immigrants and their descendants living in a non-endemic area of central Brazil. Six families were investigated. Thirty-seven relatives of the six index cases were tested by ELISA for the presence of anti-HTLV antibodies, and the positive cases were confirmed by Western blot. HTLV-1 isolates were genotyped by partial nucleotide sequencing (5' LTR) of the proviral DNA. All individuals, including index cases and relatives, were asymptomatic. In five families, at least one relative was infected with HTLV-1. In all, eight (22%) relatives (one mother, four wives, one brother, and two brothers-in-law) were infected. However, none of the 22 individuals under 55 years of age was infected. In each family, the HTLV-1 sequences from the relatives were identical or almost identical to that of the index case, except in one case. Pedigrees of the families, together with socio-demographic data of the HTLV-1 infected individuals, strongly suggested the occurrence of both vertical and sexual transmission, with breastfeeding as an important risk factor. Whether and why the virus transmission is less effective among younger generations deserves to be further investigated.

Epidemiological and molecular analysis of hepatitis B virus infection in manicurists in Central Brazil.

The aim of this study was to investigate the serological and molecular prevalence of hepatitis B virus (HBV) infection in 514 manicurists/pedicurists and identify the risk factors related with this infection. Samples were tested for HBV serological markers, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) by enzyme-linked immunosorbent assay (ELISA). Anti-HBc-positive samples were tested to investigate occult hepatitis B by PCR. HBsAg-positive samples were genotyped and the viral loads of HBV-DNA positive samples were quantified. The overall HBV prevalence was 5.6% (29/514) and of HBsAg was 0.4% (2/514). One case of occult hepatitis B was found. The genotypes A1 and F2 were identified in two HBsAg-positive samples. Low level of education, not being born in the State of Mato Grosso do Sul and working in the central region of the city were associated with the HBV infection. No single manicurist/pedicurist was infected by HCV or HIV. These findings suggest that despite the exposure to blood contact, this occupation was not associated to an increased risk of acquiring HBV, HCV, and HIV infections.

Overt and occult hepatitis B virus infection among treatment-naïve HIV-infected patients in Brazil.

Although hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co-infection is common, only few data are available on HBV among HIV patients including occult hepatitis B infection (OBI), regardless of serological markers. This study aims to determine the prevalence of OBI and overall HBV infection, associated factors, HBV genotypes, and surface (S) gene mutations in a population of treatment-naïve HIV-infected patients in Brazil. A cross-sectional study was conducted in treatment-naïve HIV-infected patients in Central Brazil. All samples were tested for HBV serological markers and HBV DNA. Sequence analysis of the S gene and overlapping polymerase gene was preformed. Overall, 25.1% (127/505) of the patients had markers of current or previous HBV infection, which was associated with age over 40 years, history of injection drug use, and homosexual sex. The hepatitis B surface antigen (HBsAg) seroprevalence was 4.9% (25/505). HBV DNA was detected in 39 out of 505 patients: 20 of them were HBsAg-positive and 19 were HBsAg-negative, resulting in an OBI prevalence of 3.8%. Patients with OBI had significantly higher HCV seropositivity rate compared to HBsAg-positive patients. Sequencing of the S gene revealed Y100C, T131N, and D144A mutations. One patient had the M204I and L180M drug-resistance mutations (polymerase). HBV genotypes A (A1, A2), D (D2, D3), and F (F2) were identified. In conclusion, OBI represented almost half of all HBV infections with detectable HBV DNA, suggesting that hepatitis B diagnosis in HIV patients should include in addition to serological markers the detection of HBV DNA.

Genetic characterization of the parvovirus full-length VP2 gene in domestic cats in Brazil.

Feline parvovirus (FPV) and canine parvovirus (CPV) are over 98% identical in their DNA sequences, and the new variants of CPV (2a/2b/2c) have gained the ability to infect and replicate in cats. The aim of this study was to determine the genetic diversity in the VP2 gene of parvovirus strains circulating in domestic cats in Brazil during a 10-year period (2008-2017). For parvovirus screening, specific PCR was performed, and 25 (34.7%) of 72 cats tested positive. The PCR-positive samples were further subjected to full-length VP2 sequencing (1755 bp), and eight sequences (36%) were characterized as FPV, seven (28%) as CPV-2a and (32%) nine (36%) as CPV-2b. One sequence (RJ1085/11) showing typical CPV amino acid (aa) at residues 80 R, 93 N, 103 A, 232 I, and 323 N could not be characterized at this time. The sequences in this study displayed aa changes previously described for FPV (A14T, A91S, I101T, N564S, and A568G) from cats and CPV-2a/2b (S297N and Y324L) from dogs. However, the Y324L mutation has not yet been reported in any CPV-2a/2b strains from cats. Phylogenetic analysis supported the division of these sequences into two well-defined clades, clade 1 for FPV and clade 2 for CPV2a/2b. Unusually, the sequence RJ1085/11 was grouped separately. Two recombination breakpoints were detected by Bootscan and 3Seq methods implemented in the RDP4. This study is the first report of CPV-2a/2b in cats in Brazil. The detection of FPV strains with mutations characteristic of CPV indicates that Brazilian FPV strains have undergone genetic changes.

Exploring genetic diversity of hepatitis D virus full-length genome in Brazil: Discovery of a novel HDV-8 subgenotype beyond African borders.

Hepatitis D virus (HDV) is currently classified into 8 genotypes (1 to 8) and several subgenotypes, with distinct distribution worldwide. However, due to the scarcity of complete genome sequences in databases, this classification is constantly being updated and tends to be regularly revisited in upcoming years as more sequence data becomes available. Aiming to increase knowledge about the genetic variability of HDV, this study presents the full-length genomes of 11 HDV samples collected in Brazil in endemic and non-endemic regions, including the first complete genomes of the genotypes 5 and 8 obtained outside Africa. We also determined the co-infecting HBV genotypes to investigate their prevalence among the HDV-infected individuals throughout the country. Whole genome sequencing confirmed our previous findings based on a partial fragment of the HDV genome, in which HDV subgenoypes 3c (9/11; 81.8 %), 5b (1/11; 9.1 %) and one HDV-8 sequence (1/11; 9.1 %) were detected. As previously observed, HDV-8 formed a distinct branch apart from subgenotypes 8a and 8b, a monophyletic clade representing a novel HDV-8 subgenotype, designated as 8c. Among HDV-3 samples, the main co-infecting HBV genotype found was HBV-F (4/8; 50 %), reflecting the higher incidence of this native South American genotype in the endemic Amazon Basin. Both samples infected with HDV-5 and HDV-8 were coinfected with HBV genotype E, also a genotype with African origin. Our findings based on complete genome sequence of HDV corroborated our results based on a partial region of the HDV genome of a novel HDV-8 subgenotype and reinforced the need to use full-length genomes to properly subdivide genotypes with very low intragroup genetic variability, such as HDV-3. The provision of these complete genomes is expected to contribute to the enrichment of sequence databases for future molecular and evolutionary investigations of HDV.

Could Herpesviridae be the cause of severe acute hepatitis of unknown origin in children?

INTRODUCTION: Severe acute hepatitis (SAH) is defined by a severe inflammation of hepatocytes in the liver parenchyma which can lead to an acute liver failure, a clinical condition with high mortality rate that can be triggered by several factors but is usually associated to hepatotropic viruses' infection. In 2022, cases of children with severe acute hepatitis of unknown origin hospitalized in Glasgow, Scotland, were reported. Possible causes of this condition include, but are not limited to, undiagnosed viral (and non-viral) infections, autoimmune hepatitis, drug and/or chemical toxicity, mitochondrial chain respiratory and metabolic disorders. AREAS COVERED: Herpesviruses can cause severe acute hepatitis, but little is known about the role and the mechanisms of herpesviruses as a causative agent of this type of hepatitis. We review the role of herpesviruses as causative agent of SAH in children and other possible mechanisms involved in this disease. EXPERT OPINION: Differential diagnosis for herpesvirus in SAH should be implemented in all settings. Alternative fluids, such as saliva and dried blood, could be used in the diagnosis to overwhelm the availability of biological specimens at sufficient volume. In the future, genetic studies could also be added to increase the knowledge about severe acute hepatitis in children.

SARS-CoV-2 and dialysis: humoral response, clinical and laboratory impacts before vaccination.

BACKGROUND: Patients with kidney disease on Hemodialysis (HD) are susceptible to Coronavirus Disease (COVID-19) due to multiple risk factors. AIM: This study aims to report the prevalence of antibodies against SARS-CoV-2 among patients on hemodialysis before vaccination in Brazil and to compare with clinical, demographic, and laboratory data. METHODS: Blood samples from 398 Chronic Kidney Disease (CKD) patients treated in three different private institutions in Rio de Janeiro State, Brazil were submitted to the total anti-SARS-CoV-2 testing. Kidney, liver, and hematological markers were also determined. Respiratory samples were tested by real-time PCR for SARS-CoV-2 RNA and positive samples were subjected to high-throughput sequencing on the MinION device. RESULTS: Overall, anti-SARS-CoV-2 prevalence was 54.5 % (217/398) and two individuals had SARS-CoV-2 RNA with variant B.1.1. High anti-SARS-CoV-2 seroprevalence was found in male gender and those with hospital admission in the last 3-months before the inclusion in the study. Lower red blood cell count was observed in the anti-SARS-CoV-2 seropositive group. High levels of anti-SARS-CoV-2 were found in those who reported symptoms, had low levels of eosinophils and low hematocrit, and who practiced physical activity. CONCLUSION: High prevalence of anti-SARS-CoV-2 was found in CKD patients before the universal immunization in Brazil suggesting that dialysis patients were highly exposed to SARS-CoV-2.

Differences in risky sexual behaviors and HIV prevalence between men who have sex with men and transgender women in the Midwest Brazil.

Men who have sex with men (MSM) and transgender women (TW) are disproportionally affected by HIV infection. This cross-sectional study evaluated the HIV-1/2 prevalence, risk factors and HIV molecular features of MSM and TW from Midwest Brazil. Four hundred and thirty participants (278 MSM and 152 TW) from Mato Grosso do Sul, Brazil, were interviewed and tested for HIV-1/2 infection between November 2011 and September 2013. Participants who were assigned male at birth, older than 18 years old and self-declared as MSM or TW were recruited from LGBT+ associations, as well as public (parks, square, streets, etc) and private [nightclubs, saunas, brothels, etc] places. The prevalence of HIV-1 was 14.4% (9.0% among MSM and 24% among TW; p<0.001). The factor independently associated with HIV-1 infection among MSM was being 30 years-old or older. Among TW, having suffered sexual coercion, lifetime syphilis infection and hepatitis C virus exposure were associated with HIV-1 infection. Phylogenetic analyses classified 65% sequences as subtype B and 35% as possible recombinants. All but one recombinant sample were from TW individuals. High HIV-1 prevalences were observed in both groups, highlighting the urgent need to devise specific HIV interventions targeting these key populations. Notably, TWs are more vulnerable to HIV infection, which was associated with sexual violence and co-infection with other STIs. With regard to MSM, being 30 years old or older was significanty associated to HIV, reinforcing the idea that MSM are less exposed [or exposed later] to STIs than TWs, although MSM are clearly more vulnerable than the general population.

Genetic variability of hepatitis B virus in acute and in different phases of chronic infection in Brazil.

The selection pressure imposed by the host immune system impacts on hepatitis B virus (HBV) variability. This study evaluates HBV genetic diversity, nucleos(t)ide analogs resistance and HBsAg escape mutations in HBV patients under distinct selective pressures. One hundred and thirteen individuals in different phases of HBV infection were included: 13 HBeAg-positive chronic infection, 9 HBeAg-positive chronic hepatitis, 47 HBeAg-negative chronic infection (ENI), 29 HBeAg-negative chronic hepatitis (ENH) and 15 acute infected individuals. Samples were PCR amplified, sequenced and genetically analyzed for the overlapping POL/S genes. Most HBV carriers presented genotype A (84/113; 74.3%), subgenotype A1 (67/84; 79.7%), irrespective of group, followed by genotypes D (20/113; 17.7%), F (8/113; 7.1%) and E (1/113; 0.9%). Clinically relevant mutations in polymerase (tL180M/M204V) and in the Major Hydrophilic Region of HBsAg (sY100C, T118A/M, sM133T, sD144A and sG145R) were observed. Our findings, however, indicated that most polymorphic sites were located in the cytosolic loops (CYL1-2) and transmembrane domain 4 (TMD4) of HBsAg. Lower viral loads and higher HBV genetic diversity were observed in ENI and ENH groups (p < 0.001), suggesting that these groups are subjected to a higher selective pressure. Our results provide information on the molecular characteristics of HBV in a diverse clinical setting, and may guide future studies on the balance of HBV quasispecies at different stages of infection.

Phylogeography and evolutionary history of hepatitis B virus genotype F in Brazil.

BACKGROUND: Hepatitis B virus (HBV) genotype F (HBV/F) is considered to be indigenous to the Americas, but its emergence and spread in the continent remain unknown. Previously, only two HBV/F complete genome sequences from Brazil were available, limiting the contribution of Brazilian isolates to the phylogenetic studies of HBV/F. The present study was carried out to assess the proportion and geographic distributions of HBV/F subgenotypes in Brazil, to determine the full-length genomic sequences of HBV/F isolates from different Brazilian geographic regions, and to investigate the detailed evolutionary history and phylogeography of HBV/F in Brazil. METHODS: Complete HBV/F genomes isolated from 12 Brazilian patients, representing the HBV/F subgenotypes circulating in Brazil, were sequenced and analyzed together with sequences retrieved from GenBank, using the Bayesian coalescent and phylogeographic framework. RESULTS: Phylogenetic analysis using all Brazilian HBV/F S-gene sequences available in GenBank showed that HBV/F2a is found at higher frequencies countrywide and corresponds to all sequences isolated in the Brazilian Amazon Basin. In addition, the evolutionary analysis using complete genome sequences estimated an older median ancestral age for the Brazilian HBV/F2a compared to the Brazilian HBV/F1b and HBV/F4 subgenotypes, suggesting that HBV/F2a represents the original native HBV of Brazil. The phylogeographic patterns suggested a north-to-south flow of HBV/F2a from Venezuela to Brazil, whereas HBV/F1b and HBV/F4 strains appeared to have spread from Argentina to Brazil. CONCLUSIONS: This study suggests a plausible route of introduction of HBV/F subgenotypes in Brazil and demonstrates the usefulness of recently developed computational tools for investigating the evolutionary history of HBV.

Genetic diversity of hepatitis B virus quasispecies in different biological compartments reveals distinct genotypes.

The selection pressure imposed by the host immune system impacts hepatitis B virus (HBV) quasispecies variability. This study evaluates HBV genetic diversity in different biological fluids. Twenty paired serum, oral fluid, and DBS samples from chronic HBV carriers were analyzed using both Sanger and next generation sequencing (NGS). The mean HBV viral load in serum was 5.19 ± 4.3 log IU/mL (median 5.29, IQR 3.01-7.93). Genotype distribution was: HBV/A1 55% (11/20), A2 15% (3/20), D3 10% (2/20), F2 15% (3/20), and F4 5% (1/20). Genotype agreement between serum and oral fluid was 100% (genetic distances 0.0-0.006), while that between serum and DBS was 80% (genetic distances 0.0-0.115). Two individuals presented discordant genotypes in serum and DBS. Minor population analysis revealed a mixed population. All samples displayed mutations in polymerase and/or surface genes. Major population analysis of the polymerase pointed to positions H122 and M129 as the most polymorphic (≥ 75% variability), followed by V163 (55%) and I253 (50%). Neither Sanger nor NGS detected any antiviral primary resistance mutations in the major populations. Minor population analysis, however, demonstrated the rtM204I resistance mutation in all individuals, ranging from 2.8 to 7.5% in serum, 2.5 to 6.3% in oral fluid, and 3.6 to 7.2% in DBS. This study demonstrated that different fluids can be used to assess HBV diversity, nonetheless, genotypic differences according to biological compartments can be observed.

High prevalence of hepatitis A and E viruses in environmental and clinical samples from West Argentina.

Environmental surveillance of water sources is important to monitoring viral hepatitis transmission in clinical settings. This study investigated the circulation of hepatitis A (HAV) and E (HEV) viruses in sewage and clinical samples from Argentina. Between 2016 and 2017, 80 raw sewage samples and 86 clinical samples (stool and serum) from suspected cases of hepatitis A and hepatitis E were obtained. HAV and HEV were tested by both real-time and nested PCR. Positive samples were sequenced for genotype determination and phylogenetic analysis. Overall, HAV was recovered in 39% of sewage samples and 61.1% of clinical samples. HEV was detected in 22.5% of sewage samples and 15.9% of clinical samples. HAV was found more frequently in sewage during the winter and in clinical samples in spring; HEV was more prevalent in sewage during summer and in clinical samples in autumn. All HAV isolates belonged to genotype IA and HEV isolates belonged to genotype 3, the most prevalent genotypes in South America. High prevalence of HAV and HEV in environmental and clinical samples in Mendoza, Argentina was observed. These findings reinforce the importance of environmental surveillance and implementation of health strategies to control the spread of HAV and HEV in developing countries.

Circulation of HDV Genotypes in Brazil: Identification of a Putative Novel HDV-8 Subgenotype.

Hepatitis D virus (HDV) is classified into 8 genotypes (1 to 8) and several subgenotypes. In Brazil, HDV-3 and HDV-1 predominate; however, most of the diagnosis efforts and molecular studies are directed to the area of endemicity of the Amazon Basin. Here, we determined the molecular epidemiological profile of circulating HDV in Brazilian HBsAg-positive patients between 2013 and 2015 in areas of endemicity and non-areas of endemicity. From 38 anti-HDV-positive individuals, 13 (34.2%) had detectable HDV-RNA and 11 (28.9%) were successfully sequenced. Partial HDAg (~320 nt) sequencing followed by phylogenetic analysis with reference sequences resulted in the identification of HDV-3 (9/11; 81.8%), HDV-5 (1/11; 9.1%), and HDV-8 (1/11; 9.1%). Most HDV-3 samples (8/9; 88.9%) were found in the endemic North region, while one was found in Central-West Brazil, a non-area of endemicity. HDV-5 and 8, genotypes native from African countries, were found in São Paulo, a cosmopolitan city from Southeast Brazil with a high circulation of immigrants. Phylogenetic analysis of HDV-8 strains indicated that the sample determined in our study, along with previously reported sequences from Brazil, formed a highly supported monophyletic clade, likely representing a putative novel HDV-8 subgenotype. IMPORTANCE Considered a neglected pathogen until the last 2 decades, an increase in the availability of genetic data of hepatitis D virus (HDV) strains around the world has been noticed recently, resulting in the proposition of different classifications. Our study aimed to determine the molecular epidemiological profile of HDV isolates circulating in areas of endemicity and non-areas of endemicity in Brazil. Based on the analyzed fragment, HDV-8 sequences clustered out of the clades formed by subgenotypes 8a and 8b might suggest the identification of a novel subgenotype, putatively designated subgenotype 8c. Our findings demonstrate the importance of continuous epidemiological surveillance to map HDV spread pathways and the introduction of imported variants. It also reinforces that as the amount of HDV genomes generated and reported increases, we will have changes in viral classification and, consequently, in our understanding of the dynamics of variability of this viral agent.

Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients.

BACKGROUND: Lamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods. RESULTS: Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies. CONCLUSIONS: YMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection.

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.

Increase in Hepatitis A Cases Linked to Imported Strains to Rio de Janeiro, Brazil: A Cross-Sectional Study.

This study aims to evaluate the epidemiological and molecular features associated with HAV transmission in adults in Rio de Janeiro during a period of increased registered cases of HAV (2017-2018). Socio-epidemiological data and serum samples from anti-HAV IgM+ individuals were obtained. HAV RNA was RT-PCR amplified and sequenced for further phylogenetic and phylogeographic analyses. From fifty-two HAV IgM+ individuals, most were men (78.85%; p = 0.024), aged 20-30 years old (84.61%; p < 0.001), resided in the Rio de Janeiro north zone (31/52; 59.62%; p = 0.001), and are men who have sex with men (MSM) (57.69%; p = 0.002). Sexual practices were more frequent (96%) than others risk factors (food-borne (44%), water-borne (42.31%), and parenteral (34.62%)). Individuals who traveled to endemic regions had a 7.19-fold (1.93-36.04; p < 0.01) increased risk of HAV. Phylogenetic analysis revealed four distinct clades of subgenotype IA, three of them comprised sequences from European/Asian MSM outbreaks and one from Brazilian endemic strains. Bayesian Inference showed that the imported strains were introduced to Brazil during large mass sportive events. Sexual orientation and sexual practices may play a role in acquiring HAV infection. Public policies targeting key populations must be implemented to prevent further dissemination of HAV and other STIs.