Full genotype characterization of Brazilian canine G3P[3] strains during a 10-year survey (2012-2021) of rotavirus infection in domestic dogs and cats.

There is a dearth of information on the molecular epidemiology of rotaviruses in pets in Brazil. The aim of this study was to monitor rotavirus infections in household dogs and cats, determine full-genotype constellations, and obtain data on evolutionary relationships. Between 2012 and 2021, 600 fecal samples from dogs and cats (516 and 84, respectively) were collected at small animal clinics in São Paulo state, Brazil. Rotavirus screening was conducted using ELISA, PAGE, RT-PCR, sequencing, and phylogenetic analysis. Rotavirus type A (RVA) was detected in 0.5% (3/600) of the animals. No non-RVA types were detected. The three canine RVA strains were found to have a novel genetic constellation, G3-P[3] -I2-R3-C2-M3-A9-N2-T3-E3-H6, which has never been reported in dogs. As expected, all of the viral genes, except those encoding NSP2 and VP7, were closely related to the corresponding genes from canine, feline, and canine-like-human RVA strains. A novel N2 (NSP2) lineage was identified, grouping together Brazilian canine, human, rat and bovine strains, suggesting that genetic reassortment had occurred. Uruguayan G3 strains obtained from sewage contained VP7 genes that were phylogenetically close to those of the Brazilian canine strains, which suggests that these strains are widely distributed in pet populations in South American countries. For the NSP2 (I2), NSP3 (T3), NSP4 (E3), NSP5 (H6), VP1 (R3), VP3 (M3), and VP6 (I2) segments, phylogenetic analysis revealed possibly new lineages. The epidemiological and genetic data presented here point out the necessity for collaborative efforts to implement the One Health strategy in the field of RVA research and to provide an updated understanding of RVA strains circulating canines in Brazil.

Salivary glands are a target for SARS-CoV-2: a source for saliva contamination.

The ability of the new coronavirus SARS-CoV-2 to spread and contaminate is one of the determinants of the COVID-19 pandemic status. SARS-CoV-2 has been detected in saliva consistently, with similar sensitivity to that observed in nasopharyngeal swabs. We conducted ultrasound-guided postmortem biopsies in COVID-19 fatal cases. Samples of salivary glands (SGs; parotid, submandibular, and minor) were obtained. We analyzed samples using RT-qPCR, immunohistochemistry, electron microscopy, and histopathological analysis to identify SARS-CoV-2 and elucidate qualitative and quantitative viral profiles in salivary glands. The study included 13 female and 11 male patients, with a mean age of 53.12 years (range 8-83 years). RT-qPCR for SARS-CoV-2 was positive in 30 SG samples from 18 patients (60% of total SG samples and 75% of all cases). Ultrastructural analyses showed spherical 70-100 nm viral particles, consistent in size and shape with the Coronaviridae family, in the ductal lining cell cytoplasm, acinar cells, and ductal lumen of SGs. There was also degeneration of organelles in infected cells and the presence of a cluster of nucleocapsids, which suggests viral replication in SG cells. Qualitative histopathological analysis showed morphologic alterations in the duct lining epithelium characterized by cytoplasmic and nuclear vacuolization, as well as nuclear pleomorphism. Acinar cells showed degenerative changes of the zymogen granules and enlarged nuclei. Ductal epithelium and serous acinar cells showed intense expression of ACE2 and TMPRSS receptors. An anti-SARS-CoV-2 antibody was positive in 8 (53%) of the 15 tested cases in duct lining epithelial cells and acinar cells of major SGs. Only two minor salivary glands were positive for SARS-CoV-2 by immunohistochemistry. Salivary glands are a reservoir for SARS-CoV-2 and provide a pathophysiological background for studies that indicate the use of saliva as a diagnostic method for COVID-19 and highlight this biological fluid's role in spreading the disease. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hepatitis B virus genotypes and subgenotypes and the natural history and epidemiology of hepatitis B.

INTRODUCTION AND OBJECTIVES: Espírito Santo state is considered a region with a higher frequency of hepatitis B virus infection. This study characterized demographic, epidemiological, laboratory, virological and clinical aspects of 587 chronic HBV carriers followed up at the University of Espírito Santo Hospital. MATERIALS AND METHODS: Demographic, epidemiological, laboratory and clinical data were extracted from medical records during the entire follow-up period. Classification of the evolutionary phases of chronic hepatitis B was defined as immunotolerant; inactive carrier; chronic active hepatitis HBeAg (+)  and HBeAg (-). Characterization of HBV genotypes/subgenotypes was performed by sequencing of overlapping surface antigens and HBV DNA polymerase genes. Phylogenetic relationships were determined using BEAST 1.8.3 software. RESULTS: and Conclusions: Genotypes found were A (132/65.3%) [A1 = 129 (63.9%) and A2 = 3 (1.5%)], D (66/32.7%) [D3 = 56 (27.7%), D4 = 8 (4.0%) and D2 = 2 (1.0%)] and F (4/2.0%) - all F2a. Subgenotypes A1 or D3 were not associated with age, sex, HIV/HCV co-infection, viral load, antiviral usage, HBeAg status or clinical stages of chronic hepatitis B. Mother -to-child-transmission (MTCT) was associated with the subgenotype A1 and intrafamilial transmission with subgenotype D3. Subgenotype A1 was more frequent than D3 among individuals born outside ES compared to those born in ES. CONCLUSIONS: The most predominant clinical phases were HBeAg (-), inactive carrier and chronic active hepatitis HBeAg (-). Subgenotypes A1 and D3 were most frequent and were associated were MTCT and intrafamilial transmission of HBV, respectively.

Analysis of the complete genome of HBV genotypes F and H found in Brazil and Mexico using the next generation sequencing method.

INTRODUCTION AND OBJECTIVES: Hepatitis B Virus is classified into ten different genotypes (A- J). Genotypes F and H cluster apart from others in phylogenetic trees and is particularly frequent in the Americas. The aim of this study was to sequence complete genomes of samples of HBV genotypes F and H from Brazil and Mexico using next generation sequencing (NGS) and to study relevant characteristics for the disease associated with this virus. MATERIALS AND METHODS: Ninety plasma samples with detectable HBV DNA belonging to the F (n=59) and H (n=31) genotypes were submitted to amplification of the complete HBV genome by three different methologies. Data analysis was developed using bioinformatics tools for quality assurance and comprehensive coverage of the genome. Sequences were aligned with reference sequences for subgenotyping and detecting variants in relevant positions. A phylogenetical tree was constructed using Bayesian methods. RESULTS: HBV genome of 31 samples were amplified and 18 of them were sequenced (HBV/F=16 and HBV/H=2). One genotype F sample was co-infected with the F1b and F3 subgenotypes, while the other samples were all F2a subgenotype. Two genotype H samples clustered with other Mexican sequences. The main variants observed were found in preS and S genes (7/18) and mutations in the precore/core region (11/18). CONCLUSIONS: A NGS methodology was applied to F and H genotypes samples from Mexico and Brazil to fully characterize their sequences. This methodology will be relevant for clinical and epidemiological studies of hepatitis B in Latin America.

Ultrasound-Guided Minimally Invasive Tissue Sampling: A Minimally Invasive Autopsy Strategy During the COVID-19 Pandemic in Brazil, 2020.

BACKGROUND: Minimally invasive autopsies, also known as minimally invasive tissue sampling (MITS), have proven to be an alternative to complete diagnostic autopsies (CDAs) in places or situations where this procedure cannot be performed. During the coronavirus disease 2019 (COVID-19) pandemic, CDAs were suspended by March 2020 in Brazil to reduce biohazard. To contribute to the understanding of COVID-19 pathology, we have conducted ultrasound (US)-guided MITS as a strategy. METHODS: This case series study includes 80 autopsies performed in patients with COVID-19 confirmed by laboratorial tests. Different organs were sampled using a standardized MITS protocol. Tissues were submitted to histopathological analysis as well as immunohistochemical and molecular analysis and electron microscopy in selected cases. RESULTS: US-guided MITS proved to be a safe and highly accurate procedure; none of the personnel were infected, and accuracy ranged from 69.1% for kidney, up to 90.1% for lungs, and reaching 98.7% and 97.5% for liver and heart, respectively. US-guided MITS provided a systemic view of the disease, describing the most common pathological findings and identifying viral and other infectious agents using ancillary techniques, and also allowed COVID-19 diagnosis confirmation in 5% of the cases that were negative in premortem and postmortem nasopharyngeal/oropharyngeal swab real-time reverse-transcription polymerase chain reaction. CONCLUSIONS: Our data showed that US-guided MITS has the capacity similar to CDA not only to identify but also to characterize emergent diseases.

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.

Pulmonary and systemic involvement in COVID-19 patients assessed with ultrasound-guided minimally invasive autopsy.

AIMS: Brazil ranks high in the number of coronavirus disease 19 (COVID-19) cases and the COVID-19 mortality rate. In this context, autopsies are important to confirm the disease, determine associated conditions, and study the pathophysiology of this novel disease. The aim of this study was to assess the systemic involvement of COVID-19. In order to follow biosafety recommendations, we used ultrasound-guided minimally invasive autopsy (MIA-US), and we present the results of 10 initial autopsies. METHODS AND RESULTS: We used MIA-US for tissue sampling of the lungs, liver, heart, kidneys, spleen, brain, skin, skeletal muscle and testis for histology, and reverse transcription polymerase chain reaction to detect severe acute respiratory syndrome coronavirus 2 RNA. All patients showed exudative/proliferative diffuse alveolar damage. There were intense pleomorphic cytopathic effects on the respiratory epithelium, including airway and alveolar cells. Fibrinous thrombi in alveolar arterioles were present in eight patients, and all patients showed a high density of alveolar megakaryocytes. Small thrombi were less frequently observed in the glomeruli, spleen, heart, dermis, testis, and liver sinusoids. The main systemic findings were associated with comorbidities, age, and sepsis, in addition to possible tissue damage due to the viral infection, such as myositis, dermatitis, myocarditis, and orchitis. CONCLUSIONS: MIA-US is safe and effective for the study of severe COVID-19. Our findings show that COVID-19 is a systemic disease causing major events in the lungs and with involvement of various organs and tissues. Pulmonary changes result from severe epithelial injury and microthrombotic vascular phenomena. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches.

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.

Lipase and factor V (but not viral load) are prognostic factors for the evolution of severe yellow fever cases.

BACKGROUND: Despite a highly efficacious vaccine, yellow fever (YF) is still a major threat in developing countries and a cause of outbreaks. In 2018, the Brazilian state of São Paulo witnessed a new YF outbreak in areas where the virus has not been detected before. OBJECTIVE: The aim is to describe the clinical and laboratorial characteristics of severe cases of YF, evaluate viral to determine markers associated with fatal outcome. METHODS: Acute severe YF cases (n = 62) were admitted to the Intensive Care Unit of a reference hospital and submitted to routine laboratorial evaluation on admission. YFV-RNA was detected in serum and urine by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and then sequenced. Patients were classified in two groups: survival or death. FINDINGS: In the univariate analysis the following variables were associated with outcome: alanin aminotransferase (ALT), aspartat aminotransferase (AST), AST/ALT ratio, total bilirubin (TB), chronic kidney disease epidemiology collaboration (CKD-EPI), ammonia, lipase, factor V, international normalised ratio (INR), lactate and bicarbonate. Logistic regression model showed two independent variables associated with death: lipase [odds ratio (OR) 1.018, 95% confidence interval (CI) 1.007 to 1.030, p = 0.002], and factor V (OR -0.955, 95% CI 0.929 to 0.982, p = 0.001). The estimated lipase and factor V cut-off values that maximised sensitivity and specificity for death prediction were 147.5 U/L [area under the curve (AUC) = 0.879], and 56.5% (AUC = 0.913). MAIN CONCLUSIONS: YF acute severe cases show a generalised involvement of different organs (liver, spleen, heart, kidneys, intestines and pancreas), and different parameters were related to outcome. Factor V and lipase are independent variables associated with death, reinforcing the importance of hemorrhagic events due to fulminant liver failure and pointing to pancreatitis as a relevant event in the outcome of the disease.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

Serological and molecular markers of hepatitis E virus infection in HIV-infected patients in Brazil.

In Brazil, the circulation of hepatitis E virus (HEV) has been demonstrated in distinct groups of individuals and some animals, but its prevalence among individuals with human immunodeficiency virus (HIV) infection is unknown. This study aimed to assess the frequency of serological and molecular HEV markers in individuals infected with HIV from São Paulo, Brazil. Serum and plasma samples of 354 HIV-infected patients collected between 2007 and 2013 were included. All samples were tested for anti-HEV IgG and IgM antibodies and HEV RNA. Anti-HEV IgG and IgM antibodies were detected in 10.7% (38/354) and 1.4% (5/354) of the samples, respectively. Both antibodies were detected simultaneously in only two samples. HEV RNA was not detected in any sample. There was no significant correlation of anti-HEV serological status (positivity to anti-HEV IgG and/or IgM) with sex, age, CD4+ T cell count, HIV viral load, antiretroviral therapy, liver enzyme levels, or coinfection with hepatitis B virus and/or hepatitis C virus. Our study provides serological evidence of past and recent HEV infections in HIV-infected patients from São Paulo, Brazil. However, the occurrence of ongoing HEV infection appears be a rare event in this population.

High Prevalence of Hepatitis B Subgenotype D4 in Northeast Brazil: an Ancient Relic from African Continent?

INTRODUCTION: Hepatitis B virus (HBV) infection leads to a chronic liver disease that is distributed worldwide. The characterization of HBV into genotypes/subgenotypes is not only a mere procedure for distinguishing different HBV strains around the world because determining their geographic distribution is crucial to understanding their spread across the world. MATERIAL AND METHODS: We characterized different HBV genotypes and subgenotypes in five municipalities located in northeastern Maranhão, in the Brazilian north Atlantic coast. 92 HBsAg-positive individuals were submitted to PCR (polymerase chain reaction). Fifty samples were sequenced using automated Sanger sequencing and classified by phylogenetic methods. RESULTS: Subgenotypes D4 and A1 were found in 42 (84%) and eight (16%) samples, respectively. To our knowledge, this is the first study to describe a high frequency of subgenotype D4 in any population. Subgenotype A1 is frequently found across Brazil, but D4 has been rarely detected and only in a few Brazilian states. This study shows the characterization of HBV subgenotypes from a population based study in the state of Maranhão, particularly in populations that do not have frequent contact with populations from other regions of the world. CONCLUSION: Our findings showed a HBV subgenotype profile that probably reflect the viruses that were brought with the slave trade from Africa to Maranhão. This study also reinforces the need to evaluate the status of HBV dispersion not only in large urban centers, but also in the hinterland, to enable the implementation of effective control and treatment measures.

Characterization of clinical predictors of naturally occurring NS3/NS4A protease polymorphism in genotype 1 hepatitis C virus mono and HIV co-infected patients.

Spontaneously occurring resistance may impair the success of protease inhibitors based regimens in HCV treatment. This study aimed to evaluate associations between amino acid substitutions in NS3/NS4A domain and clinical features of 247 HCV mono or HCV/HIV co-infected patients. Fourteen samples (5.7%) harbored at least one resistance-associated substitution (RAS). The following RASs were detected in NS3 region: T54S (6-2.4%), V55A (7-2.8%), and Q80R (2-0.8%). S122G occurred in 86.9% of HCV genotype 1b samples with either natural polymorphisms or RASs. Advanced liver fibrosis and HIV co-infection were not related to NS3/NS4A amino acid substitutions.

Mutation in the S gene a determinant of the hepatitis B virus associated with concomitant HBsAg and anti-HBs in a population in Northeastern Brazil.

Mutations in the a determinant of S gene may develop co-existence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in the serum of infected hepatitis B virus (HBV) individuals. Mutations in this region may change the antigenicity of HBsAg, which in turn, lead to escape of neutralizing action of anti-HBs antibodies. This study identified individuals with concomitant HBsAg and anti-HBs serological markers in individuals of Maranhão, Northeastern Brazil. Samples from a population-based study were evaluated for HBsAg, anti-HBs, and anti-HBc, and those that tested positive for simultaneous HBsAg and anti-HBs were submitted to HBV DNA quantification and S gene characterization by Sanger sequencing. Mutations were investigated in the a determinant located in major hydrophilic region (MHR) of the S gene. Among 3,984 samples analyzed, 92 (2.3%) were positive for HBsAg and three had the atypical HBsAg and anti-HBs-positive profile (3.26%). The frequency of HBsAg and anti-HBs co-existence was similar to previous studies. Only one individual harbored mutation in the S gene a determinant associated with this profile. Little is known about this phenomenon; however, studies as ours may contribute for future enlightenment of this important issue. J. Med. Virol. 89:458-462, 2017. © 2016 Wiley Periodicals, Inc.

Natural occurrence of NS5B inhibitor resistance-associated variants in Brazilian patients infected with HCV or HCV and HIV.

Resistance-associated variants (RAVs) represent a challenge to the success of new HCV therapies. The aim of this study was to describe the prevalence of naturally occurring NS5B RAVs in Brazilian direct acting antivirals (DAA)-naïve patients infected with HCV genotype 1, or co-infected with HIV. Patient enrollment and sample collection were performed between 2011 and 2013. Using Sanger-based sequencing, 244 sequences were obtained. RAVs detected in HCV-1a sequences were V321A (1.6 %), M414V (1.3 %), A421V (21.4-23.7 %), A421G (1.3 %) and Y448H (1.3 %); and in HCV-1b sequences were L159F (16.1 %), C316N (7.1-16.3 %) and A421V (3.2-6.3 %). Understanding the real RAVs scenario in patients is fundamental to establishing the most effective therapeutic strategy and in minimizing the risks for their selection.

Basal core promoter and precore mutations among hepatitis B virus circulating in Brazil and its association with severe forms of hepatic diseases.

BACKGROUND: In Brazil, few studies have investigated the prevalence of infection with the precore (PC) and basal core promoter (BCP) mutants of the hepatitis B virus (HBV). OBJECTIVES: This study aimed to analyse the frequency of PC and BCP mutations among patients infected with HBV and to evaluate the association between the variants and advanced hepatic disease. METHODS: A total of 161 patients infected with HBV were studied. To identify PC and BCP mutations, a 501-bp fragment of HBV DNA was amplified and sequenced. FINDINGS: PC and BCP regions from HBV strains were successfully amplified and sequenced in 129 and 118 cases, respectively. PC and BCP mutations were detected in 61.0% and 80.6% of the cases, respectively. The A1762T/G1764A variant was identified in 36.7% of the patients with grade 1 and 2 liver fibrosis (29/79) and in 81.8% of the patients with grade 3 and 4 liver fibrosis (9/11) (p < 0.01); in 76.9% of the patients with cirrhosis (10/13) and in 38.1% of the patients without cirrhosis (40/105) (p = 0.01); and in 77.8% of the patients with hepatocellular carcinoma (HCC) (7/9) and in 39.4% of the patients without HCC (43/109) (p = 0.03). MAIN CONCLUSIONS: A high prevalence of HBV PC and BCP mutants was found. The A1762T/G1764A variant was independently associated with advanced forms of liver fibrosis, hepatic cirrhosis, and HCC.

HCV inter-subtype 1a/1b recombinant detected by complete-genome next-generation sequencing.

Next-generation sequencing (NGS) provides a practical approach to HCV complete-genome sequencing, detecting low-frequency variants and allowing analysis of viral genetic diversity (quasispecies) in the sample, and so far, it is very useful for identifying preexisting drug-resistant mutants and emerging escape mutations, as well as detecting viral recombinants containing genomic regions from different genotypes and subtypes. The aim of this study was to analyze the complete coding region of hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) from patients with chronic infection who were direct-acting antiviral (DAA) naïve. Next-generation sequencing (Ion Torrent™ PGM) was used to determine the sequence of the complete coding region of 100 HCV-monoinfected DAA-naïve patients (51 and 49 subtypes 1a and 1b, respectively). We report the first description of nearly complete HCV genome sequences of subtype 1a and 1b isolates from a large population of Brazilian patients with chronic hepatitis C, and HCV-1a grouped in two different clades. Using this methodology, an inter-subtype 1a/1b recombinant was identified in this study.

Serum lipidomic profiling as a useful tool for screening potential biomarkers of hepatitis B-related hepatocellular carcinoma by ultraperformance liquid chromatography-mass spectrometry.

BACKGROUND: Chronic hepatitis B (CHB) virus infection is a major cause of hepatocellular carcinoma (HCC), as late diagnosis is the main factor for the poor survival of patients. There is an urgent need for accurate biomarkers for early diagnosis of HCC. The aim of the study was to explore the serum lipidome profiles of hepatitis B-related HCC to identify potential diagnostic biomarkers. METHODS: An ultraperformance liquid chromatography mass spectrometry (UPLC-MS) lipidomic method was used to characterize serum profiles from HCC (n = 32), liver cirrhosis (LC) (n = 30), CHB (n = 25), and healthy subjects (n = 34). Patients were diagnosed by clinical laboratory and imaging evidence and all presented with CHB while healthy controls had normal liver function and no infectious diseases. RESULTS: The UPLC-MS-based serum lipidomic profile provided more accurate diagnosis for LC patients than conventional alpha-fetoprotein (AFP) detection. HCC patients were discriminated from LC with 78 % sensitivity and 64 % specificity. In comparison, AFP showed sensitivity and specificity of 38 % and 93 %, respectively. HCC was differentiated from CHB with 100 % sensitivity and specificity using the UPLC-MS approach. Identified lipids comprised glycerophosphocolines, glycerophosphoserines and glycerophosphoinositols. CONCLUSIONS: UPLC-MS lipid profiling proved to be an efficient and convenient tool for diagnosis and screening of HCC in a high-risk population.

Multilocus sequence typing of Candida tropicalis shows the presence of different clonal clusters and fluconazole susceptibility profiles in sequential isolates from candidemia patients in Sao Paulo, Brazil.

The profiles of 61 Candida tropicalis isolates from 43 patients (28 adults and 15 children) diagnosed with candidemia at two teaching hospitals in São Paulo, Brazil, were characterized by multilocus sequence typing (MLST). For the 14 patients who had bloodstream infections, 32 isolates were serially collected from their blood and/or catheters. Thirty-nine diploid sequence types (DSTs) were differentiated. According to the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/), 36 DSTs and 23 genotypes identified from the 61 isolates had not previously been described. This report represents the first study to characterize sequential isolates of C. tropicalis from candidemia cases in South America. Microvariation in a single gene was found in the sequential isolates from 7 patients. The main polymorphisms occurred in the alleles of the XYR1 gene, specifically at nucleotide positions 215, 242, and 344. Macrovariation in six gene fragments was detected in the isolates from 3 patients. eBURST analysis added two new groups to this study (groups 6 and 18). Additionally, susceptibility tests indicate that 3 isolates were resistant to fluconazole. No correlation was found between the DSTs and susceptibility to fluconazole and/or selective antifungal pressure. Two patients were sequentially infected with resistant and susceptible strains. MLST is an important tool for studying the genetic diversity of multiple/sequential isolates of patients with candidemia, allowing the comparison of our data with those from other regions of the world, as well as allowing an analysis of the genetic relationship among several clones in sequential isolates from the same or different candidemia patient sites (blood or catheter).