ABSTRACT The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

ABSTRACT Blastocystis sp. is a protist commonly found in stool samples of humans and animals. Biological and genetic factors of this organism remain controversial. The present study aimed to develop and implement the Blastocystis in vitro culture of Brazilian human isolates for routine use. The fecal isolates (n = 20) were maintained in our laboratory by several passages in Pavlova's medium. Cultures were monitored every 72 h by light microscopy. Genomic DNA was extracted to identify the subtypes (STs). In most isolates, the vacuolar form was prevalent. The amoeboid, granular and cystic forms were observed during in vitro cultivation. STs 1, 2, 3, 4 and 7 were identified. Our preliminary results show the generation time and forms present in the in vitro culture of Blastocystis subtypes isolated from Brazilian human isolates. Therefore, we emphasize the use of in vitro culture as a tool in future studies for the better understanding of the biological aspects of Blastocystis sp.

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

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.

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.

Abstract Hepatitis B virus (HBV) is distributed worldwide, with geographical variations regarding prevalence of the different genotypes. The aim of this study was to determine the HBV genotypes and subgenotypes circulating in Southeast Brazil and compare the genetic sequences found with HBV sequences previously described in the world. Sequences from 166 chronic HBV carriers were analyzed using the fragment constituted by 1306 base pairs comprising surface and polymerase regions of the HBV genome. The sequences obtained were submitted to phylogenetic analysis. HBV subgenotypes A1, A2, D1-D4, F2a, and F4 were found. HBV genotype D was the most frequent, found in 99 patients (58.4%). Within this group, subgenotype D3 was the most prevalent, in 73 patients (42.9%). HBV genotype A was identified in 58 (36%) patients, subgenotype A1, in 48 (29.8%) subjects. Genotype F was identified in 9 (5.4%). According to the phylogenetic analysis, the sequences found were grouped with sequences from Europe, Asia and Middle East (subgenotypes D1, D2, D3) and sequences from Latin America and Africa (subgenotype A1). HBV D3 grouped in different clusters inside D3 clade, several of them with sequences isolated in Italy. We also identified eight families whose relatives were infected with the same HBV subgenotype, most with high similarity between sequences. In conclusion, the distribution of the HBV sequences obtained interweaved with sequences from other continents, corresponding to regions from where many immigrants came to this region, in accordance to the hypothesis that the HBV detected over there were brought during the colonization times.

ABSTRACT OBJECTIVE To investigate the HCV cascade of care and to identify the factors associated with loss or absence to follow-up of patients identified as infected with hepatitis C through blood donation. METHODS Blood donors from 1994 to 2012, identified with positive anti- HCV by enzyme immunoassay and immunoblot tests were invited to participate in the study, through letters or phone calls. Patients who agreed to participate were interviewed and their blood samples were collected for further testing. The following variables were investigated: demographic data, data on comorbidities and history concerning monitoring of hepatitis C. Multiple regression analysis by Poisson regression model was used to investigate the factors associated with non-referral for consultation or loss of follow-up. RESULTS Of the 2,952 HCV-infected blood donors, 22.8% agreed to participate: 394 (58.2%) male, median age 48 years old and 364 (53.8%) Caucasian. Of the 676 participants, 39.7% did not receive proper follow-up or treatment after diagnosis: 45 patients referred not to be aware they were infected, 61 did not seek medical attention and 163 started a follow-up program, but were non-adherent. The main reasons for inadequate follow-up were not understanding the need for medical care (71%) and health care access difficulties (14%). The variables showing a significant association with inadequate follow-up after multiple regression analysis were male gender (PR = 1.40; 95%CI 1.15–1.71), age under or equal to 50 years (PR = 1.36; 95%CI 1.12–1.65) and non-Caucasians (PR = 1.53; 95%CI 1.27–1.84). CONCLUSIONS About 40.0% of patients did not receive appropriate follow-up. These data reinforce the need to establish strong links between primary care and reference centers and the need to improve access to specialists and treatments.

ABSTRACT OBJECTIVE To investigate the HCV cascade of care and to identify the factors associated with loss or absence to follow-up of patients identified as infected with hepatitis C through blood donation. METHODS Blood donors from 1994 to 2012, identified with positive anti- HCV by enzyme immunoassay and immunoblot tests were invited to participate in the study, through letters or phone calls. Patients who agreed to participate were interviewed and their blood samples were collected for further testing. The following variables were investigated: demographic data, data on comorbidities and history concerning monitoring of hepatitis C. Multiple regression analysis by Poisson regression model was used to investigate the factors associated with non-referral for consultation or loss of follow-up. RESULTS Of the 2,952 HCV-infected blood donors, 22.8% agreed to participate: 394 (58.2%) male, median age 48 years old and 364 (53.8%) Caucasian. Of the 676 participants, 39.7% did not receive proper follow-up or treatment after diagnosis: 45 patients referred not to be aware they were infected, 61 did not seek medical attention and 163 started a follow-up program, but were non-adherent. The main reasons for inadequate follow-up were not understanding the need for medical care (71%) and health care access difficulties (14%). The variables showing a significant association with inadequate follow-up after multiple regression analysis were male gender (PR = 1.40; 95%CI 1.15–1.71), age under or equal to 50 years (PR = 1.36; 95%CI 1.12–1.65) and non-Caucasians (PR = 1.53; 95%CI 1.27–1.84). CONCLUSIONS About 40.0% of patients did not receive appropriate follow-up. These data reinforce the need to establish strong links between primary care and reference centers and the need to improve access to specialists and treatments.

SUMMARY Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients.

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.

Strongyloides venezuelensis é um nematódeo parasita de roedores frequentemente utilizado como modelo experimental da estrongiloidíase humana e animal. O objetivo deste estudo foi avaliar a correlação entre o diagnóstico parasitológico, efetuado por meio de exame direto e cultura das fezes em carvão e técnicas moleculares na infecção experimental por S. venezuelensis. A reação de PCR foi utilizada para detectar DNA de S. venezuelensis em amostras fecais obtidas de Rattus norvegicus infectados com este nematódeo. Os resultados demonstraram a maior sensibilidade da reação de PCR em detectar a infecção em relação aos métodos parasitológicos.

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.