Polimorfismo no gene PNPLA3 e o consumo de carne vermelha aumentam o risco de fibrose em pacientes com DHGNA comprovado por biópsia em acompanhamento médico em um centro terciário no Sudoeste do Brasil / Pnpla3 gene polymorphism and red meat consumption increased fibrosis risk in nash biopsy-proven patients under medical follow-up in a tertiary center in southwest brazil

ABSTRACT Background: Recent studies show an increase in nonalcoholic fatty liver disease (NAFLD) in populations with higher consumption of red meat, processed and cooked at high temperatures. On the other hand, the single nucleotide polymorphism rs738409 in the Patatin-like phospholipase domain containing 3 (PNPLA3) gene has been implicated in susceptibility to NAFLD and liver fibrosis. However, the synergistic effect between red meat consumption and the PNPLA3 gene polymorphism in NAFLD has not yet been evaluated. Objective: To evaluate the association between the presence of the polymorphism in the PNPLA3 gene and the consumption of macronutrients, including meat consumption and its cooking method among NAFLD patients. Methods: This was a cross-sectional study with 91 patients diagnosed with NAFLD by liver biopsy with genotyping for the polymorphism in the PNPLA3 gene were included. The consumption of calories and macronutrients was verified using the semi-quantitative food frequency questionnaire and the specific questionnaire on meat consumption. PNPLA3 gene polymorphism was analyzed by real-time polymerase chain reaction (RT-PCR) and anthropometric evaluation was realized. Results: The mean BMI was 32.38±4.58 kg/m² and the waist circumference was 107±10 cm. On liver biopsy, 42% of patients had significant fibrosis (F≥2). The odds ratio of F≥2 was 2.12 for the GG group and 1.54 for the CG group, compared to the CC group. The mean caloric intake was 1170±463.20 kcal/d. The odds ratio in the CC group concerning high red meat consumption in comparison to low consumption was 1.33. For white meat, the odds ratio was 0.8 when comparing high and low intake, also in the CC group. Conclusion: High red meat intake and PNPLA3 gene polymorphism seem to synergistically affect NAFLD and liver fibrosis, requiring confirmation in a larger number of patients and in different populations.

RESUMO Contexto: Estudos recentes mostram um aumento da doença hepática gordurosa não alcoólica (DHGNA) em populações com maior consumo de carne vermelha, processada e cozida em altas temperaturas. Por outro lado, o polimorfismo rs738409 no gene Patatin-like fosfolipase contendo 3 (PNPLA3) tem sido implicado na suscetibilidade à DHGNA e fibrose hepática. No entanto, o efeito sinérgico entre o consumo de carne vermelha e o polimorfismo no gene PNPLA3 na DHGNA ainda não foi avaliado. Objetivo: Avaliar a associação entre a presença do polimorfismo no gene PNPLA3 e o consumo de macronutrientes, incluindo o consumo de carne e seu modo de cozimento em pacientes com DHGNA. Métodos: Realizamos um estudo transversal com 91 pacientes diagnosticados com DHGNA por biópsia hepática e genotipados para o polimorfismo no gene PNPLA3. O consumo de calorias e macronutrientes foi verificado por meio do questionário de frequência alimentar semi-quantitativo (QFA) e do questionário específico sobre consumo de carnes. O polimorfismo no gene PNPLA3 foi analisado por reação em cadeia da polimerase em tempo real (RT-PCR) e a avaliação antropométrica foi realizada. Resultados: O índice de massa corporal médio foi de 32,38±4,58 kg/m² e a circunferência da cintura foi de 107±10 cm. Na biópsia hepática, 42% dos pacientes apresentavam fibrose significativa (F≥2). O odds ratio de F≥2 foi de 2,12 para o grupo GG e 1,54 para o grupo GC, comparado ao grupo CC. A ingestão calórica média foi de 1.170±463,20 kcal/d. O odds ratio para alto consumo de carne vermelha no grupo CC em comparação ao baixo consumo foi de 1,33. Para a carne branca, este valor foi de 0,8 ao comparar o alto e o baixo consumo, também no grupo CC. Conclusão: A alta ingestão de carne vermelha e o polimorfismo no gene PNPLA3 parecem afetar sinergicamente a DHGNA e a fibrose hepática, necessitando de confirmação em maior número de pacientes e em diferentes populações.

Associations of SARS-CoV-2 cycle threshold values with age, gender, sample collection setting, and pandemic period

ABSTRACT Cycle threshold (Ct) values in COVID-19 reverse-transcription polymerase chain reaction (RT-PCR) tests estimate the viral load in biological samples. Studies have investigated variables associated with SARS-CoV-2 viral load, aiming to identify factors associated with higher transmissibility. Using the results from tests performed between May/2020-July/2022 obtained from the database of a referent hospital in Sao Paulo, Brazil, we investigated associations between Ct values and patient's age, gender, sample collection setting and pandemic period according to the predominant SARS-CoV-2 variant locally. We also examined variations in Ct values, COVID-19 incidence, mortality, and vaccination coverage over time. The study sample included 42,741 tests. Gender was not significantly associated with Ct values. Age, sample collection setting and the pandemic period were significantly associated with Ct values even after adjustment to the multivariable model. Results showed lower Ct values in older groups, during the Gamma and Delta periods, and in samples collected in emergency units; and higher Ct values in children under 10 years old, home-based tests, during the Omicron period. We found evidence of a linear trend in the association between age and Ct values, with Ct values decreasing as age increases. We found no clear temporal associations between Ct values and local indicators of COVID-19 incidence, mortality, or vaccination between February/2020-November/2022. Our findings suggest that SARS-CoV-2 Ct values, a proxy for viral load and transmissibility, can be influenced by demographic and epidemiological variables.

Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil

Abstract Background Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. Methods The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. Results A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. Conclusion Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

Clinical characteristics and outcomes of patients with COVID-19 admitted to the intensive care unit during the first and second waves of the pandemic in Brazil: a single-center retrospective cohort study

ABSTRACT Objective To describe and compare the clinical characteristics and outcomes of patients admitted to intensive care units during the first and second waves of the COVID-19 pandemic. Methods In this retrospective single-center cohort study, data were retrieved from the Epimed Monitor System; all adult patients admitted to the intensive care unit between March 4, 2020, and October 1, 2021, were included in the study. We compared the clinical characteristics and outcomes of patients admitted to the intensive care unit of a quaternary private hospital in São Paulo, Brazil, during the first (May 1, 2020, to August 31, 2020) and second (March 1, 2021, to June 30, 2021) waves of the COVID-19 pandemic. Results In total, 1,427 patients with COVID-19 were admitted to the intensive care unit during the first (421 patients) and second (1,006 patients) waves. Compared with the first wave group [median (IQR)], the second wave group was younger [57 (46-70) versus 67 (52-80) years; p<0.001], had a lower SAPS 3 Score [45 (42-52) versus 49 (43-57); p<0.001], lower SOFA Score on intensive care unit admission [3 (1-6) versus 4 (2-6); p=0.018], lower Charlson Comorbidity Index [0 (0-1) versus 1 (0-2); p<0.001], and were less frequently frail (10.4% versus 18.1%; p<0.001). The second wave group used more noninvasive ventilation (81.3% versus 53.4%; p<0.001) and high-flow nasal cannula (63.2% versus 23.0%; p<0.001) during their intensive care unit stay. The intensive care unit (11.3% versus 10.5%; p=0.696) and in-hospital mortality (12.3% versus 12.1%; p=0.998) rates did not differ between both waves. Conclusion In the first and second waves, patients with severe COVID-19 exhibited similar mortality rates and need for invasive organ support, despite the second wave group being younger and less severely ill at the time of intensive care unit admission.

High prevalence of hepatitis B virus and low vaccine response in children and adolescents in Northeastern Brazil

ABSTRACT Children have an increased likelihood of becoming carriers of the chronic hepatitis B virus. A total of 1,381 children and adolescents were assessed in five municipalities of Maranhao State, Brazil, for detection of anti-HBc, HBsAg and anti-HBs serologic markers and sociodemographic and behavioral features. Among those who were HBsAg negative and anti-HBc negative, the proportion of anti-HBs positives was calculated after the individuals had completed the vaccination schedule. The robust variance of the Poisson's regression model was used in order to have adjusted tables and calculate the prevalence ratio. Multivariate analysis was performed to identify the factors associated with the prevalence of anti-HBc with or without HBsAg and the vaccine response. It was observed that 163 children were anti-HBc positive and nine individuals were HBsAg positive. The factors associated with the infection were: municipality of residence (residing in Morros municipality or Humberto de Campos municipality), residence in a rural area, aged between 13 and 15 years old, and illicit drug use. The percentage of individuals who were anti-HBc negative and received all three doses of the vaccine was 48.5%. Among these, only 276 (38.9%) had antibodies at protective concentrations. In an adjusted analysis, Morros municipality presented an increased positivity of vaccine response (p < 0.001), and the age ranging between 6 and 10 years old presented a reduced frequency of response. This study reveals a high prevalence of current and past HBV infection within the targeted age group which, in addition to the low vaccination coverage and serological responses, raises concerns about the management of prevention measures, especially the quality of vaccination in these locations.

Seroprevalence of hepatitis B, C and D markers in indigenous patients seen at the Native American Outpatient Clinic of Universidade Federal de São Paulo

ABSTRACT Objective To detect and treat cases of viral hepatitis B, C and D in patients seen at the Native American Outpatient Clinic of Universidade Federal de São Paulo. Methods This sample comprised 81 indigenous recruited between 2018 and 2020. Volunteers were aged 7 months to 70 years (mean age of 28±20 years), belonged to 26 ethnic groups spanning the Brazilian territory and answered a questionnaire, which was attached to their medical records. Peripheral blood samples (20mL) were collected, transported to the Clinical Laboratory of Hospital Israelita Albert Einstein, processed, and tested for markers of viral hepatitis B, C and D. Results In this study, 39 (48.1%) individuals were anti-HBs (+) only, 13 (16.0%) individuals were anti-HBs (+) and anti-HBc (+), and 28 (34.6%) individuals were negative for all markers. No anti-HBc IgM+ samples were found. No cases of hepatitis C and D were found. Conclusion This analysis provided evidence of previous infection by the hepatitis B virus. These findings led to prescription of vaccination against hepatitis B to all participants who were negative for all viral hepatitis B markers, given records of prior hepatitis B vaccination were unreliable.

SARS-CoV-2 and rhinovirus infections: are there differences in clinical presentation, laboratory abnormalities, and outcomes in the pediatric population?

ABSTRACT This study aims to assess COVID-19 and other respiratory viruses in pediatric patients. Between April 17 and September 30, 2020, we collected 1,566 respiratory samples from 1,044 symptomatic patients who were younger than 18 years old to assess SARS-CoV-2 infection. Of these, 919 were analyzed for other respiratory pathogens (ORP). Patients with laboratory-confirmed COVID-19 or ORP were included. We evaluated 76 pediatric COVID-19 infections and 157 other respiratory virus infections. Rhinovirus occurred in 132/157 (84%). COVID-19 patients who were significantly older, had more fevers, headaches and pneumonia than those with ORP. The median white blood cell count was lower in patients with SARS-CoV-2 than in those with ORP (6,470 versus 8,170; p=0.02). COVID-19 patients had significantly worse symptoms than those with ORP.

Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis

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.

Salivary glands are a target for SARS-CoV-2: a source for salivacontamination

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 innasopharyngeal swabs. We conducted ultrasound-guided postmortem biopsies in COVID-19 fatal cases. Samples ofsalivary glands (SGs; parotid, submandibular, and minor) were obtained. We analyzed samples using RT-qPCR, immu-nohistochemistry, electron microscopy, and histopathological analysis to identify SARS-CoV-2 and elucidate qual-itative and quantitative viral proles in salivary glands. The study included 13 female and 11 male patients, with amean age of 53.12 years (range 8­83 years). RT-qPCR for SARS-CoV-2 was positive in 30 SG samples from18 patients (60% of total SG samples and 75% of all cases). Ultrastructural analyses showed spherical 70­100 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 acluster of nucleocapsids, which suggests viral replication in SG cells. Qualitative histopathological analysis showedmorphologic alterations in the duct lining epithelium characterized by cytoplasmic and nuclear vacuolization, as wellas 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 cellsof major SGs. Only two minor salivary glands were positive for SARS-CoV-2 by immunohistochemistry. Salivaryglands are a reservoir for SARS-CoV-2 and provide a pathophysiological background for studies that indicate theuse of saliva as a diagnostic method for COVID-19 and highlight this biological uid's role in spreading the disease.© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

An autopsy study of the spectrum of severe COVID-19 in children: From SARS to different phenotypes of MIS-C

Background: COVID-19 in children is usually mild or asymptomatic, but severe and fatal paediatric cases have been described. The pathology of COVID-19 in children is not known; the proposed pathogenesis for severe cases includes immune-mediated mechanisms or the direct effect of SARS-CoV-2 on tissues. We describe the autopsy findings in five cases of paediatric COVID-19 and provide mechanistic insight into the mechanisms involved in the pathogenesis of the disease. Methods: Children and adolescents who died with COVID-19 between March 18 and August 15, 2020 were autopsied with a minimally invasive method. Tissue samples from all vital organs were analysed by histology, electron microscopy (EM), reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Findings: Five patients were included, one male and four female, aged 7 months to 15 years. Two patients had severe diseases before SARS-CoV-2 infection: adrenal carcinoma and Edwards syndrome. Three patients were previously healthy and had multisystem inflammatory syndrome in children (MIS-C) with distinct clinical presentations: myocarditis, colitis, and acute encephalopathy with status epilepticus. Autopsy findings varied amongst patients and included mild to severe COVID-19 pneumonia, pulmonary microthrombosis, cerebral oedema with reactive gliosis, myocarditis, intestinal inflammation, and haemophagocytosis. SARSCoV- 2 was detected in all patients in lungs, heart and kidneys by at least one method (RT-PCR, IHC or EM), and in endothelial cells from heart and brain in two patients with MIS-C (IHC). In addition, we show for the first time the presence of SARS-CoV-2 in the brain tissue of a child with MIS-C with acute encephalopathy, and in the intestinal tissue of a child with acute colitis. Interpretation: SARS-CoV-2 can infect several cell and tissue types in paediatric patients, and the target organ for the...(AU)

Differences in children and adolescents with SARS-CoV-2 infection: a cohort study in a Brazilian tertiary referral hospital

OBJECTIVES: To compare demographic/clinical/laboratory/treatments and outcomes among children and adolescents with laboratory-confirmed coronavirus disease 2019 (COVID-19). METHODS: This was a cross-sectional study that included patients diagnosed with pediatric COVID-19 (aged <18 years) between April 11, 2020 and April 22, 2021. During this period, 102/5,951 (1.7%) of all admissions occurred in neonates, children, and adolescents. Furthermore, 3,962 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection samples were processed in patients aged <18 years, and laboratory-confirmed COVID-19 occurred in 155 (4%) inpatients and outpatients. Six/155 pediatric patients were excluded from the study. Therefore, the final group included 149 children and adolescents (n=97 inpatients and 52 outpatients) with positive SARS-CoV-2 results. RESULTS: The frequencies of sore throat, anosmia, dysgeusia, headache, myalgia, nausea, lymphopenia, pre-existing chronic conditions, immunosuppressive conditions, and autoimmune diseases were significantly reduced in children and adolescents (p<0.05). Likewise, the frequencies of enoxaparin use (p=0.037), current immunosuppressant use (p=0.008), vasoactive agents (p=0.045), arterial hypotension (p<0.001), and shock (p=0.024) were significantly lower in children than in adolescents. Logistic regression analysis showed that adolescents with laboratory-confirmed COVID-19 had increased odds ratios (ORs) for sore throat (OR 13.054; 95% confidence interval [CI] 2.750-61.977; p=0.001), nausea (OR 8.875; 95% CI 1.660-47.446; p=0.011), and lymphopenia (OR 3.575; 95% CI 1.355-9.430; p=0.010), but also had less hospitalizations (OR 0.355; 95% CI 0.138-0.916; p=0.032). The additional logistic regression analysis on patients with preexisting chronic conditions (n=108) showed that death as an outcome was significantly associated with pediatric severe acute respiratory syndrome (SARS) (OR 22.300; 95% CI 2.341-212.421; p=0.007) and multisystem inflammatory syndrome in children (MIS-C) (OR 11.261; 95% CI 1.189-106. 581; p=0.035). CONCLUSIONS: Half of the laboratory-confirmed COVID-19 cases occurred in adolescents. Individuals belonging to this age group had an acute systemic involvement of SARS-CoV-2 infection. Pediatric SARS and MIS-C were the most important factors associated with the mortality rate in pediatric chronic conditions with COVID-19.

Long term persistence of coronavirus SARS-CoV-2 infection / infecção persistente de longo prazo pelo coronavírus SARS-CoV-2

ABSTRACT During the COVID-19 pandemic, a case of a long-term persistence of SARS-CoV-2 infection (from March 26 to May 20, 2020) was identified at a private hospital in São Paulo, SP, Brazil. The long-term positivity for SARS-CoV-2 in reverse transcriptase polymerase chain reaction tests of a patient diagnosed with COVID-19 suggests, at least part of patients who recovered, may still carry and transmit the SARS-CoV-2 virus. This fact emphasizes the importance of having at least two negative reverse transcriptase polymerase chain reaction test results for SARS-CoV-2. Serological assays were not particularly helpful in the case described, since the patient had very low antibodies titers at the end of the follow-up period. Low viral loads may not be detected by current molecular methods, leading to wrong conclusions regarding viral clearance.

RESUMO Durante a pandemia da COVID-19, um caso de persistência de longo prazo de infecção por SARS-CoV-2, de 26 de março a 20 de maio de 2020, foi identificado em um hospital privado localizado em São Paulo, SP, Brasil. A positividade de longo prazo para SARS-CoV-2 nos exames de reação em cadeia da polimerase via transcriptase reversa de uma paciente diagnosticada com COVID-19 sugere que parte dos pacientes que se recuperaram podem ser portadores e transmitir o SARS-CoV-2. Esse fato enfatiza a importância da obtenção de pelo menos dois resultados negativos para SARS-CoV-2 no exame de reação em cadeia da polimerase via transcriptase reversa. Os ensaios sorológicos não foram de grande utilidade no caso descrito, uma vez que a paciente apresentava baixos títulos de anticorpos no final do período de acompanhamento. Baixas cargas virais podem não ser detectadas pelos métodos moleculares vigentes, levando a conclusões equivocadas a respeito da eliminação do vírus.

The impact of hepatitis E infection on hepatic fibrosis in liver transplanted patients for hepatitis C infection

ABSTRACT Hepatitis E Virus (HEV) is an infection known worldwide for its asymptomatic and self-limited course in most cases. Some cases progressing to chronicity have been described in immunosuppressed patients, especially in recipients of solid organ transplants. We evaluated laboratory parameters of HEV infection (HEV RNA, anti-HEV IgM and anti-HEV IgG) through enzyme-linked immunosorbent assay (Elisa), confirmed by immunoblotting, in a cohort of 294 patients who received liver transplants at the HCFMUSP (Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo). Laboratory and demographic data were collected from the entirety of the transplanted population. Hepatic biopsies of 122 patients transplanted due liver failure secondary to hepatitis C (HCV), with or without serological or molecular markers of HEV, were analyzed according to METAVIR score. Out of 24 (8.2%) patients tested positive for anti-HEV IgG, six (2%) were positive for anti-HEV IgM and 17 (5.8%) for HEV RNA. Of the patients transplanted because of HCV infection, 95 (77.8%) had received treatment including ribavirin for at least six months before blood sample collection. Among patients transplanted due to HCV cirrhosis who tested positive for anti-HEV IgG, only three (37.5%) showed fibrosis beyond stage 2, while five (41.7%) of the HEV RNA-positive patients had liver fibrosis beyond stage 2. Overall, the prevalence of HEV in the post-hepatic transplant scenario appears to be low, and, at least histologically, seemingly not harmful. We conclude that, although some studies reported a risk of HEV chronification, patients who had their livers transplanted due to HCV and showed serological or molecular markers of HEV did not have higher levels of fibrosis compared to patients who showed no indications of HEV infection at the time of the analysis.

Detection of coronavirus-2 by real-time reverse transcription polymerase chain reaction in conjunctival swabs from patients with severe form of Coronavirus disease 2019 in São Paulo, Brazil

OBJECTIVES: To test conjunctival swabs from patients with laboratory-confirmed severe forms of coronavirus disease 2019 (COVID-19) for the presence of SARS-CoV-2 on real-time reverse-transcription polymerase chain reaction (rRT-PCR). METHODS: Fifty conjunctival swabs were collected from 50 in-patients with laboratory-confirmed severe forms of COVID-19 at the largest teaching hospital and referral center in Brazil (HCFMUSP, São Paulo, SP). The samples were tested for SARS-CoV-2 on rRT-PCR with the primers and probes described in the CDC protocol which amplify the region of the nucleocapsid N gene (2019_nCoV_N1 and 2019_nCoV_N2) of SARS-CoV-2 RNA and compared with naso/oropharyngeal swabs collected within 24 hours of the conjunctival swabs. RESULTS: Five conjunctival samples (10%) tested positive (amplification of the N1 and N2 primer/probe sets) while two conjunctival samples (4%) yielded inconclusive results (amplification of the N1 primer/probe set only). The naso/oropharyngeal swabs were positive for SARS-CoV-2 on rRT-PCR in 34 patients (68%), negative in 14 (28%) and inconclusive in 2 (4%). The 5 patients with positive conjunctival swabs had positive (n=2), negative (n=2) or inconclusive (n=1) naso/oropharyngeal swabs on rRT-PCR. Patients with negative or inconclusive naso/oropharyngeal swabs had the diagnosis of COVID-19 confirmed by previous positive rRT-PCR results or by serology. CONCLUSION: This is the first study to present conjunctival swab rRT-PCR results for SARS-CoV-2 in a Brazilian population. In our sample of 50 patients with severe forms of COVID-19, 10% had positive conjunctival swabs, most of which were correlated with positive naso/oropharyngeal rRT-PCR results.

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network

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.

Severe clinical spectrum with high mortality in pediatric patients with COVID-19 and multisystem inflammatory syndrome

OBJECTIVES: To assess the outcomes of pediatric patients with laboratory-confirmed coronavirus disease (COVID-19) with or without multisystem inflammatory syndrome in children (MIS-C). METHODS: This cross-sectional study included 471 samples collected from 371 patients (age<18 years) suspected of having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The study group comprised 66/371 (18%) laboratory-confirmed pediatric COVID-19 patients: 61 (92.5%) patients tested positive on real-time reverse transcription-polymerase chain reaction tests for SARS-CoV-2, and 5 (7.5%) patients tested positive on serological tests. MIS-C was diagnosed according to the criteria of the Center for Disease Control. RESULTS: MIS-C was diagnosed in 6/66 (9%) patients. The frequencies of diarrhea, vomiting, and/or abdominal pain (67% vs. 22%, p=0.034); pediatric SARS (67% vs. 13%, p=0.008); hypoxemia (83% vs. 23%, p=0.006); and arterial hypotension (50% vs. 3%, p=0.004) were significantly higher in patients with MIS-C than in those without MIS-C. The frequencies of C-reactive protein levels >50 mg/L (83% vs. 25%, p=0.008) and D-dimer levels >1000 ng/mL (100% vs. 40%, p=0.007) and the median D-dimer, troponin T, and ferritin levels (p<0.05) were significantly higher in patients with MIS-C. The frequencies of pediatric intensive care unit admission (100% vs. 60%, p=0.003), mechanical ventilation (83% vs. 7%, p<0.001), vasoactive agent use (83% vs. 3%, p<0.001), shock (83% vs. 5%, p<0.001), cardiac abnormalities (100% vs. 2%, p<0.001), and death (67% vs. 3%, p<0.001) were also significantly higher in patients with MIS-C. Similarly, the frequencies of oxygen therapy (100% vs. 33%, p=0.003), intravenous immunoglobulin therapy (67% vs. 2%, p<0.001), aspirin therapy (50% vs. 0%, p<0.001), and current acute renal replacement therapy (50% vs. 2%, p=0.002) were also significantly higher in patients with MIS-C. Logistic regression analysis showed that the presence of MIS-C was significantly associated with gastrointestinal manifestations [odds ratio (OR)=10.98; 95%CI (95% confidence interval)=1.20-100.86; p=0.034] and hypoxemia [OR=16.85; 95%CI=1.34-211.80; p=0.029]. Further univariate analysis showed a positive association between MIS-C and death [OR=58.00; 95%CI=6.39-526.79; p<0.0001]. CONCLUSIONS: Pediatric patients with laboratory-confirmed COVID-19 with MIS-C had a severe clinical spectrum with a high mortality rate. Our study emphasizes the importance of investigating MIS-C in pediatric patients with COVID-19 presenting with gastrointestinal involvement and hypoxemia.

Hepatitis B viremia in HIV-coinfected individuals under antiretroviral therapy

ABSTRACT Background: Antiretroviral therapy (ART) has decreased AIDS incidence and mortality, rendering comorbidities, such as hepatitis B more relevant for people living with human immunodeficiency virus (HIV). Since antiretroviral drugs may also inhibit hepatitis B virus (HBV) replication, analyzing the impact of ART on management of hepatitis B in this population is important. Objective: To assess HBV viremia among HIV/HBV coinfected individuals on ART and its associated factors. Method: For this cross-sectional study, HIV/HBV-coinfected individuals, aged over 18 years, who were on ART for over six months and receiving care at an outpatient clinic in São Paulo were recruited. Sociodemographic characteristics, information about viral exposure, clinical and laboratory data, including evaluation of liver fibrosis were obtained. Plasma HBV DNA was measured by polymerase chain reaction. Viral genome sequencing was conducted for genotyping and identification of drug resistance-conferring mutations if viral load exceeded 900 IU/mL. Results: Out of 2,946 patients who attended the clinic in 2015, 83 were eligible and 56 evaluated. Plasma HBV DNA was detected in 16 (28.6%) (95% CI: 18.0-41.3%), all on lamivudine and tenofovir treatment. HBV DNA detection was associated with lower education (p = 0.015), higher international normalized ratios (p = 0.045), history of an AIDS-defining illness [OR: 3.43 (95% CI: 1.10-11.50)], and HBeAg detection [OR: 6.60 (95% CI: 1.84-23.6)]. In contrast, a last CD4+ count above 500 cells/mm3 in the year prior to inclusion [OR: 0.18 (95% CI: 0.04-0.71)] and detection of anti-HBe [OR: 0.21 (95% CI: 0.04-0.99)] were negatively associated. Patients with HBV DNA above 900 IU/mL were infected with subgenotypes A1 (n = 3) and D2 (n = 1), and exhibited viral mutations associated with total resistance to lamivudine and partial resistance to entecavir. Conclusions: Despite being on ART, a significant proportion of HIV/HBV-coinfected individuals present HBV viremia. Characterization of factors that are associated with this finding may help professionals provide better management to these 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.

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.

Distribution of HBV subgenotypes in Ribeirão Preto, Southeastern Brazil: a region with history of intense Italian immigration

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.