Evaluation of renal markers and liver enzymes in patients infected with the Chikungunya virus.

Chikungunya virus (CHIKV) is an arbovirus (Togaviridae family, Alphavirus genus) that was first identified in 1953 in Tanzania. In 2014, the Asian and East/Central/South/African (ECSA) genotypes were identified in Brazil, although the genotype that spread the most in the following years across the Brazilian territory was the ECSA. The clinical symptoms associated with the infection caused by CHIKV include mainly fever, myalgia, headache, and arthralgia. In infections caused by other arboviruses (such as the ones caused by Dengue and West Nile viruses), changes in biochemical markers are often observed. This study aims to evaluate the biochemical markers profile of kidney and liver injury in acute patients infected with CHIKV. Two groups of correlations were found between the variables analyzed, namely, one between liver enzymes (r = 0.91), and another for kidney markers (r = 0.54-0.66). A significant elevation in the percentage of altered creatinine in CHIKV-infected patients was observed, followed by uric acid and AST. Altogether, in 8 different comparisons, it was possible to observe statistically significant differences between the levels of the markers when compared to the manifestation of symptoms (presence and absence). These noticeable changes in marker measurements could potentially be connected to the range of clinical symptoms seen in the disease.

Co-circulation of Chikungunya virus, Zika virus, and serotype 1 of Dengue virus in Western Bahia, Brazil.

Chikungunya, mayaro, dengue, zika, and yellow fever are mosquito-borne viral diseases caused, respectively, by Chikungunya virus, Mayaro virus (CHIKV and MAYV, respectively: Togaviridae: Alphavirus), Dengue virus, Zika virus, and Yellow fever virus (DENV, ZIKV, and YFV, respectively: Flaviviridae: Flavivirus). These viruses have an important epidemiological impact worldwide, especially in Brazil. Western Bahia is one of the less studied regions in that country regarding the circulation of these pathogens. In this study, we aimed to apply molecular biology assays to better know the mosquito-borne viruses circulating in Barreiras and Luís Eduardo Magalhães, two main cities of Western Bahia. From March to June 2021, we enrolled 98 patients with the clinical diagnosis of dengue. Personal information (gender and age) were retrieved at the moment of enrollment. Serum samples were obtained from volunteers and used in molecular detection of CHIKV, MAYV, DENV, ZIKV, and YFV by reverse transcription followed by real-time polymerase chain reaction as well as in genome sequencing aiming phylogenetic analysis. As the main result, we found that from the 98 patients 45 were infected by CHIKV, 32 were infected by serotype 1 of DENV (DENV-1) and six were infected by ZIKV, while 15 were negative for all arboviruses tested. In addition, phylogenetic analysis revealed that all CHIKV-positive samples were of the East/Central/South African (ECSA) genotype, while all DENV-1-positive samples were of the V genotype. These results clearly show that epidemiological surveillance cannot be based only on clinical evaluations. Laboratory diagnosis is important in arbovirus infection that are prevalent in a particular area. These findings also demonstrate the co-circulation of many arboviruses in Western Bahia in 2021.

Understanding yellow fever-associated myocardial injury: an autopsy study.

BACKGROUND: Yellow fever (YF) is a viral hemorrhagic fever, endemic in parts of South America and Africa. There is scarce evidence about the pathogenesis of the myocardial injury. The objective of this study is to evaluate the cardiac pathology in fatal cases of YF. METHODS: This retrospective autopsy study included cases from the São Paulo (Brazil) epidemic of 2017-2019. We reviewed medical records and performed cardiac tissue histopathological evaluation, electron microscopy, immunohistochemical assays, RT-qPCR for YF virus (YFV)-RNA, and proteomics analysis on inflammatory and endothelial biomarkers. FINDINGS: Seventy-three confirmed YF cases with a median age of 48 (34-60) years were included. We observed myocardial fibrosis in 68 (93.2%) patients; cardiomyocyte hypertrophy in 68 (93.2%); endothelial alterations in 67 (91.8%); fiber necrosis in 50 (68.5%); viral myocarditis in 9 (12.3%); and secondary myocarditis in 5 (6.8%). Four out of five patients with 17DD vaccine-associated viscerotropic disease presented with myocarditis. The cardiac conduction system showed edema, hemorrhages and endothelial fibrinoid necrosis. Immunohistochemistry detected CD68-positive inflammatory interstitial cells and YFV antigens in endothelial and inflammatory cells. YFV-RNA was detected positive in 95.7% of the cardiac samples. The proteomics analysis demonstrated that YF patients had higher levels of multiple inflammatory and endothelial biomarkers in comparison to cardiovascular controls, and higher levels of interferon gamma-induced protein 10 (IP-10) in comparison to sepsis (p = 0.01) and cardiovascular controls (p < 0.001) in Dunn test. INTERPRETATION: Myocardial injury is frequent in severe YF, due to multifactorial mechanisms, including direct YFV-mediated damage, endothelial cell injury, and inflammatory response, with a possible prominent role for IP-10. FUNDING: This study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, Bill and Melinda Gates Foundation, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

Severe COVID-19 patients exhibit elevated levels of autoantibodies targeting cardiolipin and platelet glycoprotein with age: a systems biology approach.

Age is a significant risk factor for the coronavirus disease 2019 (COVID-19) severity due to immunosenescence and certain age-dependent medical conditions (e.g., obesity, cardiovascular disorder, and chronic respiratory disease). However, despite the well-known influence of age on autoantibody biology in health and disease, its impact on the risk of developing severe COVID-19 remains poorly explored. Here, we performed a cross-sectional study of autoantibodies directed against 58 targets associated with autoimmune diseases in 159 individuals with different COVID-19 severity (71 mild, 61 moderate, and 27 with severe symptoms) and 73 healthy controls. We found that the natural production of autoantibodies increases with age and is exacerbated by SARS-CoV-2 infection, mostly in severe COVID-19 patients. Multiple linear regression analysis showed that severe COVID-19 patients have a significant age-associated increase of autoantibody levels against 16 targets (e.g., amyloid ß peptide, ß catenin, cardiolipin, claudin, enteric nerve, fibulin, insulin receptor a, and platelet glycoprotein). Principal component analysis with spectrum decomposition and hierarchical clustering analysis based on these autoantibodies indicated an age-dependent stratification of severe COVID-19 patients. Random forest analysis ranked autoantibodies targeting cardiolipin, claudin, and platelet glycoprotein as the three most crucial autoantibodies for the stratification of severe COVID-19 patients ≥50 years of age. Follow-up analysis using binomial logistic regression found that anti-cardiolipin and anti-platelet glycoprotein autoantibodies significantly increased the likelihood of developing a severe COVID-19 phenotype with aging. These findings provide key insights to explain why aging increases the chance of developing more severe COVID-19 phenotypes.

Phylogeographic patterns of the yellow fever virus around the metropolitan region of São Paulo, Brazil, 2016-2019.

From 2016 to 2019, the largest outbreak caused by the Yellow Fever virus (YFV) in the 21st century in the Americas occurred in southeastern Brazil. A sylvatic cycle of transmission was reported near densely populated areas, such as the large metropolitan area of the city of São Paulo. Here, we describe the origin, spread, and movement of the YFV throughout the state of São Paulo. Whole-genome sequences were obtained from tissues of two patients who died due to severe yellow fever, during 2018-2019. Molecular analysis indicated that all analyzed tissues were positive for YFV RNA, with the liver being the organ with the highest amount of viral RNA. Sequence analysis indicates that genomes belonged to the South American genotype I and were grouped in the epidemic clade II, which includes sequences from the states of Goiás, Minas Gerais, and São Paulo of previous years. The analysis of viral dispersion indicates that the outbreak originated in Goiás at the end of 2014 and reached the state of São Paulo through the state of Minas Gerais after 2016. When the virus reached near the urban area, it spread towards both the east and south regions of the state, not establishing an urban transmission cycle in the metropolitan region of São Paulo. The virus that moved towards the east met with YFV coming from the south of the state of Rio de Janeiro, and the YFV that was carried to the south reached the Brazilian states located in the south region of the country.

Chikungunya Virus E2 Structural Protein B-Cell Epitopes Analysis.

The Togaviridae family comprises a large and diverse group of viruses responsible for recurrent outbreaks in humans. Within this family, the Chikungunya virus (CHIKV) is an important Alphavirus in terms of morbidity, mortality, and economic impact on humans in different regions of the world. The objective of this study was to perform an IgG epitope recognition of the CHIKV's structural proteins E2 and E3 using linear synthetic peptides recognized by serum from patients in the convalescence phase of infection. The serum samples used were collected in the state of Sergipe, Brazil in 2016. Based on the results obtained using immunoinformatic predictions, synthetic B-cell peptides corresponding to the epitopes of structural proteins E2 and E3 of the CHIKV were analyzed by the indirect peptide ELISA technique. Protein E2 was the main target of the immune response, and three conserved peptides, corresponding to peptides P3 and P4 located at Domain A and P5 at the end of Domain B, were identified. The peptides P4 and P5 were the most reactive and specific among the 11 epitopes analyzed and showed potential for use in serological diagnostic trials and development and/or improvement of the Chikungunya virus diagnosis and vaccine design.

The impact of Zika virus exposure on the placental proteomic profile.

Zika virus (ZIKV) infection has caused severe unexpected clinical outcomes in neonates and adults during the recent outbreak in Latin America, particularly in Brazil. Congenital malformations associated with ZIKV have been frequently reported; nevertheless, the mechanism of vertical transmission and the involvement of placental cells remains unclear. In this study, we applied quantitative proteomics analysis in a floating explant model of chorionic villi of human placental tissues incubated with ZIKV and with ZIKV pre-adsorbed with anti-ZIKV envelope protein. Proteomic data are available via ProteomeXchange with identifier PXD025764. Altered levels of proteins were involved in cell proliferation, apoptosis, inflammatory processes, and the integrin-cytoskeleton complex. Antibody-opsonized ZIKV particles differentially modulated the pattern of protein expression in placental cells; this phenomenon may play a pivotal role in determining the course of infection and the role of mixed infections. The expression of specific proteins was also evaluated by immunoperoxidase assays. These data fill gaps in our understanding of early events after ZIKV placental exposure and help identify infection control targets.

Systemic dengue infection associated with a new dengue virus type 2 introduction in Brazil - a case report.

BACKGROUND: Dengue infection is caused by an arbovirus with a wide range of presentations, varying from asymptomatic disease to unspecific febrile illness and haemorrhagic syndrome with shock, which can evolve to death. In Brazil, the virus circulates since the 1980s with many introductions of new serotypes, genotypes, and lineages since then. Here we report a fatal case of dengue associated with a Dengue virus (DENV) lineage not detected in the country until now. CASE PRESENTATION: The patient, a 58-year-old man arrived at the hospital complaining of fever and severe abdominal pain due to intense gallbladder edema, mimicking acute abdomen. After 48 h of hospital admission, he evolved to refractory shock and death. DENV RNA was detected in all tissues collected (heart, lung, brain, kidney, spleen, pancreas, liver, and testis). Viral sequencing has shown that the virus belongs to serotype 2, American/Asian genotype, in a new clade, which has never been identified in Brazil before. The virus was phylogenetically related to isolates from central America [Puerto Rico (2005-2007), Martinique (2005), and Guadeloupe (2006)], most likely arriving in Brazil from Puerto Rico. CONCLUSION: In summary, this was the first fatal documented case with systemic dengue infection associated with the new introduction of Dengue type 2 virus in Brazil during the 2019 outbreak.

Origin of the Sao Paulo Yellow Fever epidemic of 2017-2018 revealed through molecular epidemiological analysis of fatal cases

The largest outbreak of yellow fever of the 21st century in the Americas began in 2016, with intense circulation in the southeastern states of Brazil, particularly in sylvatic environments near densely populated areas including the metropolitan region of São Paulo city (MRSP) during 2017­2018. Herein, we describe the origin and molecular epidemiology of yellow fever virus (YFV) during this outbreak inferred from 36 full genome sequences taken from individuals who died following infection with zoonotic YFV. Our analysis revealed that these deaths were due to three genetic variants of sylvatic YFV that belong the South American I genotype and that were related to viruses previously isolated in 2017 from other locations in Brazil (Minas Gerais, Espírito Santo, Bahia and Rio de Janeiro states). Each variant represented an independent virus introduction into the MRSP. Phylogeographic and geopositioning analyses suggested that the virus moved around the peri-urban area without detectable human-to-human transmission, and towards the Atlantic rain forest causing human spill-over in nearby cities, yet in the absence of sustained viral transmission in the urban environment.

Ultrasound-guided minimally invasive autopsy as a tool for rapid post-mortem diagnosis in the 2018 Sao Paulo yellow fever epidemic: Correlation with conventional autopsy.

BACKGROUND: New strategies for collecting post-mortem tissue are necessary, particularly in areas with emerging infections. Minimally invasive autopsy (MIA) has been proposed as an alternative to conventional autopsy (CA), with promising results. Previous studies using MIA addressed the cause of death in adults and children in developing countries. However, none of these studies was conducted in areas with an undergoing infectious disease epidemic. We have recently experienced an epidemic of yellow fever (YF) in Brazil. Aiming to provide new information on low-cost post-mortem techniques that could be applied in regions at risk for infectious outbreaks, we tested the efficacy of ultrasound-guided MIA (MIA-US) in the diagnosis of patients who died during the epidemic. METHODOLOGY/PRINCIPAL FINDINGS: In this observational study, we performed MIA-US in 20 patients with suspected or confirmed YF and compared the results with those obtained in subsequent CAs. Ultrasound-guided biopsies were used for tissue sampling of liver, kidneys, lungs, spleen, and heart. Liver samples from MIA-US and CA were submitted for RT-PCR and immunohistochemistry for detection of YF virus antigen. Of the 20 patients, 17 had YF diagnosis confirmed after autopsy by histopathological and molecular analysis. There was 100% agreement between MIA-US and CA in determining the cause of death (panlobular hepatitis with hepatic failure) and main disease (yellow fever). Further, MIA-US obtained samples with good quality for molecular studies and for the assessment of the systemic involvement of the disease. Main extrahepatic findings were pulmonary hemorrhage, pneumonia, acute tubular necrosis, and glomerulonephritis. One patient was a 24-year-old, 27-week pregnant woman; MIA-US assessed the placenta and provided adequate placental tissue for analysis. CONCLUSIONS: MIA-US is a reliable tool for rapid post-mortem diagnosis of yellow fever and can be used as an alternative to conventional autopsy in regions at risk for hemorrhagic fever outbreaks with limited resources to perform complete diagnostic autopsy.

Origin of the São Paulo Yellow Fever epidemic of 2017-2018 revealed through molecular epidemiological analysis of fatal cases.

The largest outbreak of yellow fever of the 21st century in the Americas began in 2016, with intense circulation in the southeastern states of Brazil, particularly in sylvatic environments near densely populated areas including the metropolitan region of São Paulo city (MRSP) during 2017-2018. Herein, we describe the origin and molecular epidemiology of yellow fever virus (YFV) during this outbreak inferred from 36 full genome sequences taken from individuals who died following infection with zoonotic YFV. Our analysis revealed that these deaths were due to three genetic variants of sylvatic YFV that belong the South American I genotype and that were related to viruses previously isolated in 2017 from other locations in Brazil (Minas Gerais, Espírito Santo, Bahia and Rio de Janeiro states). Each variant represented an independent virus introduction into the MRSP. Phylogeographic and geopositioning analyses suggested that the virus moved around the peri-urban area without detectable human-to-human transmission, and towards the Atlantic rain forest causing human spill-over in nearby cities, yet in the absence of sustained viral transmission in the urban environment.

Outbreak of chikungunya virus in a vulnerable population of Sergipe, Brazil-A molecular and serological survey.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging arbovirus that is causing outbreaks in several countries of the Americas. The virus was introduced in Brazil in 2014, and since then, several Brazilian states have notified autochthonous cases. OBJECTIVES: Provide additional evidence on a CHIKV outbreak and an outline of the laboratory and clinical profile of symptomatic patients in Sergipe, Brazil. STUDY DESIGN: In February 2016, we collected 142 serum samples from symptomatic patients for arboviruses in Sergipe, Brazil. All samples were submitted to qRT-PCR for the emerging arboviruses circulating in Brazil - ZIKV, CHIKV, and DENV - and later submitted to the immunoenzymatic assay. RNA positive samples were randomly selected and sequenced for characterization of the genotype involved in the outbreak. RESULTS: Our study had 75.35% (107/142) positivity for CHIKV infection, with all age groups and genera being equally infected. The virus was identified in 11 of the 13 cities studied in that state, including the ECSA genotype. Importantly, fever was the only statistically significant symptoms for CHIKV infection (p<0.05), while asthenia was significantly associated with symptomatic patients that were CHIKV-negative (p<0.05). CONCLUSIONS: Our findings support the importance of fever as a clinical marker and contribute to molecular and serological surveillance data, which may help in the understanding of CHIKV circulation, emergence and clinical description.

Outbreak of chikungunya virus in a vulnerable population of Sergipe, Brazil—a molecular and serological survey

Background Chikungunya virus (CHIKV) is a re-emerging arbovirus that is causing outbreaks in several countries of the Americas. The virus was introduced in Brazil in 2014, and since then, several Brazilian states have notified autochthonous cases. Objectives Provide additional evidence on a CHIKV outbreak and an outline of the laboratory and clinical profile of symptomatic patients in Sergipe, Brazil. Study design In February 2016, we collected 142 serum samples from symptomatic patients for arboviruses in Sergipe, Brazil. All samples were submitted to qRT-PCR for the emerging arboviruses circulating in Brazil – ZIKV, CHIKV, and DENV – and later submitted to the immunoenzymatic assay. RNA positive samples were randomly selected and sequenced for characterization of the genotype involved in the outbreak. Results Our study had 75.35% (107/142) positivity for CHIKV infection, with all age groups and genera being equally infected. The virus was identified in 11 of the 13 cities studied in that state, including the ECSA genotype. Importantly, fever was the only statistically significant symptoms for CHIKV infection (p < 0.05), while asthenia was significantly associated with symptomatic patients that were CHIKV-negative (p < 0.05). Conclusions Our findings support the importance of fever as a clinical marker and contribute to molecular and serological surveillance data, which may help in the understanding of CHIKV circulation, emergence and clinical description.