RESUMO
Metagenomic next-generation sequencing (mNGS) methodology serves as an excellent supplement in cases where diagnosis is challenging to establish through conventional laboratory tests, and its usage is increasingly prevalent. Examining the causes of infectious diseases in the central nervous system (CNS) is vital for understanding their spread, managing outbreaks, and effective patient care. In a study conducted in the state of São Paulo, Brazil, cerebrospinal fluid (CSF) samples from 500 patients with CNS diseases of indeterminate etiology, collected between 2017 and 2021, were analyzed. Employing a mNGS approach, we obtained the complete coding sequence of Pegivirus hominis (HPgV) genotype 2 in a sample from a patient with encephalitis (named IAL-425/BRA/SP/2019); no other pathogen was detected. Subsequently, to determine the extent of this virus's presence, both polymerase chain reaction (PCR) and/or real-time PCR assays were utilized on the entire collection. The presence of the virus was identified in 4.0% of the samples analyzed. This research constitutes the first report of HPgV detection in CSF samples in South America. Analysis of the IAL-425 genome (9107 nt) revealed a 90% nucleotide identity with HPgV strains from various countries. Evolutionary analyses suggest that HPgV is both endemic and extensively distributed. The direct involvement of HPgV in CNS infections in these patients remains uncertain.
RESUMO
Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417-501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs.
Assuntos
COVID-19 , Evolução Molecular , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/virologia , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.
Assuntos
Flavivirus/genética , Vigilância em Saúde Pública/métodos , Animais , Brasil , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil.
Assuntos
Betacoronavirus/genética , Doenças Transmissíveis Importadas/transmissão , Infecções por Coronavirus/transmissão , Pandemias , Pneumonia Viral/transmissão , Idoso , Brasil/epidemiologia , COVID-19 , Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/virologia , Infecções por Coronavirus/epidemiologia , Humanos , Pessoa de Meia-Idade , Filogenia , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2RESUMO
In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.
Assuntos
Culicidae/virologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Genoma Viral , Animais , Sequência de Bases , Brasil/epidemiologia , Infecções por Flaviviridae/epidemiologia , Infecções por Flaviviridae/virologia , Camundongos , Mosquitos Vetores/virologia , Filogenia , RNA Viral/genéticaRESUMO
We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil. (AU)
Assuntos
Brasil , Vigilância em Saúde Pública , SARS-CoV-2 , COVID-19 , COVID-19/transmissãoRESUMO
Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016-2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.
Assuntos
Culicidae/virologia , Surtos de Doenças/classificação , Haplorrinos/virologia , Febre Amarela/epidemiologia , Vírus da Febre Amarela/classificação , Animais , Antígenos Virais/análise , Teorema de Bayes , Brasil/epidemiologia , Linhagem Celular , Humanos , Filogenia , Filogeografia , Sequenciamento Completo do Genoma , Febre Amarela/imunologia , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/isolamento & purificação , Zoonoses/virologiaRESUMO
INTRODUCTION: Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. METHODS: The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. RESULTS: Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. CONCLUSIONS: This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.
Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Infecções por Rickettsia/epidemiologia , Rickettsia/imunologia , Adulto , Brasil/epidemiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Prevalência , Rickettsia/classificação , Infecções por Rickettsia/diagnóstico , Estudos SoroepidemiológicosRESUMO
Brazilian spotted fever (BSF) is caused by the bacterium Rickettsia rickettsii. Because of its high case-fatality rate and apparent increase in areas of transmission, it is considered to be the rickettsial illness of primary public health interest. Cases of this disease have historically occurred in Southeastern Brazil. This article reports the first fatal case of BSF in Southern Brazil. This case high lights the importance of BSF to be considered as a differential diagnosis for acute hemorrhagic fever in areas where cases of BSF may not be expected.
Assuntos
Febre Maculosa das Montanhas Rochosas/diagnóstico , Anticorpos Antibacterianos/sangue , Brasil , Criança , Evolução Fatal , Feminino , Humanos , Rickettsia rickettsii/imunologiaRESUMO
Abstract Brazilian spotted fever (BSF) is caused by the bacterium Rickettsia rickettsii. Because of its high case-fatality rate and apparent increase in areas of transmission, it is considered to be the rickettsial illness of primary public health interest. Cases of this disease have historically occurred in Southeastern Brazil. This article reports the first fatal case of BSF in Southern Brazil. This case high lights the importance of BSF to be considered as a differential diagnosis for acute hemorrhagic fever in areas where cases of BSF may not be expected.
Assuntos
Humanos , Feminino , Criança , Febre Maculosa das Montanhas Rochosas/diagnóstico , Rickettsia rickettsii/imunologia , Brasil , Evolução Fatal , Anticorpos Antibacterianos/sangueRESUMO
Abstract INTRODUCTION Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. METHODS The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. RESULTS Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. CONCLUSIONS This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.
Assuntos
Humanos , Masculino , Feminino , Adulto , Rickettsia/imunologia , Infecções por Rickettsia/epidemiologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Anticorpos Antibacterianos/sangue , Rickettsia/classificação , Infecções por Rickettsia/diagnóstico , Brasil/epidemiologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Estudos Soroepidemiológicos , Prevalência , Técnica Indireta de Fluorescência para AnticorpoRESUMO
This study presents a new recombinant protein that acts as a powerful antiviral (rAVLO-recombinant Antiviral protein of Lonomia obliqua). It was able to reduce the replication by 10(6) fold for herpes virus and by 10(4) fold for rubella virus. RT-PCR of viral RNA rAVLO treated infected cells also showed similar rate of inhibition in replication. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide and has the ability to bind to MHC class I. It was found that there are several protein binding sites with various HLA and a prevalence of α-helices in the N-terminal region (overall classified as a α/ß protein type). BLAST similarity sequence search for corresponding cDNA did not reveal a similar sequence in Genbank, suggesting that it is from a novel protein family. In this study we have observed that this recombinant protein and hemolymph has a potent antiviral action. This protein was produced in a baculovirus/Sf-9 system. Therefore, these analyses suggest that this novel polypeptide is a candidate as a broad spectrum antiviral.
RESUMO
A Febre Maculosa Brasileira (FMB), causada pela Rickettsia rickettsii, é a principal doença transmitida por carrapato com impacto em saúde pública no Brasil. Os primeiros sintomas são inespecíficos, porém a doença pode evoluir rapidamente para quadro de Síndrome Febril Hemorrágica Aguda (SFHA) com a ocorrência de óbito em poucos dias. O objetivo deste estudo foi verificar as aplicações da PCR em tempo real na rotina laboratorial para o diagnóstico etiológico da FMB, em amostras biológicas encaminhadas ao Laboratório de Riquétsias do Instituto Adolfo Lutz. Foram testados 2 protocolos de PCR em tempo real: um para detecção do gênero Rickettsia spp (gltA-TaqMan®) com detecção por sonda TaqMan® e outro para riquétsias do Grupo Febre Maculosa (OmpA-SYBR) com detecção por SYBRGreen. O protocolo de PCR em tempo real para RNAseP foi utilizado como controle interno endógeno. A amostragem foi constituída de sangue, soro, coágulo e biópsia de pele de lesão de casos fatais e não fatais com suspeita clínica de FMB. Os resultados mostraram que o protocolo OmpA-SYBR sofre interferência da matriz biológica e os melhores resultados com relação à sensibilidade foram obtidos quando utilizado em amostras de soro. Os protocolos de PCR em tempo real gltA-TaqMan® e OmpA-SYBR apresentaram concordância de resultados acima de 90%. O protocolo gltA-TaqMan® mostrou-se mais sensível do que o protocolo OmpA-SYBR, porém com menor especificidade, particularmente para amostras com CT>36. O melhor desempenho da PCR em tempo real para FMB, foi obtido quando a FMPCR, composta do três protocolos: gltA-TaqMan®, OmpA-SYBR e RNAseP humana, foi utilizada em amostras de soro para detecção do agente etiológico da FMB nos casos fatais. A PCR em tempo real para FMB apresentou baixa sensibilidade para detectar casos não fatais, confirmados pela sorologia para FMB, com positividade de 21%. Os resultados deste estudo indicam que a FMPCR apresenta sensibilidade e especificidade que permitem utilizá-la...
Brazilian Spotted Fever (BSF) is the main tick borne disease with impact on public health in Brazil. Early symptoms are nonspecific, but the disease may progress rapidly to Febrile Acute Hemorrhagic Syndrome (SFHA) with occurrence of death within a few days. The purpose of this study was to investigate the applications of real-time PCR in the routine laboratory for the etiologic diagnosis of BSF, in the biological samples sent to the laboratory. Two real-time PCR protocols were tested: one for detecting the genus Rickettsia spp (gltA-TaqMan®) with detection by TaqMan ® probe and another for specific detection of Spotted Fever Group species (OmpA-SYBR) by SYBR Green detection. The qPCR protocol for RNaseP was used as endogenous internal control. Samples used were blood, serum, blood clot and biopsy from skin lesion of fatal and nonfatal clinically suspected of BSF. The results showed that ompA-SYBR protocol suffers interference from the biological matrix and the best performance was obtained with serum samples. Protocols OmpA-SYBR and gltA-TaqMan® showed results concordance up to 90%. The protocol gltA-TaqMan® was more sensitive than OmpA-SYBR protocol, but less specificity, particularly for samples with TC> 36. The best performance for BSF qPCR assay was obtained when combining three qPCR protocols (OmpA-SYBR, gltA-TaqMan® and RnaseP), named FMPCR, were used in serum samples to detection BSF in fatal cases. FMPCR showed low sensitivity for detecting non-fatal cases positive by IFA, the positivity was 21%. The results of this study indicate that FMPCR has sensitivity and specificity to be used as a diagnostic tool for elucidation of fatal BSF...
Assuntos
Humanos , Febre Maculosa das Montanhas Rochosas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rickettsia rickettsii , Técnicas e Procedimentos DiagnósticosRESUMO
Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cut-off IgG and/or IgM ≥ 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n=86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Patologia Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rickettsia/isolamento & purificação , Febre Maculosa das Montanhas Rochosas/diagnóstico , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Brasil , Citrato (si)-Sintase/genética , DNA Bacteriano/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Rickettsia/genética , Sensibilidade e EspecificidadeAssuntos
Doenças Transmissíveis Emergentes/epidemiologia , Vírus do Sarampo/genética , Sarampo/epidemiologia , Adolescente , Adulto , Brasil/epidemiologia , Criança , Pré-Escolar , Doenças Transmissíveis Emergentes/virologia , Humanos , Lactente , Sarampo/diagnóstico , Sarampo/virologia , Vigilância da População , Adulto JovemAssuntos
Adolescente , Adulto , Criança , Pré-Escolar , Humanos , Lactente , Adulto Jovem , Doenças Transmissíveis Emergentes/epidemiologia , Vírus do Sarampo/genética , Sarampo/epidemiologia , Brasil/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Sarampo/diagnóstico , Sarampo/virologia , Vigilância da PopulaçãoRESUMO
Os autores apresentam sua experiência no diagnóstico laboratorial de InfluenzaA (H1N1) em 37.240 amostras clínicas obtidas de pacientes com suspeita de gripe, encaminhadas ao Instituto Adolfo Lutz para análise. Eles apresentam os algoritmos de testes moleculares empregados, comparam a eficiência dos mesmos quanto à sensibilidade, especificidade e custo e, finalmente sugerem um novo algoritmo para ser usado em caso de nova epidemia de Influenza A (H1N1) em 2010.
The authors present their experience with the molecular diagnosis of Influenza A (H1N1) with 37.240 clinicalsamples obtained from individuals suspected of flu, sent to Instituto Adolfo Lutz for analysis. They show the used algorithms, compare their efficiency in terms of sensitivity, specificity and cost, and suggest a new algorithm to be employed in case of an outbreak of Influenza A (H1N1) in 2010.