Pegivirus Detection in Cerebrospinal Fluid from Patients with Central Nervous System Infections of Unknown Etiology in Brazil by Viral Metagenomics.

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.

Frequency of congenital cytomegalovirus infections in newborns in the Sao Paulo State, 2010-2018.

Human cytomegalovirus (HCMV) infections remain a neglected public health issue. The aim of the present study was to evaluate the frequency of HCMV congenital infections in newborns up to 1 month in the Sao Paulo State, from 2010 to 2018. The molecular characterization of HCMV-positive samples was also undertaken. Urine samples from 275 potential congenital HCMV-infected patients were tested by real-time Polymerase Chain Reaction (qPCR). HCMV-positive samples were amplified by conventional PCR targeting the UL89 gene, sequenced and searched for mutations. A total of 32 (11.6%) positive-HCMV cases were detected (mean Ct 30.59); mean and median age of 10.3 and 6 days old, respectively. Children aged between 0-3 weeks had higher HCMV detection rates (84.4%; 27/32). UL89 gene was successfully sequenced in two samples, both classified as the human betaherpesvirus 5. No described resistance-associated mutations were identified. A routine screening in newborns coupled with the genetic characterization of key viral genes is vital to decrease sequels associated with congenital HCMV infections.

Isolation of infectious Zika virus from a urine sample cultured in SIRC cells from a patient suspected of having rubella virus.

A great variety of viruses which cause exanthema share other clinical manifestations, making the etiologic identification a very difficult task, relying exclusively on the clinical examination. Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). In the present report, we described the presence of Zika virus (ZIKV) particles in urine samples and also ZIKV isolation in SIRC cells from the urine of a patient in acute phase of suspected rubella disease. The 50-year-old unvaccinated woman living in Sao Paulo, Brazil, was admitted to the emergency room with fever, headache, rash, arthralgia and prostration. Urine samples were collected for virus isolation and RT-qPCR. SIRC and Vero cells were inoculated with urine samples during 7 days. RT-qPCR was performed using measles virus (MV) and RV primers and both were found to be negative. After this result, RT-qPCR was performed for parvovirus B19, herpes virus 6 and ZIKV. The urine sample and the isolate were positive by Real Time PCR for ZIKV and negative for all other viruses tested. The sequences isolated are from the Asiatic lineage.

Human bocavirus in hospitalized children under 5 years with acute respiratory infection, São Paulo, Brazil, 2010.

The aims of this study were to investigate the human bocavirus (HBoV) frequency and genotypes in hospitalized children <5 years presenting acute respiratory infections (ARI) within the São Paulo metropolitan area. Nasopharyngeal samples from 300 patients, previously screened for common respiratory viruses, were tested by qPCR for the NSP1 and NP-1 genes. The VP1/2 gene in positive samples was then amplified by PCR and sequenced. A total of 49 positive HBoV cases (16.3%; mean Ct value of 34.41) were detected with the mean age being 18.1 months (range 1 month to 5 years) and the median age being 1 year of age. Children aged between 0 and 12 months had higher detection rates of HBoV (69.4%; 34/49; mean Ct = 34.45) than children from other age groups (30.6%; 15/49; mean Ct = 34.34). No significant differences were observed between HBoV Ct levels and clinical illness. The occurrence was more frequently associated with fall (38.8%; 19/49) and spring (36.7%; 18/49). All 12 sequenced isolates were identified as HBoV-1, displaying minor genetic variation compared to the Swedish reference strains ST1 and ST2 (99.1-99.7% nt). The sole identification of HBoV-1 supports the hypothesis that this particular genotype is strongly related to ARI, and contributes to the role of this virus in the aetiology of respiratory diseases.

Genotype 1 of human parvovirus B19 in clinical cases.

INTRODUCTION:: Virus surveillance strategies and genetic characterization of human parvovirus B19 (B19V) are important tools for regional and global control of viral outbreak. In São Paulo, Brazil, we performed a study of B19V by monitoring the spread of this virus, which is an infectious agent and could be mistakenly reported as a rash and other types of infection. METHOD:: Serum samples were subjected to enzyme immunoassay, real time polymerase chain reaction, and sequencing. RESULTS:: From the 462 patients with suspected cases of exanthematic infections, the results of the 164 serum samples were positive for B19V immunoglobulin M. Among these cases, there were 38 patients with erythema infections and B19-associated with other infections such as encephalitis, hydrops fetalis, chronic anemia, hematological malignancies. These samples were sequenced and identified as genotype 1. CONCLUSION:: This study showed patients with infections caused by B19V and sequencing genotype 1. Continuous monitoring is necessary to detect all known genotypes, and the emergence of new genotypes of these viruses for case management in public health control activities.

Genotype 1 of human parvovirus B19 in clinical cases / Genótipo 1 do parvovírus humano em casos clínicos

Summary Introduction: Virus surveillance strategies and genetic characterization of human parvovirus B19 (B19V) are important tools for regional and global control of viral outbreak. In São Paulo, Brazil, we performed a study of B19V by monitoring the spread of this virus, which is an infectious agent and could be mistakenly reported as a rash and other types of infection. Method: Serum samples were subjected to enzyme immunoassay, real time polymerase chain reaction, and sequencing. Results: From the 462 patients with suspected cases of exanthematic infections, the results of the 164 serum samples were positive for B19V immunoglobulin M. Among these cases, there were 38 patients with erythema infections and B19-associated with other infections such as encephalitis, hydrops fetalis, chronic anemia, hematological malignancies. These samples were sequenced and identified as genotype 1. Conclusion: This study showed patients with infections caused by B19V and sequencing genotype 1. Continuous monitoring is necessary to detect all known genotypes, and the emergence of new genotypes of these viruses for case management in public health control activities.

Resumo Introdução: Estratégias de vigilância para o parvovírus humano B19 e caracterização genética são ferramentas importantes para o controle regional e global do surto viral. Em São Paulo, Brasil, foi realizado um estudo de parvovírus B19, monitorando a disseminação desse vírus, que é um agente infeccioso e poderia ser erroneamente relatado como uma erupção cutânea e outros tipos de infecções. Método: As amostras de soro foram submetidas ao ensaio imunoenzimático, PCR quantitativo em tempo real e sequenciamento. Resultados: Dos 462 pacientes com casos suspeitos de infecções exantemáticas, os resultados das 164 amostras de soro foram positivos para parvovírus B19 imunoglobulina M. Entre eles, 38 pacientes com eritema infeccioso apresentaram B19 associado com outras infecções, como encefalite, hidropisia fetal, anemia crônica, doenças hematológicas malignas. Essas amostras foram sequenciadas e identificadas como genótipo 1. Conclusão: Os pacientes foram infectados com parvovírus B19 e apresentaram genótipo 1. Monitoração contínua é necessária para detectar todos os genótipos conhecidos e o surgimento de novos genótipos para o controle de casos em saúde pública.

Rubella virus genotype 1G and echovirus 9 as etiologic agents of exanthematous diseases in Brazil: insights from phylogenetic analysis.

The aim of the present study was to identify the rubella virus (RV) and enterovirus (EV) genotypes detected during the Epidemiological Surveillance on Exanthematic Febrile Diseases (VIGIFEX) study and to perform phylogenetic analysis. Ten RV- and four EV-positive oropharyngeal samples isolated from cell culture were subjected to RT-PCR and sequencing. Genotype 1G and echovirus 9 (E-9) was identified in RV- and EV-positive samples, respectively. The RV 1G genotype has been persisting in Brazil since 2000-2001. No evidence of E-9 being involved in exanthematic illness in Brazil has been reported previously. Differential laboratory diagnosis is essential for management of rash and fever disease.

Molecular characterization of human parvovirus B19 associated with neuromyelitis / Caracterização molecular do parvovirus humano B19 associado com neuromielite

INTRODUCTION: Human parvovirus B19 (B19V) is associated with a wide range of clinical symptoms. An acute infection can lead to anemia, erythema infectiosum, or hydrops fetalis, as well as arthritis and other clinical manifestations. Among other diseases associated with B19V, several demyelinating disorders may occur as a result of B19V infection. Phylogenetic analysis of partial erythrovirus sequences resulted in three possible B19V genotype groups. Our study involved analysis of samples from a patient with neuromyelitis that enabled the molecular characterization of human parvovirus B19. OBJECTIVE: To study B19V infection associated with a neurological manifestation and to perform phylogenetic analysis.METHODS: Serum and cerebrospinal fluid samples were tested for B19V infection. The serological assay for B19V was a commercial IgG and IgM enzyme immunoassay kit. Nucleic acid detection was performed using a PCR assay. The phylogenetic analyses were performed using PAUP and other software programs. RESULTS: Our study reports that the topology of viral isolate sequences from a patient with neuromyelitis fell within a clade consisting of B19V genotype 1.CONCLUSION: This result indicates that this virus genotype group has been circulating in Sao Paulo, Brazil. This report represents a rare case in which B19V might be the responsible agent for central nervous system infection.

INTRODUÇÃO: O parvovírus B19 humano (B19V) é associado a vários sintomas clínicos. Uma infecção aguda pode levar à ocorrência de anemia, eritema infeccioso ou hidropsia fetal, bem como artrite e outras manifestações clínicas. Infecções por B19V podem também ocasionar várias doenças desmielinizantes. A análise filogenética das sequências parciais de eritrovírus resultou em três possíveis grupos de genótipos de B19V. Este estudo consistiu na análise de amostras de uma paciente com neuromielite, o que possibilitou a caracterização molecular do parvovírus B19 humano. OBJETIVO: Estudar a infecção por B19V associada a uma manifestação neurológica e realizar sua análise filogenética. MÉTODOS: Amostras de soro e de líquido cefalorraquidiano foram testadas para a observação de infecção por B19V. O teste sorológico utilizado para a pesquisa de B19V foi um kit comercial de ensaio imunoenzimático para IgG e IgM. A detecção de ácido nucleico foi realizada utilizando-se um teste de PCR. As análises filogenéticas foram realizadas utilizando PAUP e outros programas. RESULTADOS: Este artigo registra que a topologia de sequências de isolados virais de uma paciente com neuromielite foram inseridas em um clado constituído de grupos dentro do genótipo 1 de B19V. CONCLUSÃO: Esses achados indicam que este genótipo do vírus tem se mantido circulante em São Paulo, Brasil. Este relato representa um caso raro no qual o B19V pode ter sido o agente responsável pela infecção no sistema nervoso central.

Investigation of measles IgM-seropositive cases of febrile rash illnesses in the absence of documented measles virus transmission, State of São Paulo, Brazil, 2000-2004.

INTRODUCTION: To review measles IgM-positive cases of febrile rash illnesses in the State of São Paulo, Brazil, over the five-year period following interruption of measles virus transmission. METHODS: We reviewed 463 measles IgM-positive cases of febrile rash illness in the State of São Paulo, from 2000 to 2004. Individuals vaccinated against measles < or = 56 days prior to specimen collection were considered to be exposed to the vaccine. Serum from the acute and convalescent phases was tested for evidence of measles, rubella, parvovirus B19 and human herpes virus-6 infection. In the absence of seroconversion to measles immunoglobulin-G, measles IgM-positive cases were considered false positives in individuals with evidence of other viral infections. RESULTS: Among the 463 individuals with febrile rash illness who tested positive for measles IgM antibodies during the period, 297 (64%) were classified as exposed to the vaccine. Among the 166 cases that were not exposed to the vaccine, 109 (66%) were considered false positives based on the absence of seroconversion, among which 21 (13%) had evidence of rubella virus infection, 49 (30%) parvovirus B19 and 28 (17%) human herpes virus-6 infection. CONCLUSIONS: Following the interruption of measles virus transmission, thorough investigation of measles IgM-positive cases is required, especially among cases not exposed to the vaccine. Laboratory testing for etiologies of febrile rash illness aids interpretation of these cases.

Investigation of measles IgM-seropositive cases of febrile rash illnesses in the absence of documented measles virus transmission, State of São Paulo, Brazil, 2000-2004 / Investigação dos casos de doenças febris exantemáticas com IgM reagente contra o sarampo na ausência de transmissão documentada do vírus do sarampo, Estado de São Paulo, 2000-2004

INTRODUCTION: To review measles IgM-positive cases of febrile rash illnesses in the State of São Paulo, Brazil, over the five-year period following interruption of measles virus transmission. METHODS: We reviewed 463 measles IgM-positive cases of febrile rash illness in the State of São Paulo, from 2000 to 2004. Individuals vaccinated against measles < 56 days prior to specimen collection were considered to be exposed to the vaccine. Serum from the acute and convalescent phases was tested for evidence of measles, rubella, parvovirus B19 and human herpes virus-6 infection. In the absence of seroconversion to measles immunoglobulin-G, measles IgM-positive cases were considered false positives in individuals with evidence of other viral infections. RESULTS: Among the 463 individuals with febrile rash illness who tested positive for measles IgM antibodies during the period, 297 (64 percent) were classified as exposed to the vaccine. Among the 166 cases that were not exposed to the vaccine, 109 (66 percent) were considered false positives based on the absence of seroconversion, among which 21 (13 percent) had evidence of rubella virus infection, 49 (30 percent) parvovirus B19 and 28 (17 percent) human herpes virus-6 infection. CONCLUSIONS: Following the interruption of measles virus transmission, thorough investigation of measles IgM-positive cases is required, especially among cases not exposed to the vaccine. Laboratory testing for etiologies of febrile rash illness aids interpretation of these cases.

INTRODUÇÃO: Revisar os casos de doenças febris exantemáticas com IgM reagente contra o sarampo, no Estado de São Paulo, Brasil, durante os cinco anos seguidos a interrupção da transmissão do vírus do sarampo. MÉTODOS: Nós revisamos 463 casos de doenças febris exantemáticas com IgM reagente contra o sarampo, no Estado de São Paulo, Brasil, de 2000 a 2004. Indivíduos vacinados contra o sarampo 56 dias antes da coleta de amostra foram considerados expostos à vacina. Soros da fase aguda e de convalescença foram testados para a evidência de infecção de sarampo, rubéola, parvovírus B19 e herpes vírus 6. Na ausência de soroconversão para imunoglobulina G contra o sarampo, casos com IgM reagente contra o sarampo foram considerados falsos positivos em pessoas com evidência de outras infecções virais. RESULTADOS: Entre as 463 pessoas com doenças febris exantemáticas que testaram positivo para anticorpos IgM contra o sarampo durante o período, 297 (64 por cento) pessoas foram classificadas como expostas à vacina. Entre os 166 casos não expostos à vacina, 109 (66 por cento) foram considerados falsos positivos baseado na ausência de soroconversão, dos quais 21 (13 por cento) tiveram evidência de infecção por vírus da rubéola, 49 (30 por cento) parvovírus B19 e 28 (17 por cento) infecção por herpes vírus humano 6. CONCLUSÕES: Após a interrupção da transmissão do vírus do sarampo é necessária exaustiva investigação dos casos com IgM reagente contra o sarampo, especialmente dos casos não expostos à vacina. Testes laboratoriais para etiologias das doenças febris exantemáticas ajudam na interpretação destes casos.

Growth of rubella virus into SIRC (rabbit cornea cell line) using various culture media

The present study evaluated the efficacy of various culture media for performing the isolation and growth of the rubella virus inoculated into SIRC cells. Rubella virus RA-27/3 strain and RVi/São Paulo/BRA99 wild type strain (Gen Bank number DQ458965) were inoculated into SIRC cell line and cultivated in 199, DMEM, MEM and RPMI media. The inoculated cells when examined on phase contrast microscopy showed the characteristic rounded and multipolar cells. The CPE was observed at the first 48 hours cultivation in the respective tested media. The curve of the infectivity increase was higher in the cultures maintained in DMEM and RPMI media. Hence, the SIRC cellular lineage cultivated in DMEM or RPMI media is an excellent substratum for performing the rubella virus isolation. These findings are relevant since the SIRC has been one of the few cell lines described in the literature which presents a cytopathic effect, and on that account it can be useful for carrying out the virus isolation from clinical specimens.

Detection of human parvovirus B19 in cases of hydrops fetalis in São Paulo, Brazil / Detecção de parvovírus humano B19 em casos de hydropsia fetal em São Paulo, Brasil

Human parvovirus B19 infection is known to be one of the causes of hydrops fetalis. The maternal infection caused by the virus may be symptomatic or asymptomatic. In this study, 40 pregnant women with gestational age of approximately 25 weeks, prenatal diagnosis of non immune hydrops fetalis and suspected of human parvovirus B19 infection were studied between January 1999 and December 2005. Serology results and detection of DNA in the maternal serum, foetal serum and amniotic fluid confirmed that 20 pregnant women had been infected by human parvovirus B19. The ultrasound examination demonstrated foetal hydrops, anaemia, hepatosplenomegaly, ascites, cardiopathy and amniotic fluid disorders. Among the positive cases, there were three fatal losses, one by miscarriage and two by intrauterine foetal death.

A infecção por parvovírus humano B19 é um dos responsáveis pela hidropsia fetal. A infecção materna causada pelo vírus pode ser sintomática ou assintomática. Neste estudo 40 mulheres com idade gestacional de aproximadamente 25 semanas, diagnóstico pré-natal de hidropsia fetal e suspeita de infecção por parvovírus humano B19 foram avaliadas durante o período de janeiro de 1999 a dezembro de 2005. Os resultados de sorologia e detecção de DNA no soro materno, fetal e fluido amniótico confirmaram 20 mulheres grávidas com infecção por parvovírus humano B19. A análise de ultra-som demonstrou hidropsia fetal, anemia, hepatosplenomegalia, ascite, cardiopatia e desordens amnióticas. Entre os casos positivos, ocorreram três perdas fetais: uma por aborto e duas por morte fetal intra-uterina.

Surveillance epidemiological: parvovirus B19 genotype 1

O parvovirus humano B19 foi isolado e caracterizado de amostra clínica de um paciente, infectado no Japão, e que apresentou os sintomas de febre e erupção cutânea após sua chegada ao Brasil. A infecção por parvovírus foi confirmada por meio de seguintes ensaios: Elisa para detecção de anticorpos IgM antiparvovirus B19 e técnica de polymerase chain reaction (PCR). Um fragmento da região NS1-VP1 foi diretamente submetido ao seqüenciamento do nucleotídeo. A análise filogenética parcial do B19, frente às várias seqüências disponíveis no GenBank, indicou que PV B19 isolado correspondeu ao genótipo 1.

Human parvovirus B19 was identified and characterized in sample collected from a patient who was infected in Japan, and the symptoms as fever and rash appeared after arriving to Brazil. The occurrence of virus infection was confirmed by both assays: Elisa parvovirus B19-specific IgM antibody detection andpolymerase chain reaction (PCR). A fragment of NS1-VP1 region was directly submitted to nucleotide sequencing. Partial phylogenetic analysis of B19 sequences, including several sequences available in GenBank, indicated that the isolated HPV B19 corresponded to genotype 1.

Molecular surveillance of an imported measles virus infection in São Paulo, Brazil

No estado de São Paulo, Brasil, em função da eficiente estratégia para a vigilância do vírus do sarampo(VS), não houve registro de casos nativos de sarampo no período de 2001 a 2007. No estado de São Paulo foram registrados casos de sarampo importados, sendo 01 paciente em 2001, outro em 2002 e em 2005 foi alvo de investigação uma criança não vacinada, de 18 meses de idade com exantema e febre, que foi admitida em hospital privado. O Centro de Vigilância Epidemiológica descobriu que o irmão desta criança teve uma doença semelhante uma semana antes. A infecção pelo vírus do sarampo foi confirmada no Instituto Adolfo Lutz pela detecção de anticorpo IgM anti-VS, isolamento do vírus por meio de cultivo em células Vero/hSLAM e amplificação de RNA viral por RT-PCR. A região do gene da nucleoproteína do vírus isolado foi amplificada. O resultado da análise filogênica mostrou que o vírus isolado correspondeu ao genótipo D5. Este genótipo circula no continente da Ásia e há relatos sobre sua anterior circulação em São Paulo.

Owing to the efficient strategies for measles virus (MV) surveillance in São Paulo State, Brazil, no circulation of native measles virus was registered during the period from 2001 to 2007. In Sao Paulo State the imported measles cases were registered, being one in 2001, one in 2002, and in 2005 an unvaccinated 18-month-oldchild presenting fever and exanthema admitted to a private hospital was the target of epidemiological study. The Center of Epidemiological Surveillance found out that a brother of this child had had a similar disease one week before. The measles virus infection was confirmed at Adolfo Lutz Institute by detecting the MV-specific IgM antibody, by virus isolation on Vero/hSLAM cells culture, and by means of MV-RNA amplification on RT-PCR technique. A region of nucleoprotein gene from isolated virus was amplified. The phylogenetic analysis data showed that the isolated virus corresponded to genotype D5. This genotype circulates in the...

Frequência de anticorpos específicos para herpes simples e citomegalovírus em soro e líquido cefalorraquidiano de pacientes com síndrome de imunodeficiência adquirida - AIDS / Herpes simplex virus and cytomegalovirus antibodies frequency in serum and cerebrospinal fluid from patients with Acquired Immunodeficiency Syndrome - AIDS

De janeiro a dezembro de 1988, foram submetidas a exame 865 amostras de soro e 475 amostras de líquido céfalo-raquidiano -LCR de pacientes com Síndrome de Imunodeficiência Adquirida-AIDS, para pesquisa de anticorpos específicos para herpes simples e citomegalovírus, respectivamente pelas técnicas de imunofluorescência indireta e fixaçäo do complemento. Dos pacientes estudados, 87,4% eram do sexo masculino e 65,7% pertenciam a faixa etária de 20 a 39 anos; 61,5% das amostras de soro e 7,5% das amostras de LCR apresentaram anticorpos para herpesvírus. Para citomegalovírus, registrou-se a presença de anticorpos específicos em 42,6% das amostras de soro e em 2,7% das amostras de LCR. As frequências de anticorpos específicos para ambos os vírus nas amostras de soro estudadas foram significativamente menores (p<0,05) que os valores indicados pela literatura especializada como padroes da populaçäo normal. A presença de anticorpos específicos para herpesvírus e citomegalovírus no LCR sugere uma possível infecçäo por esses vírus no Sistema Nervoso Central dos pacientes com AIDS.