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.

Antiviral activity of geopropolis extract from Scaptotrigona Aff. postica against rubella virus

The search for functional foods, which possess bioactive substances, is a new trend for the obtention of alternative and more effective treatments of many diseases with fewer side effects. Geopropolis, elaborated by stingless bees, is a mixture of plant resin sources, wax and soil. In the geopropolis from Scaptotrigona affinis postica (Latreille, 1807), (Hymenoptera, Apidae, Meliponini) was not observed the presence of soil. In a previous study, the extract of geopropolis provided by the beekeeper, from S. postica of Barra do Corda, Maranhão State, exhibited potent antiviral activity against herpes simplex virus. In this study, the propolis extract was prepared experimentally and characterized by RP-HPLC-DAD-ESI-MS/MS. The objective of this study was to evaluate the antiviral activity of an experimentally prepared geopropolis extract from S. postica against Rubella Virus infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Rubella virus infection of susceptible women during the first trimester of pregnancy, often results in a combination of birth defects in newborns. There is not an effective treatment for rubella virus infection. Different protocols were carried out to evaluate, the antiviral effect of geopropolis extract on the viral replication of infectious RV. Cell viability and cell proliferation assays indicated that this geopropolis was not toxic to cultured SIRC cells. In the viral binding assay, antiviral assay, real-time PCR, and transmission electron microscopy, was observed that different concentrations of geopropolis (17, 34 and 68 µg/mL) was able to inhibit the binding of virions to the cell receptor and the production of infectious RV particles in post treated and pre treated infected SIRC cells. The antiviral activity could to be attributed to the high contents of the apigenin derivatives, vicenin-2 and schaftoside. As far as we know, this is the first report about the antiviral activity of geopropolis from Scaptotrigona postica against a Togaviridae virus.

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.

The antiviral effect of mollusk mucus on measles virus.

Measles is a viral disease highly contagious spread by respiratory transmission. Although infection can be controlled by vaccination, numerous cases of measles have been registered in many areas of the world, highlighting the need for additional interventions. Terrestrial gastropods exude mucus on their body surface when traveling, to protect the body from mechanical injury, desiccation or contact with harmful substances. The mucus of mollusks has been studied as a source of new natural compounds with diverse biological activities. In this study, the antiviral activity of the mucus of the land slug P. boraceiensis was demonstrated in vitro using Vero cells infected with measles virus. The crude sample and four fractions were tested in cultures infected with measles virus and the antiviral activity was assessed by the cytopathic effect in infected cell cultures as well as by immunofluorescence and qPCR. Fractions 39 and 50 of the mucus from P. boraceiensis were analyzed by HPLC-DAD-ESI-MS/MS and infrared spectroscopy. A mixture of polyunsaturated fatty acids was found in the two fractions. A reduction in the growth of the measles virus was observed, measured by qPCR, with a protection index of 80% in Vero cells infected with measles and treated with fraction 39. Fraction 39 exhibited the best antiviral action in vitro and high contents of hydroxy-tritriacontapentaenoic acid and hydroxy-pentatriacontapentaenoic acid were found in this fraction.

Prenatal diagnosis of congenital rubella infection in São Paulo / Diagnóstico pré-natal de infecção congênita por rubéola, em São Paulo, Brasil

Objective: rubella during the early stages of pregnancy can lead to severe birth defects known as congenital rubella syndrome (CRS). Samples collected from pregnant women with symptoms and suspected of congenital rubella infection between 1996 and 2008 were analyzed. Methods: a total of 23 amniotic fluid samples, 16 fetal blood samples, 1 product of conception and 1 placenta were analyzed by serology and RT-PCR. Results: all patients presented positive serology for IgG / IgM antibodies to rubella virus. Among neonates, 16 were IgG-positive, 9 were IgM-positive and 4 were negative for both antibodies. Of the 25 samples analyzed in this study, 24 were positive by RT-PCR. Changes in ultrasound were found in 15 (60%) of 25 fetuses infected with rubella virus. Fetal death and miscarriage were reported in 10 (40%) of the 25 cases analyzed. The rubella virus was amplified by PCR in all fetuses with abnormal ultrasound compatible with rubella. Fetal death and abortion were reported in 10 of 25 cases analyzed. Conclusion: this study, based on primary maternal rubella infection definitely confirms the good sensitivity and specificity of RT-PCR using amniotic fluid and ultrasound. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome. .

Objetivo: a rubéola, durante os primeiros estágios da gravidez, pode levar a graves defeitos congênitos, conhecidos como síndrome da rubéola congênita (SRC). Amostras de gestantes com sintomas e suspeitas da rubéola congênita foram coletadas entre 1996 e 2008. Métodos: um total de 23 amostras de fluido amniótico, 16 amostras de sangue fetal, um produto da concepção e uma placenta foram analisados por sorologia e PCR. Resultados: todas as gestantes apresentaram sorologia positiva para IgG/IgM para o vírus da rubéola. Entre os recém-nascidos, 14 apresentaram anticorpos IgG positivos e 11 foram os anticorpos IgM positivos. Das 25 amostras analisadas neste estudo, 24 eram positivas por RT-PCR. Alterações na ultrassonografia foram encontradas em 15 (60%) dos 25 fetos infectados com o vírus da rubéola. Morte fetal e aborto espontâneo foram reportados em 10 (40%) dos 25 casos analisados. O vírus da rubéola foi amplificado por PCR em todos os fetos que apresentaram alterações na ultrassonografia, compatíveis com a rubéola. Morte fetal e aborto foram relatados em 10 dos 25 casos analisados. Conclusão: os resultados mostraram que os ensaios moleculares são ferramentas importantes para o diagnóstico precoce da rubéola e da síndrome da rubéola congênita. .

Sequence analysis of the 2009 pandemic influenza A H1N1 virus haemagglutinin gene from 2009-2010 Brazilian clinical samples.

In this paper, we analysed the haemagglutinin (HA) gene identified by polymerase chain reaction from 90 influenza A H1N1 virus strains that circulated in Brazil from April 2009-June 2010. A World Health Organization sequencing protocol allowed us to identify amino acid mutations in the HA protein at positions S220T (71%), D239G/N/S (20%), Y247H (4.5%), E252K (3.3%), M274V (2.2%), Q310H (26.7%) and E391K (12%). A fatal outcome was associated with the D239G mutation (p < 0.0001). Brazilian HA genetic diversity, in comparison to a reference strain from California, highlights the role of influenza virus surveillance for study of viral evolution, in addition to monitoring the spread of the virus worldwide.

Sequence analysis of the 2009 pandemic influenza A H1N1 virus haemagglutinin gene from 2009-2010 Brazilian clinical samples

In this paper, we analysed the haemagglutinin (HA) gene identified by polymerase chain reaction from 90 influenza A H1N1 virus strains that circulated in Brazil from April 2009-June 2010. A World Health Organization sequencing protocol allowed us to identify amino acid mutations in the HA protein at positions S220T (71 percent), D239G/N/S (20 percent), Y247H (4.5 percent), E252K (3.3 percent), M274V (2.2 percent), Q310H (26.7 percent) and E391K (12 percent). A fatal outcome was associated with the D239G mutation (p < 0.0001). Brazilian HA genetic diversity, in comparison to a reference strain from California, highlights the role of influenza virus surveillance for study of viral evolution, in addition to monitoring the spread of the virus worldwide.

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.

Gunshot upper extremity injury reconstructed with a fibula osteoseptocutaneous free flap (case report).

The management of upper extremity gunshot wound with soft tissue and bone injuries remains a remarkable problem and often requires sophisticated reconstructive strategies.There are limited reconstructive options for the treatment of segmental bone defects of the upper extremity exceeding 6 cm in length, especially when associated with soft tissue loss. Among the limited treatment options, the osteoseptocutaneous fibular transplantation is well established. The vascularized fibula flap has become a major tool in upper limb reconstruction but still is an uncommon procedure and continues to challenge reconstructive surgeons.In this paper, we report a complex combined skeletal and soft tissue involvement of an upper extremity case successfully treated with fibula osteoseptocutaneous free flap. The bone defect measured 12 cm. In severe injuries of the upper extremity, free transfer of the fibula flap not only provides replacement of the resulting composite defect but may also offer salvage of the extremity.

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...