Zoonotic potential of a novel bat morbillivirus.

Bats are significant reservoir hosts for many viruses with zoonotic potential1. SARS-CoV-2, Ebola virus, and Nipah virus are examples of such viruses that have caused deadly epidemics and pandemics when spilled over from bats into human and animal populations2,3. Careful surveillance of viruses in bats is critical for identifying potential zoonotic pathogens. However, metagenomic surveys in bats often do not result in full-length viral sequences that can be used to regenerate such viruses for targeted characterization4. Here, we identify and characterize a novel morbillivirus from a vespertilionid bat species (Myotis riparius) in Brazil, which we term myotis bat morbillivirus (MBaMV). There are 7 species of morbilliviruses including measles virus (MeV), canine distemper virus (CDV) and rinderpest virus (RPV)5. All morbilliviruses cause severe disease in their natural hosts6-10, and pathogenicity is largely determined by species specific expression of canonical morbillivirus receptors, CD150/SLAMF111 and NECTIN412. MBaMV used Myotis spp CD150 much better than human and dog CD150 in fusion assays. We confirmed this using live MBaMV that was rescued by reverse genetics. Surprisingly, MBaMV replicated efficiently in primary human myeloid but not lymphoid cells. Furthermore, MBaMV replicated in human epithelial cells and used human NECTIN4 almost as well as MeV. Our results demonstrate the unusual ability of MBaMV to infect and replicate in some human cells that are critical for MeV pathogenesis and transmission. This raises the specter of zoonotic transmission of a bat morbillivirus.

NS1-based ELISA test efficiently detects dengue infections without cross-reactivity with Zika virus.

OBJECTIVES: The aim of this study was to achieve greater specificity of dengue virus (DENV) serological tests based on a recombinant antigen derived from non-structural protein 1 (ΔNS1) with regard to cross-reactive Zika virus (ZIKV) anti-NS1 antibody responses. This is of relevance in endemic regions for the serological discrimination of both DENV and ZIKV, such as Brazil and other tropical countries. METHODS: The ΔNS1 proteins were obtained as recombinant antigens and were evaluated as solid-phase-bound antigens in the ELISA test to detect anti-NS1 IgG antibodies. The performance of the ∆NS1-based DENV IgG ELISA was assessed with both mouse and human serum samples previously exposed to DENV or ZIKV. RESULTS: The ∆NS1-based DENV IgG ELISA detected anti-DENV NS1 IgG without cross-reactivity with ZIKV-positive serum samples. The sensitivity and specificity of the assay determined using samples previously characterized by real-time PCR (qRT-PCR) or plaque reduction neutralization assay (PRNT) were 82% and 93%, respectively. CONCLUSION: The ∆NS1-based DENV IgG ELISA conferred enhanced diagnostic specificity for anti-DENV serological tests and may be particularly useful for serological analyses in endemic regions for both DENV and ZIKV transmission.

Prevalence of Measles Antibodies in São José do Rio Preto, São Paulo, Brazil: A serological survey model.

Measles is an acute and highly contagious but vaccine-preventable infectious disease. Despite years of being considered eliminated, decreased vaccination rates have produced virus reemergence in several countries, including Brazil. Measles can be controlled through immunization programs, through which aim to achieve 95% coverage with two doses of the vaccine. Measles can also be controlled if suspected cases can be properly identified in order to contain outbreaks. This cross-sectional study determined the prevalence of measles antibodies and their correlation with rubella antibodies (resulting from the combination vaccine used in Brazil's public immunization program) in individuals aged higher 10 years old in São José do Rio Preto, São Paulo State, Brazil, participants of a prospective cohort of arbovirosis surveillance before virus reemergence in the country. Our findings presented that 32.9% of individuals aged 10-40 years old had not antibodies against measles; 39.3% of total individuals with documented evidence of measles vaccination did not have anti-measles IgG, though only 20.2% of individuals with documented evidence of rubella vaccination lacked anti-rubella IgG. Besides, the most of measles cases reported in the city, following the virus spreading in the country, occurred especially in groups defined by us as susceptible. Because the combination MMR vaccine is part of Brazil's national vaccine schedule, the possible reasons for this relatively high rate of seronegativity need to be investigated further, once that it reflects outbreak risk.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure.

INTRODUCTION AND OBJECTIVES: Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). MATERIALS AND METHODS: Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. RESULTS: Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. CONCLUSIONS: Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Discovery of novel astrovirus and calicivirus identified in ruddy turnstones in Brazil.

Birds are the natural reservoir of viruses with zoonotic potential, as well as contributing to the evolution, emergence, and dissemination of novel viruses. In this study, we applied a high-throughput screening approach to identify the diversity of viruses in 118 samples of birds captured between October 2006 to October 2010 in the North and Northeast regions of Brazil. We found nearly complete genomes of novel species of astrovirus and calicivirus in cloacal swabs of ruddy turnstones (Arenaria interpres) collected in Coroa do Avião islet, Pernambuco State. These viruses are positive-sense single-stranded RNA with a genome of ~7 to 8 kb, and were designated as Ruddy turnstone astrovirus (RtAstV) and Ruddy turnstone calicivirus (RTCV), respectively. Phylogenetic analysis showed that RtAstV and RTCV grouped in a monophyletic clade with viruses identified from poultry samples (i.e., chicken, goose, and turkey), including viruses associated with acute nephritis in chickens. Attempts of viral propagation in monkey and chicken cell lines for both viruses were unsuccessful. Also, we found genomes related with viral families that infect invertebrates and plants, suggesting that they might be ingested in the birds' diet. In sum, these findings shed new light on the diversity of viruses in migratory birds with the notable characterization of a novel astrovirus and calicivirus.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure

Introduction and objectives Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). Materials and methods Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. Results Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. Conclusions Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Sofosbuvir inhibits yellow fever virus in vitro and in patients with acute liver failure

Introduction and objectives Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). Materials and methods Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. Results Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. Conclusions Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.

Cacipacore virus as an emergent mosquito-borne Flavivirus.

INTRODUCTION:: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS:: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS:: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS:: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Cacipacore virus as an emergent mosquito-borne Flavivirus

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Serological and molecular investigation of viral agents in freeliving jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil / Investigação sorológica e molecular de agentes virais em onças-pintadas de vida livre no Pantanal de Mato Grosso, Brasil

This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest ­ SFIMT and Rapid Fluorescent Focus Inhibition Test ­ RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region.(AU)

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência ­ RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onçaspintadas nesta região.(AU)

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations.

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota--LS--(vaccine strain) and Sao Joao do Meriti--SJM--strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity.

[Epidemiological characteristics of Lyme-like disease in children]. / Características clínico-epidemiológicas da doença Lyme-símile em crianças.

BACKGROUND: To determine the prevalence, age distribution, seasonality and clinical characteristics of Lyme-simile disease in Brazilians less than 15 years of age. METHODS. From July, 1998 to November, 2000, a cross-sectional study was conducted in 333 patients with skin rash and fever. Paired blood samples were collected for identification of the pathogens. Only 193 samples which were negative for other pathogens (Parvovirus B19 Human, Herpesvirus 6 Human, Measles, Rubella, Dengue, Scarlet fever and Enterovirus), were tested for borreliosis by Enzyme-Linked Immunosorbent Assay and Western-blotting. Other clinical, socioeconomic, demographic and climatic variables were studied. RESULTS: Prevalence of the disease was 6.2%(12/193). Of the variables studied, there was predominance in: <6 years old (83.2%); females (66.7%); being from the city of Franco da Rocha (58.3 %); and a summer/fall seasonality. The duration of care was 4 days. Signs and symptoms with statistical significance were itching; absence of lip notch and ocular pain; irritability and good clinical condition. Other clinical data presented were: pruritus (90%), irritability (80%) and fever (38 masculineC) (58.3%) with a duration of 1 to 3 days. Erythema was maculo-papular (40%), urticaria-like (25%) and scarlatiniform (16.7%), occurring predominately on the trunk (60%). There were no primary clinical evidences of Lyme-simile disease in the patients under study. The sensitivity and specificity of the clinical diagnosis as opposed to the laboratory diagnosis was zero. There was no initial clinical suspicion of the disease in the 10 cases studied and followed up for two years that showed no evidence of cardiologic or neurological complications. This is the first study of Lyme-simile in Brazilian children. CONCLUSION: Prevalence of Lyme-simile disease was low, and it was not remembered at the initial diagnosis of those with skin rash. However, practical knowledge is necessary, demanding increased medical attention.

Características clínico-epidemiológicas da doença Lyme-símile em crianças / Epidemiological characteristics of Lyme-like disease in children

OBJETIVO: Determinar a prevalência, distribuição etária, sazonalidade, características clínicas da doença Lyme-símile em menores de 15 anos. MÉTODOS: De julho/1998 a dezembro/2000 foi conduzido um estudo transversal em 333 pacientes, com exantema e febre. Foram coletadas amostras pareadas de sangue para a identificação de patógenos. Somente em 193 amostras, negativas aos outros patógenos (Parvovirus B19, Herpesvírus 6 humano, Sarampo, Rubéola, Dengue, Escarlatina e Enterovírus), foram realizadas a pesquisa da borreliose pelos métodos de Enzyme-Linked Immunosorbent Assay e Western-blotting. Outras variáveis clínicas, socioeconômicas, demográficas e climáticas foram estudadas. RESULTADOS: A prevalência da doença foi de 6,2 por cento(12/193). Das variáveis estudadas, houve predomínio em <6anos(83,2 por cento); sexo feminino (66,7 por cento); procedência da cidade de Franco da Rocha (58,3 por cento); com sazonalidade no outono-verão. O intervalo de atendimento foi de quatro dias. Sinais e sintomas com significância estatística: prurido, ausência da fissura labial e bom estado clínico. Outros dados presentes foram: irritabilidade (80 por cento); febre (?38ºC) (58,3 por cento) com duração de um a três dias. O exantema foi do tipo máculo-papular (33,3 por cento), urticariforme (25 por cento) e escarlatiniforme (16,7 por cento); predominando em tronco (60 por cento). Não houve apresentação clínica característica para diagnóstico da doença de Lyme-símile nestes pacientes. A sensibilidade e especificidade para o diagnóstico clínico contraposta com o diagnóstico laboratorial foi zero. O acompanhamento de 10 casos durante dois anos não evidenciou complicações cardiológicas ou neurológicas. Este é o primeiro estudo desta doença em crianças brasileiras. CONCLUSÃO: A prevalência da doença Lyme-símile foi baixa, não tendo sido lembrada no diagnóstico inicial dos exantemas, mas seu conhecimento é necessário, necessitando maior atenção médica.

BACKGROUND: To determine the prevalence, age distribution, seasonality and clinical characteristics of Lyme-simile disease in Brazilians less than 15 years of age. METHODS. From July, 1998 to November, 2000, a cross-sectional study was conducted in 333 patients with skin rash and fever. Paired blood samples were collected for identification of the pathogens. Only 193 samples which were negative for other pathogens (Parvovirus B19 Human, Herpesvirus 6 Human, Measles, Rubella, Dengue, Scarlet fever and Enterovirus), were tested for borreliosis by Enzyme-Linked Immunosorbent Assay and Western-blotting. Other clinical, socioeconomic, demographic and climatic variables were studied. RESULTS: Prevalence of the disease was 6.2 percent(12/193). Of the variables studied, there was predominance in: <6 years old (83.2 percent); females (66.7 percent); being from the city of Franco da Rocha (58.3 percent); and a summer/fall seasonality. The duration of care was 4 days. Signs and symptoms with statistical significance were itching; absence of lip notch and ocular pain; irritability and good clinical condition. Other clinical data presented were: pruritus (90 percent), irritability (80 percent) and fever (?38ºC) (58.3 percent) with a duration of 1 to 3 days. Erythema was maculo-papular (40 percent), urticaria-like (25 percent) and scarlatiniform (16.7 percent), occurring predominately on the trunk (60 percent). There were no primary clinical evidences of Lyme-simile disease in the patients under study. The sensitivity and specificity of the clinical diagnosis as opposed to the laboratory diagnosis was zero. There was no initial clinical suspicion of the disease in the 10 cases studied and followed up for two years that showed no evidence of cardiologic or neurological complications. This is the first study of Lyme-simile in Brazilian children. CONCLUSION: Prevalence of Lyme-simile disease was low, and it was not remembered at the initial diagnosis ...

Genetic diversity of environmental Aspergillus flavus strains in the state of São Paulo, Brazil by random amplified polymorphic DNA.

Aspergillus flavus is a very important toxigenic fungus that produces aflatoxins, a group of extremely toxic substances to man and animals. Toxigenic fungi can grow in feed crops, such as maize, peanuts, and soybeans, being thus of high concern for public health. There are toxigenic and non-toxigenic A. flavus variants, but the necessary conditions for expressing the toxigenic potential are not fully understood. Therefore, we have studied total-DNA polymorphism from toxigenic and non toxigenic A. flavus strains isolated from maize crops and soil at two geographic locations, 300 km apart, in the Southeast region of Brazil. Total DNA from each A. flavus isolate was extracted and subjected to polymerase chain reaction amplification with five randomic primers through the RAPD (random amplified polymorphic DNA) technique. Phenetic and cladistic analyses of the data, based on bootstrap analyses, led us to conclude that RAPD was not suitable to discriminate toxigenic from non toxigenic strains. But the present results support the use of RAPD for strain characterization, especially for preliminary evaluation over extensive collections.

Genetic diversity of environmental Aspergillus flavus strains in the state of São Paulo, Brazil by random amplified polymorphic DNA

Aspergillus flavus is a very important toxigenic fungus that produces aflatoxins, a group of extremely toxic substances to man and animals. Toxigenic fungi can grow in feed crops, such as maize, peanuts, and soybeans, being thus of high concern for public health. There are toxigenic and non-toxigenic A. flavus variants, but the necessary conditions for expressing the toxigenic potential are not fully understood. Therefore, we have studied total-DNA polymorphism from toxigenic and non toxigenic A. flavus strains isolated from maize crops and soil at two geographic locations, 300 km apart, in the Southeast region of Brazil. Total DNA from each A. flavus isolate was extracted and subjected to polymerase chain reaction amplification with five randomic primers through the RAPD (random amplified polymorphic DNA) technique. Phenetic and cladistic analyses of the data, based on bootstrap analyses, led us to conclude that RAPD was not suitable to discriminate toxigenic from non toxigenic strains. But the present results support the use of RAPD for strain characterization, especially for preliminary evaluation over extensive collections.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results.

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors.

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04 percent) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

O uso de testes de ácidos nucleicos (NAT) na rotina de triagem de doadores de sangue tornou-se uma realidade ao final da década de 1990. Descreve-se aqui uma metodologia de RT-PCR multiplex "in-house" que permite a detecção simultânea dos RNAs dos vírus HIV e HCV além de uma molécula artificial de RNA usada como controle externo. O método detecta todos os subtipos de HIV do grupo M e também do grupo N e O, com uma sensibilidade de 500 UI/mL. Após validação, este teste substituiu o do antígeno p24, até então na rotina de triagem em nosso laboratório, desde 1996. De julho de 2001 a fevereiro de 2006 foram testadas 102.469 doações e 41 (0.04 por cento) foram NAT reativas. Uma doação NAT isoladamente reativa (anticorpo não-reativa) foi detectada com soroconversão subseqüente do doador, portanto, o rendimento do NAT nesta população até o presente momento é de 1:102.469. Este número contrasta com a experiência obtida internacionalmente, onde taxas de 1:600.000 - 1:3.100.000 foram descritas.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95 percent hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23 percent) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83 percent. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Desenvolveu-se uma metodologia própria ("in-house") baseada em RT-PCR, que permite detectar simultaneamente o RNA do vírus HCV e de um RNA artificial empregado como controle externo. As amostras são analisadas em pools de 6-12 doações, cada doação sendo incluída em dois pools diferentes, um horizontal e um vertical, permitindo a identificação imediata de uma doação reativa, sem a necessidade de desmembrar-se um pool reativo. O processo todo consumiu de 6-8 horas diárias e os resultados foram emitidos em paralelo à sorologia. O método detectou os seis genótipos de HCV, com um limite de sensibilidade de 500 UI/mL (95 por cento hit rate). Até julho de 2005 haviam sido testadas 139.678 doações com a detecção de 315 (0,23 por cento) doações reativas para HCV-RNA. Exceto cinco falso-positivas, todas estas doações também apresentavam o respectivo anticorpo, portanto não se detectou nenhuma doação em janela imunológica. A especificidade foi de 99,83 por cento. A detecção de amostra em janela imunológica, nesta população de doadores, provavelmente demandará a análise de um número maior de doações, espelhando-se na experiência internacional que tem mostrado a detecção de amostras HCV-RNA isoladas em 1:200.000 - 1:500.000 doações.

Infeccao nosocomial pelo virus respiratorio sincicial em enfermaria de pediatria / Nosocomial respiratory syncytial virus infection at pediatric ward

Objetivo : avaliar a taxa de infeccao nosocomial causada por virus no trato respiratorio inferior de criancas internadas em uma enfermaria geral de pediatria durante a epidemia...