Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Experimental infection of Rio Mamore hantavirus in Sigmodontinae rodents

This study shows an experimental spillover infection of Sigmodontinae rodents with Rio Mamore hantavirus (RIOMV). Necromys lasiurus and Akodon sp were infected with 103 RNA copies of RIOMV by intraperitoneal administration. The viral genome was detected in heart, lung, and kidney tissues 18 days after infection (ai), and viral excretion in urine and faeces began at four and six ai, respectively. These results reveal that urine and faeces of infected rodents contain the virus for at least 18 days. It is possible that inhaled aerosols of these excreta could transmit hantavirus to humans and other animals.

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

Development of a novel plaque reduction neutralisation test for hantavirus infection

In the Americas, hantaviruses cause severe cardiopulmonary syndrome (HCPS) with a high fatality rate. Hantavirus infection is commonly diagnosed using serologic techniques and reverse transcription-polymerase chain reaction. This paper presents a novel plaque reduction neutralisation test (PRNT) for detecting antibodies to Brazilian hantavirus. Using PRNT, plaque detection was enhanced by adding 0.6% of dimethyl sulfoxide into the overlay culture medium of the infected cells. This procedure facilitated clear visualisation of small plaques under the microscope and provided for easy and accurate plaque counting. The sera from 37 HCPS patients from the city of Ribeirão Preto, Brazil was evaluated for the Rio Mamoré virus (RIOMV) using PRNT. Six samples exhibited neutralising antibodies; these antibodies exhibited a low titre. The low level of seropositive samples may be due to fewer cross-reactions between two different hantavirus species; the patients were likely infected by Araraquara virus (a virus that has not been isolated) and RIOMV was used for the test. This assay offers a new approach to evaluating and measuring neutralising antibodies produced during hantavirus infections and it can be adapted to other hantaviruses, including viruses that will be isolated in the future.

Antibody levels to hantavirus in inhabitants of western Santa Catarina State, Brazil / Níveis de anticorpos para hantavírus em habitantes da região Oeste de Santa Catarina, Brasil

Hantavirus cardiopulmonary syndrome (HCPS) is an infectious disease caused by hantaviruses of the family Bunyaviridae, and is transmitted by aerosols of excreta of infected rodents. The aim of the present study was to determine antibody levels to hantavirus in the population that lives at frontier of Brazil and Argentina. Participated of the study 405 individuals living in the municipalities of Bandeirante, Santa Helena, Princesa and Tunapolis, state of Santa Catarina, Brazil. IgG antibodies to hantavirus were analyzed in sera by an ELISA that uses a recombinant N protein of Araraquara hantavirus as antigen. The results were also confirmed by immunofluorescent test. Eight individuals showed antibodies to hantavirus (1.97% positivity), with serum titers ranging from 100 to 800. Six seropositives were males, older than 30 years and farmers. Our results reinforce previous data on hantavirus circulation and human infections in the southern border of Brazil with Argentina.

Síndrome Cardiopulmonar por Hantavírus (HCPS) é uma doença emergente, causada pelo gênero hantavírus membro da família Bunyaviridae, e são transmitidos aos humanos por aerossol de roedores infectados. O objetivo principal deste estudo foi determinar os níveis de anticorpos para hantavírus em uma população de residentes na fronteira do Brasil com a Argentina. Participaram deste estudo 405 indivíduos que moravam nos municípios de Bandeirante, Santa Helena, Princesa e Tunapólis, no estado de Santa Catarina, Brasil. Os anticorpos IgG para hantavírus foram analisados no soro por um ELISA que usa a nucleoproteína recombinante do vírus Araraquara como antígeno, posteriormente confirmados por imunofluorescência. Oito indivíduos apresentavam anticorpos para hantavírus (1.97% positivo), com titulo entre 100 a 800. Seis soropositivos foram homens, com idade superior a 30 anos e agricultores. Nossos resultados reforçam a circulação do hantavírus e infecção humana na fronteira do Brasil com a Argentina.

Serosurvey of hantavirus infection in humans in the border region between Brazil and Argentina / Estudo sorológico de infecção por hantavírus em humanos na região de fronteira, entre Brasil e Argentina

INTRODUCTION: According to reports by the Ministry of Health, in the far western region of the State of Santa Catarina, there have been no reports of hantavirus pulmonary syndrome, a zoonotic disease transmitted by feces of infected rodents. A seroepidemiological study of residents of this region, was conducted, with the aim of determining the presence of hantavirus infections. A total of 340 volunteers of both genus, from the towns of Belmonte and Paraíso, were studied. METHODS: The serum of these patients was collected and used to detect IgG antibodies against recombinant N protein of Araraquara hantavirus, by ELISA assay. The positive samples were then titrated and confirmed by immunofluorescence assay. RESULTS: This study demonstrated the presence of IgG antibodies against hantavirus N protein in 3.5 percent of the population. The most frequent occupation was farm worker, 81 percent had direct and indirect contact with rodents, 91.7 percent of positive cases were farm workers, indicating that the probable cause of infection occurred during barn cleaning. These antibodies are noteworthy, given that the levels of antibodies were verified in individuals whose contact with hantavirus may have occurred many years ago. CONCLUSIONS: This study shows the circulation of hantavirus in the region, a fact that until now, had not reported. All the serum reagents had contact with the pathogen, but did not develop pulmonary and cardiovascular syndrome. It is important to remain alert, because hantavirus is a serious and emerging disease of some relevance.

INTRODUÇÃO: De acordo com relatórios do Ministério da Saúde, na região do extremo oeste do Estado de Santa Catarina, não há relatos de síndrome pulmonar por hantavírus, doença zoonótica transmitida por excretas de roedores contaminados. Com a finalidade de demosntrar a infecção por hantavírus, um estudo soroepidemiológico de moradores da região foi realizado. Assim, foi estudado um total de 340 voluntários de ambos os gêneros, dos municípios de Belmonte e Paraíso. MÉTODOS: O soro destes pacientes foi coletado e usado para a detecção de anticorpos IgG contra a proteína N recombinante de hantavírus Araraquara, pelo teste de ELISA. As amostras positivas foram tituladas e confirmadas por imunofluorescência indireta. RESULTADOS: Este estudo demonstrou a presença de anticorpos IgG contra a proteína N hantavírus em 3,5 por cento da população. A ocupação de lavrador foi a mais frequente, e 81 por cento tiveram contato direto e indireto com os roedores, 91,7 por cento dos casos positivos foram os agricultores, a causa provável da infecção foi através da limpeza de celeiros. Estes anticorpos são notáveis, dado que os níveis de anticorpos são encontrados nos indivíduos cujo contato com o hantavírus pode ter ocorrido há muitos anos. CONCLUSÕES: Este estudo mostra a circulação de hantavírus na região, um fato que até então não havia relatado, todos os reagentes soro tiveram contato com o patógeno, mas não desenvolveram a síndrome pulmonar e cardiovascular. É preciso estar alerta, porque é uma doença grave e emergente com grande importância.