Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection

Abstract Human adenoviruses comprise an important group of etiologic agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2, 3, 5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice.

A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil

A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed.

Adenoviruses of canine and human origins in stool samples from free-living pampas foxes (Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) in São Francisco de Paula, Rio dos Sinos basin / Adenovírus de origens canina e humana em fezes de graxains (Lycalopex gymnocercus) e cachorros-do-mato (Cerdocyon thous) de vida livre em São Francisco de Paula, bacia do Rio dos Sinos

The spread of enteric viruses of domestic animals and human beings to wild species can be facilitated by the resistance of these viruses on the environment and their ability to be transmitted by water and contaminated food. The health status of the populations of pampas foxes (Lycalopex gymnocercus) and crab-eating foxes (Cerdocyon thous) is largely unknown and the landscapes occupied by these animals in southern Brazil have been threatened by human occupation and expansion of agriculture. In this work, the search of genomes of human and canine adenoviruses in feces from these wild carnivores was used to track the dissemination of domestic animals and human pathogens to the free-living populations in a wildlife reserve located in southern Brazil. This was performed by virus-specific differential real-time polymerase chain reactions (qPCR) on stool specimens, avoiding capture and additional stress to the animals. Genus-specific conventional reverse-transcriptase PCR (RT-PCR) was complementarily performed aiming the detection of enteroviruses (EV) and rotaviruses (RV) on these same samples. HAdV genomes were found on 14 out of the 17 (82.35%) stool samples analysed, whereas CAV was found co-infecting 5 of these samples. RV genomes were detected on 7 of the 17 samples (41.18%) and all samples were negative for EV. The results point to the dispersion of HAdV and RV at a high rate to these species of South American wild carnivores, which can be an effect of growing anthropisation of the habitat of these animals.

A disseminação de vírus entéricos de animais domésticos e seres humanos para espécies selvagens pode ser facilitada pela resistência desses vírus no ambiente e sua capacidade de ser transmitida por água e alimentos contaminados. O estado de saúde das populações de Graxains-do-campo (Lycalopex gymnocercus) e Cachorros-do-mato (Cerdocyon thous) é em grande parte desconhecida e as paisagens ocupadas por estes animais no sul do Brasil têm sido ameaçadas pela ocupação humana e a expansão da agricultura. Neste trabalho, utilizou-se a pesquisa de genomas de adenovírus humanos (HAdV ) e caninos (CAV-1 e -2) em amostras fezes desses carnívoros selvagens com vistas a diagnosticar a disseminação de patógenos de animais domésticos e seres humanos às populações de vida livre em uma reserva de vida selvagem, localizado no sul do Brasil. Foram realizadas reações em cadeia da polimerase diferenciais e em tempo real (qPCR) de adenovírus específicos em amostras de fezes, evitando a captura e estresse adicional para os animais. PCRs gênero-específicas convencionais com transcrição reversa prévia (RT-PCR) foram ainda realizadas visando a detecção de enterovírus (EV) e rotavírus (RV) nestas mesmas amostras. Genomas de HAdV foram encontrados em 14 a 17 amostras de fezes (82.35%) analisados, Considerando que o CAV foi encontrado coinfectando 5 destas amostras. Genomas de RV foram detectados em 7 das 17 amostras (41.18%) e todas as amostras foram negativas para EV. Os resultados apontam para a dispersão de HAdV e RV em uma taxa elevada para estas espécies de carnívoros selvagens sul-americanas, que podem ser um efeito da crescente antropização do habitat desses animais.

Rotavirus infection in children and adult patients attending in a tertiary Hospital of São Paulo, Brazil

During the period of January 2003 to December 2005, 3,768 stool samples were received in the Microbiology Laboratory for rotavirus antigen detection from outpatients and inpatients of Albert Einstein Hospital, SP. Fresh stool samples from children and adults were analyzed by two methodologies: during 2003 and 2004 by latex agglutination (Slidex Rotavirus, Biomerieux) and 2005 by an immunochromatographic assay for the combined detection of rotavirus and adenovirus (Vikia Rota-Adeno, Biomerieux). Rotavirus group A was detected in 755 (20 percent) samples. The annual prevalence was 19.8 percent in 2003, 21.7 percent in 2004, and 18.7 percent in 2005. Rotavirus was detected every month during the period of the study, with peak of positivity between June and August (>35 percent). The prevalence in hospitalized patients was 26.1 percent (352/1,350) and in outpatients was 16.7 percent (403/2,418). For hospitalized patients most of the rotavirus infections were diagnosed in Pediatric setting, age range of 0 to 10 years (prevalence of 55.3 percent, 295/534). Overall positivity was up to 30 percent in patients between six months and five years of age (67 percent of all positive patients), all other age groups had at least 10 percent positive tests. Rotavirus infection is common in Sao Paulo, and besides the expected higher frequency in children it is also frequent in adults.

Virus Respiratorios en Menores de Diez Años con Infección Respiratoria en el Hospital Militar Central de Bogotá 2000-2001 / Respiratory virus in children aged less than 10 years old suffering from respiratory infection in the Hospital Militar Central in Bogota from 2000-2001

Objetivo: Se llevo a cabo un estudio en niños que consultaban por infección respiratoria aguda al Hospital Militar (Hosmil) de Bogota, Colombia, con el objetivo de estimar la frecuencia de enfermedad respiratoria aguda en esa institución producida por algunos virus respiratorios. Métodos: Durante 18 meses, de Marzo de 2000 a Septiembre de 2001, se seleccionaron semanalmente tres a cinco niños con infección respiratoria de aquellos que consultaban al servicio de emergencia o de consulta ambulatoria del Hospital. De cada uno de ellos se obtuvo muestras nasofaringeas que fueron examinadas por inmunofluorescencia para virus sincitial respiratorio (VSR), influenza, adenovirus y parainfluenza. Resultados: Se estudiaron 139 niños de los cuales el 40 por ciento fue positivo para alguno de los virus estudiados. El agente viral mas frecuentemente encontrado fue el VSR (27 por ciento), seguido por el virus de la influenza (5 por ciento) y adenovirus (3 por ciento). La circulación de VSR coincidía con los picos de hospitalizaciones por IRA y por neumonía y del hospital, era mas frecuente en los niños menores de 3 años y se encontraba presente durante todo el año. Conclusiones: Los virus respiratorios causan gran parte de la enfermedad respiratoria moderada o severa en el Hosmil y el VSR es el agente más importante entre ellos.

Objective: A study was carried out on children seeking medical care at the Hospital Militar (Hosmil) in Bogota due to acute respiratory infection; it was aimed at estimating the frequency of respiratory virus-associated acute respiratory disease (ARD) in children aged under 10. Methods: Three to five children aged less than 10 years old were selected every week from the hospital's emergency ward or ambulatory services over an 18-month period from March 2000 to September 2001. Nasopharyngeal samples were obtained from them and processed using indirect immunofluorescence. The relative frequency of respiratory syncytial virus (RSV), influenza, adenovirus and parainfluenza were calculated. Results: Around 40 percent of the 139 children examined proved positive for at least one respiratory virus. RSV was the most frequently found virus (27 percent positivity: 38 patients) followed by influenza (5 percent: 7 patients) and adenovirus (3 percent: 4 patients). RSV circulation peaks coincided with peaks related to ARD and pneumonia in the hospital: it was found most frequently in children aged less than 3 and was present throughout the year. Conclusion: Respiratory viruses were an important cause of moderate to severe respiratory illness in children seeking health care at Hosmil; RSV was the leading agent amongst such viruses.