Carmen Ricarte

Viral etiology of acute respiratory infections in hospitalized and outpatient children in Buenos Aires, Argentina.

Objectives: To determine and compare the viral frequency, seasonality and clinical-demographic features in 2 groups of children (hospitalized versus outpatients) with acute respiratory infections. Material and Methods: A cross-sectional, descriptive study was performed from 2008 to 2010 in 620 children <6 years of age with acute respiratory infection. Respiratory samples were studied for classical respiratory viruses by immunofluorescence and for human rhinoviruses (HRV) by real-time reverse transcription polymerase chain reaction. Clinical and demographic data were recorded. Results: Viral detection by immunofluorescence was 48% in 434 inpatients and 37% in 186 outpatients. Viral diagnosis increased to 83% and 62%, respectively, when testing for HRV. HRV (41%) and respiratory syncytial virus (RSV) (27%) were most common viruses identified, followed by metapneumovirus (9%), influenza A and parainfluenza (3%), adenovirus and influenza B (2%). HRV frequency was significantly higher in hospitalized patients (47%) than in outpatients (27%) (P < 0.001). Coinfection was detected in 12% of hospitalized and 4% of outpatients (P < 0.031). HRV and adenovirus circulated throughout the entire year. RSV, influenza A and B predominated in winter, whereas metapneumovirus and parainfluenza predominated in spring. Of 362 patients with bronchiolitis, 84% had a virus identified; HRV (42%) and RSV (38%) were predominant. Of 77 patients with pneumonia, 84% had a virus detected with HRV (43%) and RSV (29%) predominating. Conclusions: HRV were significant pathogens associated with bronchiolitis and pneumonia, especially in hospitalized patients. Both, HRV and coinfections, were risk factors for hospitalization. These findings support the importance of including HRV detection in children with acute respiratory infection.

Diagnóstico de virus respiratorios utilizando un sistema automatizado de PCR múltiples (FilmArray) y su comparación con métodos convencionales

Acute respiratory infections, which are commonly caused by viruses, are an important cause of morbidity and mortality in children. In Argentina, national surveillance programs for the detection of respiratory viruses are usually performed by using immunofluorescence (IF) assays, although it is well known that molecular methods are more sensitive. An automated multiplex PCR device, the FilmArray-Respiratory Panel (FilmArray-RP), can detect 17 viral and 3 bacterial pathogens in a closed system that requires only 5 min of hands-on time and 1h of instrumentation time. A total of 315 respiratory samples from children under 6 years of age suffering from acute respiratory infections, were studied by IF for 8 respiratory viruses and by RT-PCR for rhinoviruses. Later, these samples were tested by the FilmArray-RP. The positivity frequency obtained for the 9 viruses tested was 75% by IF/RT-PCR and 92% by the FilmArray-RP. The positive and negative percent agreement between both methods was 70.5% whereas the negative percent agreement was 99.6% (95% confidence interval:65.5-75.1 and 99.2-99.8 respectively). The FilmArray-RP allowed a higher positive diagnosis (97%) and detected other viruses such as coronavirus NL63, 229E, OC43, HKU1 (10%) and bocavirus (18%). In addition, this method identified multiple coinfections (39%) with 2, 3, 4 and up to 5 different viruses. At present, IF is still the most frequently used method in most Latin American countries for respiratory viruses diagnosis due to its low cost, its capability to process a high number of samples simultaneously and the fast determination of results for the most frequent viruses, which are available within 5h. However, the coming incorporation of molecular methods in routine procedures will significantly increase the diagnostic yield of these infections. Resumen Las infecciones respiratorias agudas producen una importante morbimortalidad y comúnmente son causadas por virus. En Argentina, los programas de vigilancia epidemiológica se basan en la detección de antígenos virales por inmunofluorescencia (IF), aunque es bien conocido que los métodos moleculares son más sensibles. El panel respiratorio (PR) FilmArray (PR-FilmArray) es un equipo comercial automatizado de PCR múltiples que detecta 17 virus respiratorios y 3 bacterias, en un sistema cerrado que requiere 5min de procesamiento y una 1h de instrumentación. Se evaluó un total de 315 muestras respiratorias de niños menores de 6 años con infecciones respiratorias agudas por IF para 8 virus respiratorios y por RT-PCR para rinovirus. Posteriormente, estas muestras se estudiaron con el PR-FilmArray. La frecuencia de positividad al considerar los 9 virus estudiados por IF y RT-PCR fue del 75%; por PR-FilmArray fue del 92%. El porcentaje de acuerdo positivo entre ambas metodologías fue del 70,5% y el de acuerdo negativo fue del 99,6% (intervalo de confianza 95%: 65,5-75,1 y 99,2-99,8, respectivamente). El PR-FilmArray permitió obtener un mayor diagnóstico positivo (97%) y detectó otros virus, como los coronavirus NL63, 229E, OC43 y HKU1 (10%) y los bocavirus (18%). Además, permitió identificar coinfecciones múltiples (39%) con 2, 3, 4 y hasta 5 virus. Actualmente, la IF continúa siendo el método más utilizado en los países latinoamericanos para el diagnóstico de virus respiratorios por su bajo costo, por su capacidad para procesar un alto número de muestras simultáneamente y porque los resultados de los virus más frecuentes están disponibles en 5h. Sin embargo, la futura incorporación de métodos moleculares aumentaría notablemente la capacidad diagnóstica. Abstract Acute respiratory infections, which are commonly caused by viruses, are an important cause of morbidity and mortality in children. In Argentina, national surveillance programs for the detection of respiratory viruses are usually performed by using immunofluorescence (IF) assays, although it is well known that molecular methods are more sensitive. An automated multiplex PCR device, the FilmArray-Respiratory Panel (FilmArray-RP), can detect 17 viral and 3 bacterial pathogens in a closed system that requires only 5min of hands-on time and 1h of instrumentation time. A total of 315 respiratory samples from children under 6 years of age suffering from acute respiratory infections, were studied by IF for 8 respiratory viruses and by RT-PCR for rhinoviruses. Later, these samples were tested by the FilmArray-RP. The positivity frequency obtained for the 9 viruses tested was 75% by IF/RT-PCR and 92% by the FilmArray-RP. The positive and negative percent agreement between both methods was 70.5% whereas the negative percent agreement was 99.6% (95% confidence interval:65.5-75.1 and 99.2-99.8 respectively). The FilmArray-RP allowed a higher positive diagnosis (97%) and detected other viruses such as coronavirus NL63, 229E, OC43, HKU1 (10%) and bocavirus (18%). In addition, this method identified multiple coinfections (39%) with 2, 3, 4 and up to 5 different viruses. At present, IF is still the most frequently used method in most Latin American countries for respiratory viruses diagnosis due to its low cost, its capability to process a high number of samples simultaneously and the fast determination of results for the most frequent viruses, which are available within 5h. However, the coming incorporation of molecular methods in routine procedures will significantly increase the diagnostic yield of these infections.

Real time PCR for rapid determination of susceptibility of adenovirus to antiviral drugs.

Human adenoviruses (HAdV) are associated with respiratory, ocular and gastrointestinal infections as well as potentially fatal disseminated disease in highly immunocompromised patients. Although there is no specific FDA approved treatment for HAdV infections, some antivirals are used in certain patients. The in vitro antiviral assays for HAdV are not standardized and are usually time consuming. The objective of this study was to evaluate a real time PCR assay for rapid determination of susceptibility of HAdV to antiviral drugs. The nucleoside analogue stavudine (d4T) was used as test drug in A549 cells infected with HAdV5. The antiviral assay measured the reduction of the HAdV DNA levels in culture supernatants by real time PCR using specific primers that amplify a conserved region of the hexon gene. This real time PCR assay demonstrated that stavudine was a selective inhibitor for HAdV5, since the effective concentration 50% (EC(50)) ranged from 0.08 to 0.12 mM at multiplicity of infection between 0.001 and 1. Furthermore, EC(50) showed a high correlation with plaque reduction and virus yield inhibition assays (r(2)=0.9938 and r(2)=0.9468, respectively). In conclusion, the real time PCR-based antiviral assay is rapid, reproducible and could replace classical and more labor-intensive infectivity assays.