Current diagnostic standards involve severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection in nasopharyngeal swabs (NPS), but saliva is an attractive and noninvasive option for ...diagnosis. The objectives were to determine the performance of saliva in comparison with NPS for detecting SARS‐CoV‐2 and to compare the optimized home brew reverse‐transcription polymerase chain reaction (RT‐PCR) with a commercial RT‐PCR. Paired NPS and saliva specimens were prospectively collected and tested by RT‐PCR from patients presenting at an emergency room with signs and symptoms compatible with coronavirus disease‐2019. A total of 348 samples from 174 patients were tested by RT‐PCR assays. Among 174 patients with symptoms, 63 (36%) were SARS‐CoV‐2 positive in NPS using the optimized home‐brew PCR. Of these 63 patients, 61 (98%) were also positive in saliva. An additional positive SARS‐CoV‐2 saliva was detected in a patient with pneumonia. Kappa Cohen's coefficient agreement between NPS and saliva was 0.96 (95% confidence interval CI, 0.90–0.99). Median Ct values in NPS versus saliva were 18.88 (interquartile range IQR, 15.60–23.58; range, 11.97–38.10) versus 26.10 (IQR, 22.75–30.06; range, 13.78–39.22), respectively (p < .0001). The optimized home‐brew RT‐PCR demonstrated higher analytical and clinical sensitivity compared with the commercial RT‐PCR assay. A high sensitivity (98%) and agreement (kappa 0.96) in saliva samples compared to NPS was demonstrated when using an optimized home‐brew PCR even when the viral load in saliva was lower than in NPS. This noninvasive sample is easy to collect, requires less consumable and avoids discomfort to patients. Importantly, self‐collection of saliva can diminish exposure to healthcare personnel.
To determine and compare the viral frequency, seasonality and clinical-demographic features in 2 groups of children (hospitalized versus outpatients) with acute respiratory infections.
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.
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.
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.
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.
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.
•Saliva screening was helpful for identifying SARS-CoV-2 infection in HCWs.•Six percent of asymptomatic/pre-symptomatic HCWs from high/middle area, were positive in saliva.•Five out of six HCWs were ...positive in a subsequent nasopharyngeal swab.•Four out of six HCWs developed signs and symptoms compatible with COVID-19.•Three out of 4 symptomatic seroconverted while asymptomatic HCWs remained seronegative.
Health care workers (HCWs) are at high risk for SARS-CoV-2. In addition, pre-symptomatic or asymptomatic transmission accounts for around half of the cases. Saliva testing is an option to detect SARS-CoV-2 infection. To determine the performance of saliva samples for screening, HCWs were tested for SARS-CoV-2 by RT-PCR. Those with a positive result in saliva were tested by nasopharyngeal swabbing for viral RNA detection and blood collection to search for the presence of specific antibodies. In September–October 2020, 100 HCWs were enrolled and followed up. Six subjects (6%) tested positive in saliva. Of them, 5/6 were positive in a subsequent nasopharyngeal swab and 4/6 developed signs and symptoms compatible with COVID-19. Among the latter, 3 seroconverted while asymptomatic HCWs remained seronegative. Saliva screening was helpful for identifying SARS-CoV-2 infection in HCWs. This screening permitted rapid personnel isolation avoiding further transmission of the virus in the hospital setting.
El personal de salud (PS) tiene un alto riesgo de contraer SARS-CoV-2. La transmisión presintomática/asintomática representa alrededor de la mitad de los casos y el análisis a partir de muestras de saliva puede ser una opción para detectar la infección. Para determinar el rendimiento de estas muestras, 100 voluntarios del PS se sometieron a la detección de SARS-CoV-2 por RT-PCR en muestras de saliva en el período septiembre-octubre de 2020. De aquellos con resultado positivo en saliva, se tomaron hisopados nasofaríngeos para detectar ARN viral y muestras de suero para evaluar anticuerpos específicos. Se detectó ARN viral en la saliva de seis individuos (6%). De ellos, 5/6 fueron SARS-CoV-2 positivos en hisopado nasofaríngeo y 4/6 desarrollaron signos y síntomas compatibles con COVID-19. Entre estos últimos, tres seroconvirtieron, en tanto que los voluntarios asintomáticos permanecieron seronegativos. La muestra de saliva fue útil para identificar la infección por SARS-CoV-2 en esta cohorte del personal de salud y así proceder al rápido aislamiento de los individuos infectados, lo que evitó una mayor transmisión del virus en el ámbito hospitalario.
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.
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.
Human rhinoviruses (HRV), the major cause of common colds, have a significant genetic diversity and are classified into 3 species (A, B, C) with more than 100 serotypes. HRV species C, described in ...2006, can only be detected using molecular methods. The objectives of this paper were to adapt a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for HRV detection and to further determine the frequency of HRV in respiratory samples from children under 2 years of age, with acute respiratory infection (ARI), from Buenos Aires, Argentina. Two real-time RT-PCR assays amplifying the 207 base pair of the 5' non-coding region were compared. The original protocol includes locked nucleic acid analogues and a pyrimidine derivative in the forward primer, while the adapted protocol avoided those molecules. Of 67 respiratory samples, 17 (25.4 %) were positive with the original protocol, and 20 (29.9 %) with the adapted one. Discrepant results were confirmed by sequencing analysis. An expanded gold standard was defined to determine the performance of both assays, and was used to describe the clinical characteristics of positive patients. Better sensitivity and specificity were obtained with the adapted protocol. Considering the expanded gold standard, HRV were detected in 23/67 (34.3 %) patients with ARI: 8/18 (44.4 %) outpatients and 15/49 (30.6 %) hospitalized. Wheezing episodes were more frequent in HRV positive patients (43.5 %) than in HRV negative patients (18.2 %) (p = 0.041). This study describes the utility and clinical sensitivity of an adapted real-time RT-PCR assay for HRV detection.
Human rhinoviruses (HRV), the major cause of common colds, have a significant genetic diversity and are classified into 3 species (A, B, C) with more than 100 serotypes. HRV species C, described in ...2006, can only be detected using molecular methods. The objectives of this paper were to adapt a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for HRV detection and to further determine the frequency of HRV in respiratory samples from children under 2 years of age, with acute respiratory infection (ARI), from Buenos Aires, Argentina. Two real-time RT-PCR assays amplifying the 207 base pair of the 5' non-coding region were compared. The original protocol includes locked nucleic acid analogues and a pyrimidine derivative in the forward primer, while the adapted protocol avoided those molecules. Of 67 respiratory samples, 17 (25.4 %) were positive with the original protocol, and 20 (29.9 %) with the adapted one. Discrepant results were confirmed by sequencing analysis. An expanded gold standard was defined to determine the performance of both assays, and was used to describe the clinical characteristics of positive patients. Better sensitivity and specificity were obtained with the adapted protocol. Considering the expanded gold standard, HRV were detected in 23/67 (34.3 %) patients with ARI: 8/18 (44.4%) outpatients and 15/49 (30.6 %) hospitalized. Wheezing episodes were more frequent in HRV positive patients (43.5 %) than in HRV negative patients (18.2 %) (p = 0.041). This study describes the utility and clinical sensitivity of an adapted real-time RT-PCR assay for HRV detection.Los rinovirus humanos (RVH) constituyen la principal causa de resfrío común y poseen una gran diversidad genética, con más de 100 serotipos clasificados en tres especies (A, B, C). Los RVH C fueron descritos en 2006 y solo pueden detectarse utilizando métodos moleculares. El objetivo del presente trabajo fue adaptar un protocolo de transcripción reversa seguida de reacción en cadena de polimerasa (RT-PCR) en tiempo real para detectar RVH y posteriormente determinar su frecuencia en muestras de niños menores de 2 años con infección respiratoria aguda (IRA). Se compararon dos protocolos de RT-PCR en tiempo real, que amplifican 207 pares de bases de la región 5' no codificante. El protocolo original incluyó un cebador directo con análogos de nucleótidos bloqueados (LNA) y un derivado pirimidínico en su secuencia, mientras que el protocolo adaptado no los incluyó. De 67 muestras, 17 (25,4 %) fueron positivas con el protocolo original y 20 (29,9 %) con el protocolo adaptado; los resultados discrepantes se confirmaron por secuenciación. Se definió un gold standard expandido para determinar el desempeño de ambos ensayos y describir las características clínicas de los pacientes RVH positivos. La mejor sensibilidad y especificidad se obtuvo con el protocolo adaptado. Considerando el gold standard expandido, se detectó RVH en 23/67 (34,3 %) pacientes con IRA: 44,4 % (8/18) ambulatorios y 30,6 % (15/49) internados. Los episodios de sibilancias fueron más frecuentes en pacientes RVH positivos (43,5 %) que en RVH negativos (18,2 %) (p = 0,041). El presente estudio describe la utilidad y la sensibilidad clínica de esta RT-PCR en tiempo real adaptada para detectar RVH.
PDF
