Detección del SARS-CoV-2 mediante RT-qPCR utilizando saliva en pacientes ambulatorios con estudio de COVID-19

Resumen Introducción: La pandemia de COVID-19 ha afectado a millones de personas en todo el mundo. La identificación de sujetos infectados ha sido importante para el control. Objetivo: Evaluar el rendimiento de una reacción de polimerasa en cadena (RPC) cuantitativa en tiempo real (en inglés: RT-qPCR) para SARS-CoV-2, utilizando saliva como matriz en comparación con un hisopado nasofaríngeo (HNF). Metodología: Se reclutaron adultos en atención ambulatoria, la mayoría sintomáticos. Fueron estudiadas 530 muestras pareadas de saliva e HNF con RT-qPCR. Resultados: Fueron positivas 59 muestras de HNF y 54 de saliva. La sensibilidad con saliva fue 91%, especificidad 100%, el valor predictor positivo (VPP) 100%, valor predictor negativo (VPN) 98%. El índice Kappa fue de 0,95 y LR-0,08. En promedio, el umbral de ciclo (en inglés cycle threshold-CT) de la saliva fue 3,99 puntos más alto que los de HNF (p < 0,0001) mostrando que la carga viral (CV) es menor en saliva. La carga viral en ambas disminuyó con el tiempo después del inicio de los síntomas. El muestreo de saliva fue preferido por los sujetos en lugar de HNF. Conclusión: Este estudio demuestra que la RPC para SARS-CoV-2 utilizando saliva, es adecuada para el diagnóstico de COVID-19 en adultos ambulatorios, especialmente en la etapa temprana de los síntomas.

Abstract Background: The COVID-19 pandemic has affected millions of people around the world. Part of control strategies is testing a large proportion of the population to identify and isolate the infected subjects. Aim: To evaluate the SARS-CoV-2 detection by the performance of a reverse transcription and quantitative polymerase chain reaction (RT-qPCR) against SARS-CoV-2, using saliva as a matrix compared to a nasopharyngeal swab (NPS) to simplify obtaining a diagnostic sample. Methods: Adults in outpatient care were recruited, 95% of them symptomatic. We studied 530 paired saliva and NPS samples by SARS-CoV-2 RT-qPCR. Results: Fifty-nine individuals tested positive in NPS and 54 in saliva samples. Sensitivity for saliva sample was 91%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value (NPV) 98%. The Kappa index was 0.95 and LR-0.08. On average, the cycle threshold (CT) of saliva was 3.99 points higher than those of NPS (p < 0.0001) showing that viral load (VL) is lower in saliva than in NPS. Viral load in both decreased over the time after onset of symptoms. Saliva sampling was preferred by subjects instead of NPS. Conclusion: This study demonstrates that SARS-CoV-2 RT-qPCR using saliva, even with lower VL, is suitable for the diagnosis of COVID-19 in outpatient adults, especially at early stage of symptoms.

Aporte de la biología molecular en el diagnóstico de infecciones respiratorias agudas / Contribution of multiplex respiratory molecular panel to the diagnosis of acute respiratory infections

Objective: To assess the performance of multiplex-PCR for diagnosis of respiratory viruses in parallel with direct fluorescence assay (DFA). We assessed the performance and co-infection diagnosis of molecular respiratory panel PCR (MRP-PCR) and DFA in hospitalized and outpatients. Results: 8535 samples were included, 1792 tested by MRP-PCR (46.9% positive) and 6743 by DFA (35.1% positive). MRP-PCR diagnosed co-infection in 21.3% and DFA in 1.8% of the samples. Rhinovirus was the most common virus in any age group. In 210 patients both tests were done; 100 were positive by MRP-PCR and 18 by DFA. Positive concordance value was 6.2%. 85 samples were positive only by MRP-PCR and in 42 of them only novel respiratory viruses were identified. Performance of MRP-PCR was statistically significant compared DFA for traditional respiratory viruses. Discussion: Multiplex PCR has shown better sensitivity, may expand the etiologic spectrum of respiratory infections and detect a higher number of co-infections.

Objetivo: Evaluar la contribución del panel respiratorio molecular por reacción en cadena de la polimerasa-multiplex (PRM-RPC) en paralelo a la de inmunofluorescencia directa (IFD) al diagnóstico de infecciones respiratorias. Analizamos y comparamos el rendimiento y diagnóstico de co-infección de PRM-RPC con IFD en pacientes hospitalizados y ambulatorios. Resultados: Se analizaron 8535 muestras; 1792 por PRM-RPC (46,9% positivas) y 6743 por IFD (35,1% positivas). La co-infección fue 21,3% por PRM-RCP y 1,8% por IFD. El virus más frecuente fue rinovirus a toda edad. Se analizaron 210 pacientes por ambos métodos; resultaron positivas 100 por PRM-RPC y 18 por IFD, concordancia positiva de 6,2%. 85 muestras fueron solo positivas por PRM-RPC, 42 diagnosticaron nuevos virus respiratorios. El rendimiento de PRM-RPC fue significativamente mayor que el de IFD para virus respiratorios tradicionalmente diagnosticados. Conclusiones: La RCP-multiplex tiene mejor sensibilidad, podría expandir el espectro etiológico de infecciones respiratorias y detectar un mayor número de co-infecciones comparado a IFD.

Bocavirus humano en Chile: características clínicas y epidemiológicas en niños con infecciones respiratorias / Human bocavirus in Chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections

Background: Human bocavirus (HBoV) is a newly discovered parvovirus found in children with acute respiratory tract infections (ARTI). Objectives: To describe the epidemiological and clinical profile of children < 5 years old consulting for ARTI, comparing cases of HBoV monoinfection and coinfection with other known respiratory viruses. Furthermore, we aimed to estimate the prevalence of viral shedding in asymptomatic children and perform phylogenetic analysis. Patients and Methods: We investigated the presence of HBoV in nasopharyngeal secretions from children consulting for AlRTI and among asymptomatic controls, between 2007 and 2008, by polymerase chain reaction. Results: HBoV was detected in 79 (21.8 percent) of 362 nasopharyngeal swabs obtained from children with ARTI. In 60/79 (76 percent), coinfection with other respiratory viruses was confirmed. Most common symptoms were cough, fever and rhinorrhea. Children infected only with HBoV showed significantly lower frequencies of respiratory distress, oxygen requirements and hospital admission than those with coinfection. HBoV was detected in 6/16 (37.5 percent) samples from asymptomatic children. The phylogenetic analysis of viruses from Chilean patients revealed that circulating HBoV was closely related to original strains. Conclusions: HBoV was found either in symptomatic and asymptomatic children. The severity of the disease was greater when HBoV was associated to other respiratory viruses.

Introducción: Bocavirus humano (HBoV) es un nuevo parvovirus encontrado en niños con infecciones respiratorias agudas (IRA). Objetivos: Describir la epidemiología y perfil clínico en niños < 5 años con IRA, comparando aquellos con HBoV como único agente identificado, con los que tenían co-infección con otro virus respiratorio. Además se evaluó su prevalencia en niños asintomáticos, y se realizó análisis filogenético. Materiales y Métodos: Se investigó la presencia de HBoV, por medio de reacción de polimerasa en cadena, en muestras de secreción nasofaríngea obtenida en niños con IRA y en controles asintomáticos, entre 2007 y 2008. Resultados: Se detectó HBoV en 79 (21,8 por ciento) de 362 muestras obtenidas en pacientes con IRA. En 60/79 (76 por ciento), se demostró co-infección. Los síntomas más frecuentes fueron tos, fiebre y rinorrea. Los pacientes con HBoV como único agente identificado mostraron frecuencias significativamente menores de dificultad respiratoria, requerimiento de oxígeno y hospitalización, comparado con los co-infectados. HBoV se detectó en 6/16 (37,5 por ciento) muestras de niños asintomáticos. El análisis filogenético de las cepas chilenas demuestra estrecha relación con las cepas originales. Conclusiones: HBoV está presente en niños chilenos con IRA y asintomáticos. La gravedad de la enfermedad fue mayor en el grupo con co-infección.

[Human bocavirus in Chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections]. / Bocavirus humano en Chile: características clínicas y epidemiológicas en niños con infecciones respiratorias.

BACKGROUND: Human bocavirus (HBoV) is a newly discovered parvovirus found in children with acute respiratory tract infections (ARTI). OBJECTIVES: To describe the epidemiological and clinical profile of children < 5 years old consulting for ARTI, comparing cases of HBoV monoinfection and coinfection with other known respiratory viruses. Furthermore, we aimed to estimate the prevalence of viral shedding in asymptomatic children and perform phylogenetic analysis. PATIENTS AND METHODS: We investigated the presence of HBoV in nasopharyngeal secretions from children consulting for AlRTI and among asymptomatic controls, between 2007 and 2008, by polymerase chain reaction. RESULTS: HBoV was detected in 79 (21.8%) of 362 nasopharyngeal swabs obtained from children with ARTI. In 60/79 (76%), coinfection with other respiratory viruses was confirmed. Most common symptoms were cough, fever and rhinorrhea. Children infected only with HBoV showed significantly lower frequencies of respiratory distress, oxygen requirements and hospital admission than those with coinfection. HBoV was detected in 6/16 (37.5%) samples from asymptomatic children. The phylogenetic analysis of viruses from Chilean patients revealed that circulating HBoV was closely related to original strains. CONCLUSIONS: HBoV was found either in symptomatic and asymptomatic children. The severity of the disease was greater when HBoV was associated to other respiratory viruses.

[Pandemic influenza one year after the first wave: what did we learn?]. / Influenza pandémica a un año de la primera ola. ¿Qué podemos decir ahora?

During year 2009 our nation experimented the first influenza pandemic wave due to the novel influenza A (H1N1) 2009 virus that emerged in the Northern hemisphere at the end of April, 2009. Estimated attack rate was about 6 to 12% affecting mainly to schoolchildren who presented with a mild disease. Age groups with highest risk of hospitalization were elderly people and children under 5 years old. Elderly patients and patients with co-morbidities had the highest risk to die. We have learned that clinical diagnosis of influenza has a laboratory confirmation in about 80% of cases but its correlation is lower in kids under 5 years old, especially in infants when RSV co-circulates with influenza virus. Laboratory diagnostic methods like DFA and immuno-chromatography have about 80% of sensitivity but a significantly lower rate in elderly patients compared to PCR. The clinical impact of this new virus justifies the recommendation to vaccinate traditionally established risk groups and to prescribe antiviral treatment to patients that acquire severe influenza or have risk factors to progress to complications.

Influenza pandémica a un año de la primera ola: ¿qué podemos decir ahora? / Pandemic influenza one year after the first wave: what did we learn?

During year 2009 our nation experimented the first influenza pandemic wave due to the novel influenza A (H1N1) 2009 virus that emerged in the Northern hemisphere at the end of April, 2009. Estimated attack rate was about 6 to 12 percent affecting mainly to schoolchildren who presented with a mild disease. Age groups with highest risk of hospitalization were elderly people and children under 5 years old. Elderly patients and patients with co-morbidities had the highest risk to die. We have learnt that clinical diagnosis of influenza has a laboratory confirmation in about 80 percent of cases but its correlation is lower in kids under 5 years old, especially in infants when RSV co-circulates with influenza virus. Laboratory diagnostic methods like DFA and immuno-cromatography have about 80 percent of sensitivity but a significantly lower rate in elderly patients compared to PCR. The clinical impact of this new virus justifies the recommendation to vaccinate traditionally established risk groups and to prescribe antiviral treatment to patients that acquire severe influenza or have risk factors to progress to complications.

Durante el año 2009 nuestro país vivió la primera ola pandémica causada por el nuevo virus de influenza A (H1N1) 2009 aparecido en el hemisferio norte a fines de abril del ese año. La tasa de ataque estimada fue entre 6 a 12 por ciento afectando más frecuentemente a los escolares quienes presentaron una enfermedad leve. Los grupos etáreos con mayor riesgo de hospitalización fueron los adultos mayores y los niños bajo 5 años de edad. Tuvieron mayor riesgo de fallecer los adultos mayores y aquellas personas con una co-morbilidad asociada. Aprendimos que el diagnóstico clínico de influenza se confirma por laboratorio en alrededor del 80 por ciento pero tiene una baja correlación en los niños bajo 5 años de edad, especialmente en lactantes bajo 1 año de edad, al circular concomitantemente con VRS. Los métodos de diagnóstico como la IFD y los inmunocromatográficos tienen una sensibilidad que no supera el 80 por ciento y es muy baja en los adultos mayores cuando se compara con PCR. Dado el impacto clínico de este nuevo virus se justifica el uso de vacunación en los grupos de riesgo previamente descritos y el tratamiento con antivirales en los pacientes que cursan con enfermedad grave o con riesgo de evolucionar a una enfermedad más complicada.

[Seroepidemiological evidence of human exposure to Anaplasma sp in Santiago, Chile]. / Evidencia seroepidemiológica de exposición humana a Anaplasma sp en Santiago, Chile.

OBJECTIVES AND METHODS: To find more evidence of human exposure to Anaplasma sp in Chile, we studied 108 contacts of dogs with canine ehrlichiosis (CE) (risk group) and 61 persons without tick or CE cases contact (control group). A survey including risk factors and history of diseases compatible with ehrlichiosis/ anaplasmosis was applied to the risk group. Serum IgG anti-Anaplasma sp antibodies were determined in both groups. RESULTS: A significant difference was found in the prevalence of anti-Anaplasma sp antibodies in the risk group compared with the control group (18,5 versus 3,3%), p < 0,005. No risk factors associated to seropositivity were found, nor persons with history suggesting ehrlichiosis/anaplasmosis. Ninety four percent of the houses of the risk group had tick infestation. DISCUSSION: A greater risk of exposition to Anaplasma sp is documented in people living in close contact with CE cases and in houses with tick infestation.

Evidencia seroepidemiológica de exposición humana a Anaplasma sp en Santiago, Chile / Seroepidemiological evidence of human exposure to Anaplasma sp in Santiago, Chile

Objectives and Methods: To find more evidence of human exposure to Anaplasma sp in Chile, we studied 108 contaets of dogs with canine ehrlichiosis (CE) (risk group) and 61 persons without tick or CE cases contact (control group). A survey including risk factors and history of diseases compatible with ehrlichiosis/ anaplasmosis was applied to the risk group. Serum IgG anti-Anaplasma sp antibodies were determined in both groups. Results: A significant difference was found in the prevalence of anti-Anaplasma sp antibodies in the risk group compared with the control group (18,5 versus 3,3 percent), p < 0,005. No risk factors associated to seropositivity were found, ñor persons with history suggesting ehrlichiosis/anaplasmosis. Ninety four percent of the houses of the risk group had tick infestation. Discussion: A greater risk of exposition to Anaplasma sp is documented in people living in cióse contact with CE cases and in houses with tick infestation.

Objetivos y Método: Con el propósito de buscar mayor evidencia de exposición humana a Anaplasma sp en Chile, se estudiaron 108 personas en contacto con perros con ehrlichiosis canina (EC) (grupo de riesgo) y 61 personas sin antecedente de contacto con garrapatas ni con perros con EC (grupo control). Se aplicó encuesta sobre factores de riesgo e historia de cuadros sugerentes de ehrlichiosis/anaplasmosis al grupo de riesgo. En ambos grupos se determinó presencia de IgG anti-Anaplasma sp. Resultados: Se encontró significativa mayor prevalencia de anticuerpos anti-Anaplasma sp en el grupo de riesgo que en el grupo control (18,5 versus 3,3 por ciento), p < 0,005. No se encontraron factores de riesgo asociados a sero-positividad, ni personas con historia sugerente de ehrlichiosis/anaplasmosis clínica. Noventa y cuatro por ciento de las viviendas del grupo de riesgo presentaba infestación por garrapatas. Discusión: Se evidencia mayor riesgo de exposición humana a Anaplasma sp en personas en contacto cercano con perros con EC y que habitan viviendas con infestación por garrapatas.

Influenza aviar y riesgo de pandemia / Pandemic risk of avian influenza

Influenza is a common season pathology that occasionally presents pandemia, caused by a new Influenza A virus subtype that results from the genomic recombination of human virus with virus from other species. During the last years, there is a worldwide alert situation in terms of a new pandemia, due to the existence of Influenza A virus subtype H5N1 in birds from Southeast Asia, Europe and Africa. There are some sporadic cases in humans produced by close exposure with infected birds. The present article reviews the virologic characteristics of Influenza A H5N1 virus in humans and the chilean guidelines for a potential pandemia. Influenza is a respiratory disease produced by Influenza virus A,B,C, being the A type the most important due to its capacity to change structure and cause epidemia or pandemia. The last pandemias were classified as Spamsh flu in 1918-1919 (H1N1), Asian flu in 1957 (H2N2) and the Hong-Kong flu in 1967 (H3N2), with the biggest death population in 1918. In template countries, Influenza presents in epidemia affecting the winter months; in tropical countries, the virus circulation occurs during the whole year.

Influenza es una enfermedad común que se presenta en Chile en forma estacional. Ocasionalmente ocurren pandemias las que se generan cuando aparece un nuevo subtipo de virus influenza A en la humanidad producto de la recombinación de genomas de virus de influenza humano con virus de influenza de otras especies. En los últimos años la humanidad se encuentra en una situación de alerta de una nueva pandemia dada la existencia de la más grande epizootia por influenza A, subtipo H5N1 en aves que se extiende desde el Sudeste Asiático a Europa Oriental, Occidental y África. Se han documentado casos esporádicos en humanos por contacto cercano con aves infectadas. El presente artículo revisa las características virológicas del virus de influenza A, la situación actual de la epizootia por H5N1, las características de esta infección en humanos y el estado de preparación que se encuentra Chile frente a una eventual pandemia.

[Disseminated and fatal adenovirus infection in an immunocompromised child]. / Infección diseminada por adenovirus de curso fatal en un niño inmunocomprometido.

Severe adenovirus (ADV) infections have become increasingly important in immunocompromised patients, mainly in pediatric stem cell transplant recipients. We report a case of disseminated ADV infection leading to death in a 12-year-old stem cell transplant recipient. The diagnosis was confirmed by viral isolation and viral genome detection in tissues and blood. Main issues associated with infection, diagnosis and therapeutic alternatives are reviewed. This case should alert clinicians to suspect and study this agent in high risk patients and highlights the importance of having antiviral drugs for ADV available in Chile.

Infección diseminada por adenovirus de curso fatal en un niño inmunocomprometido / Disseminated and fatal adenovirus infection in an immunocompromised child

Las infecciones graves por adenovirus (ADV) tienen una importancia creciente en pacientes inmuno-comprometidos, en especial en niños sometidos a trasplante de precursores hematopoyéticos (TPH). Se reporta un caso de infección por ADV inicialmente gastrointestinal y luego diseminada, de curso fatal, en un niño de 12 años, post LPH. El diagnóstico se confirmó mediante aislamiento viral y detección de genoma viral en tejidos y sangre. Se revisan los principales aspectos de la infección por ADV, su diagnóstico y las posibilidades terapéuticas. Este caso debe alertar a los médicos clínicos para sospechar y estudiar este agente viral en pacientes de alto riesgo y enfatiza la importancia de disponer en Chile de antivirales para su tratamiento.

Severe adenovirus (ADV) infections have become increasingly important in immunocompromised patients, mainly in pediatric stem cell transplant recipients. We report a case of disseminated ADV infection leading to death in a 12-year-old stem cell transplant recipient. The diagnosis was confirmed by viral isolation and viral genome detection in tissues and blood. Main issues associated with infection, diagnosis and therapeutic alternatives are reviewed. This case should alert clinicians to suspect and study this agent in high risk patients and highlights the importance of having antiviral drugs for ADV available in Chile.

[Human metapneumovirus as hospitalization cause in children under 3 years old with acute respiratory infections during 2004]. / Metapneumovirus humano como causa de hospitalización en niños bajo 3 años de edad, con infección respiratoria aguda, durante el año 2004.

Human metapneumovirus was detected in 15 of 123 children (12%) younger than 3 years of age hospitalized for treatment of acute respiratory infection between July and November 2004. The virus was detected by RT-PCR directly from nasopharyngeal swabs and/or from supernatants after cell culture. Children infected with hMPV were mostly younger than one year of age (67%), all presenting with fever and cough. The main cause for hospitalization was the need for oxygen therapy (73%). Four hMPV positive children had an identifiable co-morbid condition but had a similar clinical evolution when compared to previously healthy infants. Chest radiography showed an increase in interstitial infiltrates with focal consolidation in 6 children. Obstructive bronchial syndrome and bronchiolitis, with or without pneumonia, were the most frequent diagnosis associated with hMPV positivity. A rapid and sensitive diagnostic method is required to improve diagnosis and treatment of these patients.

Metapneumovirus humano como causa de hospitalización en niños bajo 3 años de edad, con infección respiratoria aguda, durante el año 2004 / Human metapneumovirus as hospitalization cause in children under 3 years old with acute respiratory infections during 2004

Metapneumovirus humano (MPVh) fue detectado entre julio y noviembre en 15 de 123 niños bajo 3 años de edad hospitalizados por infección respiratoria aguda (12 por ciento). Las muestras fueron estudiadas mediante técnicas de biología molecular (RPC-TR de muestra de hisopado nasofaríngeo y/o de sobrenadante de cultivo). El 67 por ciento de los niños hospitalizados con MPVh tenían menos de 1 año de edad, todos ellos presentaron tos y fiebre y el principal motivo de hospitalización fue el requerimiento de oxígeno en 73 por ciento de los casos. Si bien un tercio de los pacientes tenía patología previa, su evolución clínica no fue diferente respecto de los niños previamente sanos. El patrón radiológico mostró aumento de la trama intersticial, con focos de consolidación en 6 casos (40 por ciento). El diagnóstico más frecuente fue síndrome bronquial obstructivo o bronquiolitis, asociado o no a neumonía. Destaca la necesidad de un método de diagnóstico rápido para optimizar el diagnóstico diferencial, manejo y control de infecciones en estos pacientes.

Human metapneumovirus was detected in 15 of 123 children (12 percent) younger than 3 years of age hospitalized for treatment of acute respiratory infection between July and November 2004. The virus was detected by RT-PCR directly from nasopharyngeal swabs and/or from supernatants after cell culture. Children infected with hMPV were mostly younger than one year of age (67 percent), all presenting with fever and cough. The main cause for hospitalization was the need for oxygen therapy (73 percent). Four hMPV positive children had an identifiable co-morbid condition but had a similar clinical evolution when compared to previously healthy infants. Chest radiography showed an increase in interstitial infiltrates with focal consolidation in 6 children. Obstructive bronchial syndrome and bronchiolitis, with or without pneumonia, were the most frequent diagnosis associated with hMPV positivity. A rapid and sensitive diagnostic method is required to improve diagnosis and treatment of these patients.

Vacuna anti-pertussis para uso en adolescentes y adultos / Pertussis vaccine for adolescents and adults

A pesar de la vacunación para prevenirla, la tos convulsiva es una enfermedad aún presente, afectando principalmente a la población de lactantes pequeños con alta morbi-mortalidad, pero también a los adolescentes y adultos que han perdido el efecto de la vacunación inicial; este segundo grupo se ha transformado en el principal reservorio y fuente de transmisión de Bordetella pertussis a lactantes. Las nuevas vacunas anti-pertussis, especialmente formuladas para este grupo etario, tienen un buen perfil de inmunogenicidad y de seguridad y su eficacia alcanza a 92%. La utilización universal de esta vacuna podría contribuir al control de la tos convulsiva en los grupos más susceptibles.

[Pertussis vaccine for adolescents and adults]. / Vacuna anti-pertussis para uso en adolescentes y adultos.

After the introduction of the pertussis vaccine the number of cases decreased substantially, albeit, the disease remains present affecting mostly young infants in whom it causes severe complications, adolescents and adults. Adults are the main reservoir of Bordetella pertussis and serve as vehicle of transmission reservoir to infants. The new pertussis vaccines, manufactured for adolescents and adults, have good immunogenicity, safety and efficacy (92%) profile. The introduction of this vaccine into a universal vaccination program for adolescents and adults, would decrease the incidence of the disease in this population and more importantly, transmission of the microorganism to infants.