Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Direct costs and clinical impact of adenovirus genotype 8 conjunctivitis outbreak in a neonatology unit.

OBJECTIVE: To report a conjunctivitis outbreak in a neonatology intensive care unit (NICU) and determine the associated economic impact. DESIGN: Prospective observational study. SETTING: Centro de Educación Médica e Investigaciones Clínicas (CEMIC) University Hospital, a private, tertiary-care healthcare institution in Buenos Aires, Argentina. PARTICIPANTS: The study included 52 NICU neonates and 59 NICU-related healthcare workers (HCWs) from CEMIC hospital. METHODS: Neonates and HCWs were swabbed for real-time polymerase chain reaction (PCR) testing, viral culture, and typing by sequencing. Infection control measures, structural and logistic changes were implemented. Billing records were analyzed to determine costs. RESULTS: From January 30 to April 28, 2018, 52 neonates were hospitalized in the NICU. Among them, 14 of 52 (21%) had bilateral conjunctivitis with pseudomembranes. Symptomatic neonates and HCWs were HAdV-D8 positive. Ophthalmological symptoms had a median duration of 18 days (IQR, 13-24.5). PCR positivity and infectious range had a median duration of 18.5 days. As part of containment measures, the NICU and the high-risk pregnancy unit were closed to new patients. The NICU was divided into 2 areas for symptomatic and asymptomatic patients; a new room was assigned for the general nursery, and all deliveries from the high-risk pregnancy unit were redirected to other hospitals. The outbreak cost the hospital US$205,000: implementation of a new nursery room and extra salaries cost US$30,350 and estimated productivity loss during 1 month cost US$175,000. CONCLUSIONS: Laboratory diagnosis confirmed the cause of this outbreak as HAdV-D8. The immediate adoption and reinforcement of rigorous infection control measures limited the nosocomial viral spread. This outbreak represented a serious institutional problem, causing morbidity, significant economic loss, and absenteeism.

Nosocomial Transmission and Genetic Diversity of Rhinovirus in a Neonatal Intensive Care Unit.

Rhinoviruses were detected as sole pathogens in 6 preterm infants who developed severe respiratory infections while hospitalized in a neonatal intensive care unit. We confirmed 2 nosocomial rhinovirus transmission episodes and describe the genetic diversity of rhinovirus strains that circulated simultaneously during a winter season.

Incidence of viral respiratory infections in a prospective cohort of outpatient and hospitalized children aged ≤5 years and its associated cost in Buenos Aires, Argentina.

BACKGROUND: Although information about the incidence of viral respiratory illnesses and their associated cost can help health officials explore the value of interventions, data are limited from middle-income countries. METHODS: During 2008-2010, we conducted a prospective cohort study and followed ~1,800 Argentinian children aged ≤5 years to identify those children who were hospitalized or who sought care at an emergency room with any acute respiratory infection sign or symptom (e.g., rhinorrhea, cough, wheezing, tachypnea, retractions, or cyanosis). Respiratory samples were obtained for respiratory syncytial virus, influenza, parainfluenza, adenovirus, and metapneumovirus testing by immunofluorescence and for rhinovirus by real-time reverse transcription polymerase chain reaction. RESULTS: The incidence of respiratory syncytial virus (24/1000 children-years), human metapneumovirus (8/1000 children-years), and influenza (8/1000 children-years) illnesses was highest among hospitalized children aged <6 months and decreased among older children. In contrast, the incidence of rhinovirus was highest (12/1000 children-years) among those aged 6-23 months. In the emergency room, the incidence of rhinovirus was 459; respiratory syncytial virus 352; influenza 185; parainfluenza 177; metapneumovirus 130; and adenovirus 73/1,000 children-years. The total cost of hospitalization was a median of US$529 (Interquartile range, US$362-789). CONCLUSIONS: Our findings indicate that respiratory viruses, in particular rhinovirus, respiratory syncytial virus, metapneumovirus, and influenza may be associated with severe illness causing substantial economic burden.

Genetic diversity and clinical impact of human rhinoviruses in hospitalized and outpatient children with acute respiratory infection, Argentina.

BACKGROUND: Human rhinoviruses (HRV) are recognized as a cause of upper and lower acute respiratory infections (ARI). The circulating species and their clinical impact were not described in Argentina. OBJECTIVES: To describe the molecular epidemiology of HRV in children and to determine the association of HRV species with outcome and severity. STUDY DESIGN: Hospitalized and outpatients children <6 years old with ARI without comorbidities (n=620) were enrolled (2008-2010). Demographic, clinical data and outcome were analyzed. HRV were identified by RT-PCR. Phylogenetic analysis and demographic reconstruction for HRV were performed in selected samples. RESULTS: HRV were detected in 252/620 (40.6%) of children; 8.5% in viral coinfection. Bronchiolitis (55%) and pneumonia (13%) were the most frequent clinical diagnosis. Of 202 inpatients with HRV: 72% required oxygen supplementation, 11% intensive care unit and 3% mechanical ventilation. HRV were identified as a risk factor for hospitalization (OR: 2.47). All three HRV species were detected being HRV-A (55%) and HRV-C (43%) the most frequent; HRV-B was infrequent (2%). Of 44 sequenced HRV, 30 genotypes were detected. Seven of them were the most prevalent and circulated during limited periods of time. The demographic reconstruction revealed a constant population size and a high turnover rate of genotypes. Demographic and clinical outcome were similar for HRV-A and HRV-C infections. CONCLUSION: This study highlights the clinical impact of HRV in children without comorbidities as a cause of lower ARI and hospitalization. The high frequency of HRV infections may be associated with the simultaneous circulation of genotypes and their high turnover rate.

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.

[Acute viral respiratory infection in children under 5 years: Epidemiological study in two centers in Buenos Aires, Argentina]. / Infección respiratoria aguda viral en niños menores de 5 años: Estudio epidemiológico en dos centros de Buenos Aires, Argentina.

INTRODUCTION: Acute respiratory infections (ARI) are a major cause of hospitalization particularly at the extreme ages of life. OBJECTIVES: To determine the viral diagnosis in children < 5 years old with ARI, seasonality, clinical and epidemiological characteristics. POPULATION AND METHODS: A cross-sectional, descriptive and multicenter study was performed. Respiratory samples from patients with ARI hospitalized or outpatients with < 5 days of respiratory symptoms from June 2008 to May 2009 were studied for respiratory viruses. Clinical and demographic data were recorded. RESULTS: A total of 347 patients were enrolled: 234 hospitalized and 112 outpatients. Hospitalized patients were younger compared with outpatients (8 vs. 19 months, p <0.001) and presented more frequently bronchiolitis (74% hospitalized vs. 24% outpatients) and pneumonia (14% hospitalized vs. 5% outpatients). Age was statistically associated with hospitalization (p= 0.01). Viral diagnosis was achieved in 81% hospitalized and 57% of outpatients. Rhinovirus was the most frequent followed by respiratory syncytial virus. The rest of respiratory viruses were observed with lower frequency. Viral circulation was observed throughout the whole year. The median length of stay was 3.5 days. Intensive care was required in 11% of hospitalized patients and 3% required mechanical ventilation. No deaths were recorded. CONCLUSIONS: The use of viral diagnostic techniques allowed the identification of an etiologic agent in most of the hospitalized patients and more than half of outpatients. The addition of RT-PCR for rhinovirus, allowed the identification of this etiologic agent. Viral circulation was observed throughout the whole year. Hospitalized patients presented bronchiolitis and pneumonia more frequently than outpatients.

Infección respiratoria aguda viral en niños menores de 5 años: Estudio epidemiológico en dos centros de Buenos Aires, Argentina / Acute viral respiratory infection in children under 5 years: Epidemiological study in two centers in Buenos Aires, Argentina

Introducción. Las infecciones respiratorias agudas (IRA) son la mayor causa de hospitalización en edades extremas de la vida. Objetivos. Identifcar los agentes etiológicos de IRA viral en niños < 5 años hospitalizados y ambulatorios; describir estacionalidad y características clínico-epidemiológicas de la enfermedad. Población y métodos. Estudio descriptivo, transversal y multicéntrico en dos centros privados de Buenos Aires, en niños con IRA alta y baja. Se registraron antecedentes, cuadros clínicos y estudios virológicos. Resultados. Se estudiaron 347 pacientes: 235 hospitalizados; 112 ambulatorios. Los hospitalizados fueron menores que los ambulatorios (8 vs. 19 meses, p <0,001), presentaron más frecuentemente bronquiolitis (74% hospitalizados vs. 24% ambulatorios) y neumonía (14% hospitalizados vs. 5% ambulatorios). Solo la edad se asoció signifcativamente a hospitalización (p= 0,01). Se identifcó etiología viral en 81% de los hospitalizados y 57% de los ambulatorios. El virus más frecuente fue rinovirus seguido por virus respiratorio sincicial. Se observó circulación viral durante todo el año, especialmente en otoño e invierno. En pacientes hospitalizados, la mediana de hospitalización fue 3,5 días. Requirieron cuidados intensivos 25 (11%) pacientes, y 7 (3%) recibieron asistencia ventilatoria mecánica. No hubo letalidad. Conclusiones. Las técnicas de diagnóstico virológico permitieron identifcar agentes etiológicos en la mayoría de los pacientes hospitalizados y en más de la mitad de los ambulatorios con IRA. La incorporación de RT-PCR para rinovirus, permitió la identifcación de este agente etiológico. La circulación viral se observó a lo largo de todo el año. La bronquiolitis fue causa de internación en 174/201 (86,5%) niños con IRA y la neumonía en 33/39 (84,6%) niños.

Introduction. Acute respiratory infections (ARI) are a major cause of hospitalization particularly at the extreme ages of life. Objectives. To determine the viral diagnosis in children < 5 years old with ARI, seasonality, clinical and epidemiological characteristics. Population and methods. A cross-sectional, descriptive and multicenter study was performed. Respiratory samples from patients with ARI hospitalized or outpatients with < 5 days of respiratory symptoms from June 2008 to May 2009 were studied for respiratory viruses. Clinical and demographic data were recorded. Results. A total of 347 patients were enrolled: 234 hospitalized and 112 outpatients. Hospitalized patients were younger compared with outpatients (8 vs. 19 months, p <0.001) and presented more frequently bronchiolitis (74% hospitalized vs. 24% outpatients) and pneumonia (14% hospitalized vs. 5% outpatients). Age was statistically associated with hospitalization (p= 0.01). Viral diagnosis was achieved in 81% hospitalized and 57% of outpatients. Rhinovirus was the most frequent followed by respiratory syncytial virus. The rest of respiratory viruses were observed with lower frequency. Viral circulation was observed throughout the whole year. The median length of stay was 3.5 days. Intensive care was required in 11% of hospitalized patients and 3% required mechanical ventilation. No deaths were recorded. Conclusions. The use of viral diagnostic techniques allowed the identifcation of an etiologic agent in most of the hospitalized patients and more than half of outpatients. The addition of RT-PCR for rhinovirus, allowed the identifcation of this etiologic agent. Viral circulation was observed throughout the whole year. Hospitalized patients presented bronchiolitis and pneumonia more frequently than outpatients.