RESUMEN
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a current public health concern. Rapid diagnosis is crucial, and reverse transcription polymerase chain reaction (RT-PCR) is presently the reference standard for SARS-CoV-2 detection. OBJECTIVE: Automated RT-PCR analysis (ARPA) is a software designed to analyze RT-PCR data for SARSCoV-2 detection. ARPA loads the RT-PCR data, classifies each sample by assessing its amplification curve behavior, evaluates the experiment's quality, and generates reports. METHODS: ARPA was implemented in the R language and deployed as a Shiny application. We evaluated the performance of ARPA in 140 samples. The samples were manually classified and automatically analyzed using ARPA. RESULTS: ARPA had a true-positive rate = 1, true-negative rate = 0.98, positive-predictive value = 0.95, and negative-predictive value = 1, with 36 samples correctly classified as positive, 100 samples correctly classified as negative, and two samples classified as positive even when labeled as negative by manual inspection. Two samples were labeled as invalid by ARPA and were not considered in the performance metrics calculation. CONCLUSIONS: ARPA is a sensitive and specific software that facilitates the analysis of RT-PCR data, and its implementation can reduce the time required in the diagnostic pipeline.
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COVID-19/diagnóstico , Diagnóstico por Computador , SARS-CoV-2/aislamiento & purificación , Programas Informáticos , Prueba de COVID-19 , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saliva/virologíaRESUMEN
As part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of the viral genome through reverse transcription-quantitative PCR (RT-qPCR) is the gold standard for this virus; however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is less expensive and can be as efficient as the classical protocol, which involves column purification of the viral RNA. In addition, it bypasses the need for swab sampling, decreases the risk of the health care personnel involved in the testing process, and accelerates the diagnostic procedure.
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Betacoronavirus/aislamiento & purificación , Saliva/virología , Manejo de Especímenes/métodos , Betacoronavirus/genética , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Pruebas Diagnósticas de Rutina , Genoma Viral/genética , Humanos , Nasofaringe/virología , Orofaringe/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Carga ViralRESUMEN
Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.
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Anticuerpos Neutralizantes/aislamiento & purificación , Antígenos Virales/inmunología , Infecciones por Astroviridae/inmunología , Mamastrovirus/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/genética , Infecciones por Astroviridae/virología , Células CACO-2 , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Variación Genética , Humanos , Inmunización , Mamastrovirus/genética , Ratones , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
BACKGROUND: Most of the studies characterizing the incidence of rhinovirus (RV) have been carried out in hospitalized children and in developed countries. In those studies, RV-C has been associated with more severe respiratory tract infections than RV species A and B. In this study we determined the frequency and diversity of RV strains associated with upper and lower respiratory tract infections (URTI, LRTI) in Mexico, and describe the clinical characteristics of the illness associated with different RV species. METHODS: A prospective surveillance of 526 and 250 children with URTI and LRTI was carried out. Respiratory samples were analyzed by RT-PCR for viruses. The 5' untranslated region of the RV genome was amplified and sequenced. RESULTS: In the case of URTI, 17.5% were positive for RV, while this virus was found in 24.8% of LRTI. The RV species was determined in 73 children with URTI: 61.6% were RV-A, 37% RV-C and, 1.4% RV-B; and in 43 children with LRTI: 51.2% were RV-A, 41.8% RV-C, and 7% RV-B. No significant differences in clinical characteristics were found in patients with RV-A or RV-C infections. A high genetic diversity of RV strains was found in both URTI and LRTI. CONCLUSIONS: Both RV-A and RV-C species were frequently found in hospitalized as well as in outpatient children. This study underlines the high prevalence and genetic diversity of RV strains in Mexico and the potential severity of disease associated with RV-A and RV-C infections.
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Infecciones por Picornaviridae/virología , Infecciones del Sistema Respiratorio/virología , Rhinovirus/fisiología , Niño , Preescolar , Femenino , Hospitalización , Humanos , Lactante , Masculino , México/epidemiología , Infecciones por Picornaviridae/epidemiología , Estudios Prospectivos , Infecciones del Sistema Respiratorio/epidemiología , Rhinovirus/genética , Rhinovirus/aislamiento & purificaciónRESUMEN
BACKGROUND: While the Northern Hemisphere experiences the effects of the 2009 pandemic influenza A (H1N1) virus, data from the recent influenza season in the Southern Hemisphere can provide important information on the burden of disease in children. METHODS: We conducted a retrospective case series involving children with acute infection of the lower respiratory tract or fever in whom 2009 H1N1 influenza was diagnosed on reverse-transcriptase polymerase-chain-reaction assay and who were admitted to one of six pediatric hospitals serving a catchment area of 1.2 million children. We compared rates of admission and death with those among age-matched children who had been infected with seasonal influenza strains in previous years. RESULTS: Between May and July 2009, a total of 251 children were hospitalized with 2009 H1N1 influenza. Rates of hospitalization were double those for seasonal influenza in 2008. Of the children who were hospitalized, 47 (19%) were admitted to an intensive care unit, 42 (17%) required mechanical ventilation, and 13 (5%) died. The overall rate of death was 1.1 per 100,000 children, as compared with 0.1 per 100,000 children for seasonal influenza in 2007. (No pediatric deaths associated with seasonal influenza were reported in 2008.) Most deaths were caused by refractory hypoxemia in infants under 1 year of age (death rate, 7.6 per 100,000). CONCLUSIONS: Pandemic 2009 H1N1 influenza was associated with pediatric death rates that were 10 times the rates for seasonal influenza in previous years.
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Brotes de Enfermedades , Hospitalización/estadística & datos numéricos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/epidemiología , Adolescente , Distribución por Edad , Argentina/epidemiología , Niño , Preescolar , Comorbilidad , Femenino , Humanos , Hipoxia/etiología , Hipoxia/mortalidad , Lactante , Recién Nacido , Gripe Humana/clasificación , Gripe Humana/complicaciones , Gripe Humana/mortalidad , Masculino , Neumonía Bacteriana/epidemiología , Neumonía Bacteriana/etiología , Índice de Severidad de la Enfermedad , Staphylococcus/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
Introduction. Lateral flow test (LFTs) have been used as an alternative to reverse transcription quantitative PCR (RT-qPCR) in point-of-care testing. Despite their benefits, the sensitivity of LFTs may be low and is affected by several factors. We have previously reported the feasibility of using direct lysis of individual or pools of saliva samples from symptomatic and asymptomatic patients as a source of viral genomes for detection by RT-qPCR.Hypothesis. Direct lysed saliva is more sensitive than antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples from children.Aim. Our goals here were to valuate the specificity and sensitivity of the PanBio COVID-19 antigen rapid test device (Ag-RTD) compared with RT-qPCR of direct lysed saliva.Methodology. We evaluated the performance of the PanBio COVID-19 Ag-RTD in comparison to RT-qPCR direct lysed saliva from paired samples of 256 symptomatic and 242 asymptomatic paediatric patients.Results. Overall, although there were no differences in the specificity (96.6%), we found a lower sensitivity (64.3%) of the PanBio Ag-test RTD compared to saliva in both symptomatic and asymptomatic patients. In addition, the sensitivity of PanBio was not correlated with the viral load present in the samples.Conclusion. Our data highlight the benefits of using RT-qPCR and saliva samples for SARS-CoV-2 detection, particularly in paediatric patients.
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COVID-19 , SARS-CoV-2 , Humanos , Niño , SARS-CoV-2/genética , Prueba de COVID-19 , Saliva , COVID-19/diagnóstico , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.
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Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Saliva/virología , COVID-19/epidemiología , COVID-19/prevención & control , Prueba de Ácido Nucleico para COVID-19/normas , Humanos , Tamizaje Masivo/métodos , Tamizaje Masivo/normas , Cuarentena/normas , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidad , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Two quality controlled inter-laboratory exercises were organized within the EU project 'Realizing the European Network of Biodosimetry (RENEB)' to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. MATERIALS AND METHODS: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. RESULTS: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. CONCLUSIONS: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.
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Bioensayo/métodos , Aberraciones Cromosómicas/efectos de la radiación , Pruebas de Micronúcleos/métodos , Garantía de la Calidad de Atención de Salud , Exposición a la Radiación/análisis , Monitoreo de Radiación/métodos , Bioensayo/normas , Europa (Continente) , Humanos , Linfocitos/efectos de la radiación , Monitoreo de Radiación/normas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.
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Receptores de Superficie Celular/metabolismo , Rotavirus/fisiología , Proteínas de Uniones Estrechas/metabolismo , Internalización del Virus , Animales , Línea Celular , Humanos , Macaca mulatta , Ratones , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Superficie Celular/genética , Proteínas de Uniones Estrechas/genéticaRESUMEN
BACKGROUND: Acute respiratory tract infections are the leading cause of morbidity and mortality in children worldwide. Many studies have described the frequency of viruses in hospitalized patients, but studies describing the prevalence of viruses in the community setting are limited, particularly in developing countries, where most of the deaths from serious respiratory diseases occur. The aim of this study was to evaluate the diversity of respiratory viruses in the community setting using molecular diagnostic tools, as well as the clinical characteristics of respiratory viral infections in the general pediatric practice in Mexico. METHODS: Children with respiratory tract infections attending private pediatric practices during a 10-month period in five cities of the state of Veracruz were included. Nasal swabs were taken and processed by a multiplex detection kit for 15 respiratory viruses. RESULTS: 525 children were included from July 2011 to May 2012; 44% were female, mean age was 45 months. The 3 most frequent clinical diagnosis were: rhinopharyngitis 68%, pharyngitis 18%, and 3.3% influenza-like illness. 71.5% of the samples were positive for virus. The five most frequent pathogens were respiratory syncycitial virus in 18.3% of the children, rhinovirus in 17.5%, influenza A 9.1%, adenovirus 7.2%, and enterovirus 3.4%, although all 15 viruses were detected; there were viral coinfections in 14.1%, and 28.5% of the samples were negative. CONCLUSIONS: A large proportion of respiratory infections in the community setting in Mexico was associated to viruses. Although testing for common respiratory pathogens in children with acute respiratory tract infections may lead to a better understanding of the role of viral pathogens in, and eventually to improvement in the management of, individual patients, additional prospective studies are required to study the need of routinely using such tests in general pediatric practices in resource-limited countries.
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Infecciones del Sistema Respiratorio/virología , Virus/aislamiento & purificación , Adolescente , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Lactante , Masculino , México/epidemiología , Prevalencia , Estudios Prospectivos , Infecciones del Sistema Respiratorio/epidemiología , Estaciones del Año , Virus/clasificaciónRESUMEN
Viruses are the most frequent cause of respiratory disease in children. However, despite the advanced diagnostic methods currently in use, in 20 to 50% of respiratory samples a specific pathogen cannot be detected. In this work, we used a metagenomic approach and deep sequencing to examine respiratory samples from children with lower and upper respiratory tract infections that had been previously found negative for 6 bacteria and 15 respiratory viruses by PCR. Nasal washings from 25 children (out of 250) hospitalized with a diagnosis of pneumonia and nasopharyngeal swabs from 46 outpatient children (out of 526) were studied. DNA reads for at least one virus commonly associated to respiratory infections was found in 20 of 25 hospitalized patients, while reads for pathogenic respiratory bacteria were detected in the remaining 5 children. For outpatients, all the samples were pooled into 25 DNA libraries for sequencing. In this case, in 22 of the 25 sequenced libraries at least one respiratory virus was identified, while in all other, but one, pathogenic bacteria were detected. In both patient groups reads for respiratory syncytial virus, coronavirus-OC43, and rhinovirus were identified. In addition, viruses less frequently associated to respiratory infections were also found. Saffold virus was detected in outpatient but not in hospitalized children. Anellovirus, rotavirus, and astrovirus, as well as several animal and plant viruses were detected in both groups. No novel viruses were identified. Adding up the deep sequencing results to the PCR data, 79.2% of 250 hospitalized and 76.6% of 526 ambulatory patients were positive for viruses, and all other children, but one, had pathogenic respiratory bacteria identified. These results suggest that at least in the type of populations studied and with the sampling methods used the odds of finding novel, clinically relevant viruses, in pediatric respiratory infections are low.
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Virus ADN/fisiología , Virus ARN/fisiología , Infecciones del Sistema Respiratorio/virología , Niño , Niño Hospitalizado , Preescolar , Coronavirus/genética , Coronavirus/fisiología , Virus ADN/clasificación , Virus ADN/genética , ADN Viral/análisis , Femenino , Humanos , Lactante , Masculino , Nasofaringe/virología , Filogenia , Neumonía/diagnóstico , Neumonía/virología , Virus ARN/clasificación , Virus ARN/genética , ARN Viral/análisis , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/fisiología , Infecciones del Sistema Respiratorio/patología , Rhinovirus/genética , Rhinovirus/fisiología , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: The immune response to dengue virus (DENV) primary infection in infants and young children is not well characterized. In Northern Argentina, >90% of the population was DENV-naïve before the 2009 outbreak, allowing evaluation of age-dependent primary responses to infection. METHODS: We conducted a comparative study of the immune response to DENV in 27 infected infants, young children and their mothers. Lymphocyte T helper (Th) 1, Th2, Th17 and inflammatory responses were assayed in blood during the 2009 DENV-1 epidemic. RESULTS: The immune response to DENV-1 was significantly biased to Th2 in infected infants and young children, compared to infants with other febrile illnesses (for IL-4 p < 0.001) and to their infected mothers (for IL-4 p < 0.01). In addition, IL-17 suppression was observed in the memory response to DENV-1 in infected infants (p < 0.01 vs placebo). CONCLUSION: Age-related differences in the primary response to DENV, characterized by an immature Th2 polarization and Th17 suppression in infants, should be studied further in order to expand our understanding of the mechanism of dengue pathogenesis.
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Virus del Dengue/inmunología , Dengue/inmunología , Interleucina-17/inmunología , Células Th2/inmunología , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/análisis , Argentina/epidemiología , Preescolar , Citocinas/inmunología , Epidemias , Femenino , Humanos , Inmunidad Celular/inmunología , Inmunoglobulina G/sangre , Lactante , Recién Nacido , Masculino , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Los divertículos faringoesofágicos son relativamente infrecuentes, pero deben considerarse siempre dentro del diagnóstico diferencial de disfagia, halitosis y enfermedades respiratorias por broncoaspiración. Con el fin de informar la experiencia con 8 casos operados entre 1991 y 2005, se revisan los expedientes clínicos y se encuentran 6 hombres y 2 mujerescon edad promedio de 63 años y con síntomas más frecuentes de disfagia, tos y halitosis, a quienes se les confirmó el diagnóstico con un trago de bario. Uno de los pacientes había sido tratado por otro grupo, mediante resección del divertículo sin miotomía, y se presentó con recidiva. En todos los casos de este grupo se realizó una miotomía amplia. En 3 que eran pequeños se invirtió el divertículo con una bolsa de tabaco; en 4 se resecó el divertículo y en 1 se realizó diverticulopexia. Uno de los pacientes presentó una fístula que cerró en 2 semanas y todos evolucionaro bien, sin presentar otras complicaciones ni secuelas.