RESUMEN
Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically genotype A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of this virus in metropolitan and regional wastewater samples in Queensland, Australia. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log10 GC/2 µL of nucleic acid) down to a dilution of 1 × 10-5 pg/µL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10-4 pg/µL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations ranging from 3.60 to 6.23 log10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial.
Asunto(s)
Monitoreo del Ambiente , Kobuvirus , Técnicas de Amplificación de Ácido Nucleico , Aguas Residuales , Aguas Residuales/virología , Kobuvirus/genética , Queensland , Técnicas de Amplificación de Ácido Nucleico/métodos , Monitoreo del Ambiente/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.
Asunto(s)
COVID-19 , Virus de la Influenza A , Virus de la Influenza B , Reacción en Cadena de la Polimerasa Multiplex , Ribonucleasa P , SARS-CoV-2 , Humanos , Ribonucleasa P/genética , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , COVID-19/diagnóstico , COVID-19/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Diagnóstico Diferencial , Gripe Humana/diagnóstico , Gripe Humana/virología , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Límite de Detección , ARN Viral/genética , ARN Viral/análisisRESUMEN
In situ RT-PCR presents advantages over other expression analysis methods due to its rapid processing and low-cost equipment. However, this technique is not without its challenges. A protocol based on a capsule made from centrifuge tubes that offers advantages over slides is presented. This capsule protects histological sections from drying out, and its easy assembly reduces time pauses between incubations. In addition, the container size where the sample is deposited allows the addition and withdrawal of the different solutions. The capsule does not need previous sealing after each incubation, and, above all, it is a low-cost and accessible material. A guideline for tissue sectioning using a cryostat that offers advantages over other sectioning methods is also described.
Asunto(s)
Centrifugación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Centrifugación/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Plantas/genética , ARN de Planta/genéticaRESUMEN
Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.
Asunto(s)
Cartilla de ADN , Enfermedades de las Plantas , Enfermedades de las Plantas/virología , Cartilla de ADN/genética , Ecuador , Proteínas de la Cápside/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Especificidad de la Especie , ColombiaRESUMEN
As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.
Asunto(s)
COVID-19 , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , SARS-CoV-2 , Saliva , Manejo de Especímenes , Humanos , Saliva/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , COVID-19/virología , COVID-19/orina , Técnicas de Amplificación de Ácido Nucleico/métodos , Manejo de Especímenes/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , ARN Viral/análisis , ARN Viral/orina , ARN Viral/genética , ARN Viral/aislamiento & purificación , Prueba de Ácido Nucleico para COVID-19/métodos , Sensibilidad y Especificidad , Puerto Rico/epidemiología , Prueba de COVID-19/métodosRESUMEN
PURPOSE: This study aimed to evaluate the pre-sence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the ocular surface of individuals clinically suspected of coronavirus disease 2019 (COVID-19) and determine the accuracy of different approaches of molecular testing on the ocular surface based on the nasopharyngeal positivity status for COVID-19. METHODS: A total of 152 individuals with suspected COVID-19 symptoms who simultaneously underwent nasopharyngeal and two different tear film collection techniques for quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) were included. Tears were collected and randomized: one eye had the filter strip for the Schirmer test and the contralateral eye had conjunctival swab/cytology in the inferior fornix. All patients underwent slit lamp biomicroscopy. The accuracy of various ocular surface collection techniques used for the detection of SARS-CoV-2 RNA was determined. RESULTS: Of the 152 patients enrolled in the study, 86 (56.6%) had COVID-19 confirmed by nasopharyngeal PCR. Both tear film collection techniques detected viral particles: the Schirmer test was positive in 16.3% (14/86) and the conjunctival swab/cytology in 17.4% (15/86), with no statistically significant differences. No positive ocular tests were found among those with negative nasopharyngeal PCR tests. The overall agreement of the ocular tests was 92.7%, and in combination, the sensitivity would increase to 23.2%. The mean cycle threshold values in the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests were 18.2 ± 5.3, 35.6 ± 1.4, and 36.4 ± 3.9, respectively. Compared with the nasopharyngeal test, the Schirmer (p=0.001) and conjunctival swab/cytology (p<0.001) tests had significantly different Ct values. CONCLUSION: The Schirmer (16.3%) and conjunctival swab (17.4%) tests were comparably capable of detecting SARS-CoV-2 RNA in the ocular surface by RT-PCR accurately based on nasopharyngeal status and demonstrated indistinct sensitivity and specificity. Simultaneous specimen sampling and processing from the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests demonstrated significantly lower viral load in both ocular surface approaches than in the nasopharyngeal test. Ocular manifestations detected by slit lamp biomicroscopy were not associated with ocular RT-PCR positivity.
Asunto(s)
COVID-19 , Conjuntiva , Nasofaringe , ARN Viral , SARS-CoV-2 , Lágrimas , Carga Viral , Humanos , COVID-19/diagnóstico , COVID-19/virología , SARS-CoV-2/aislamiento & purificación , Femenino , Masculino , Lágrimas/virología , Adulto , Persona de Mediana Edad , ARN Viral/análisis , Conjuntiva/virología , Nasofaringe/virología , Prueba de Ácido Nucleico para COVID-19/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Anciano , Sensibilidad y Especificidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto Joven , Manejo de Especímenes/métodos , Reproducibilidad de los ResultadosRESUMEN
SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.
Asunto(s)
COVID-19/diagnóstico , Análisis Mutacional de ADN/métodos , Genoma Viral/genética , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Pandemias/prevención & control , Reproducibilidad de los Resultados , SARS-CoV-2/fisiología , Sensibilidad y EspecificidadRESUMEN
The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/epidemiología , Genes Virales , Pandemias/prevención & control , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/genética , Análisis de Secuencia de ARN/métodos , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Bases , Brasil/epidemiología , COVID-19/virología , Pruebas Diagnósticas de Rutina/métodos , Electroforesis en Gel de Agar/métodos , Monitoreo Epidemiológico , Humanos , Mutación , ARN Viral/genética , ARN Viral/aislamiento & purificaciónRESUMEN
BACKGROUND: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. METHODS AND RESULTS: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage. CONCLUSION: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.
Asunto(s)
Bovinos/sangre , Estabilidad del ARN , ARN Mensajero/sangre , ARN Mensajero/química , Transcriptoma/genética , Animales , Recolección de Muestras de Sangre/métodos , Frío , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
BACKGROUND: The viral dynamics and the role of children in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not completely understood. Our aim was to evaluate reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) values among children with confirmed SARS-CoV-2 compared with that of adult subjects. METHODS: Patients (from 2 months to ≤18 years of age and adults) with signs and symptoms of acute SARS-CoV-2 infection for less than 7 days were prospectively enrolled in the study from May to November 2020. All participants performed RT-PCR assay for SARS-CoV-2 detection; Ct values of ORF1ab, N and S gene targets and the average of all the 3 probes were used as surrogates of viral load. RESULTS: There were 21 infants (2 months to <2 years), 40 children (≥2 to <12 years), 22 adolescents (≥12 to <18 years) and 293 adults of 376 participants with confirmed SARS-CoV-2 infections. RT-PCR Ct values from all participants less than 18 years of age, as well as from all childhood subgroups, were not significantly different from adults, comparing ORF1ab, N, S and all the gene targets together (P = 0.453). CONCLUSIONS: Ct values for children were comparable with that of adults. Although viral load is not the only determinant of SARS-CoV-2 transmission, children may play a role in the spread of coronavirus disease 2019 in the community.
Asunto(s)
Prueba de COVID-19 , COVID-19/diagnóstico , COVID-19/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , Adolescente , Adulto , Factores de Edad , Brasil , Niño , Estudios Transversales , Humanos , Lactante , ARN Viral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Carga ViralRESUMEN
The Covid-19 pandemic, a disease transmitted by the SARS-CoV-2 virus, has already caused the infection of more than 120 million people, of which 70 million have been recovered, while 3 million people have died. The high speed of infection has led to the rapid depletion of public health resources in most countries. RT-PCR is Covid-19's reference diagnostic method. In this work we propose a new technique for representing DNA sequences: they are divided into smaller sequences with overlap in a pseudo-convolutional approach and represented by co-occurrence matrices. This technique eliminates multiple sequence alignment. Through the proposed method, it is possible to identify virus sequences from a large database: 347,363 virus DNA sequences from 24 virus families and SARS-CoV-2. When comparing SARS-CoV-2 with virus families with similar symptoms, we obtained [Formula: see text] for sensitivity and [Formula: see text] for specificity with MLP classifier and 30% overlap. When SARS-CoV-2 is compared to other coronaviruses and healthy human DNA sequences, we obtained [Formula: see text] for sensitivity and [Formula: see text] for specificity with MLP and 50% overlap. Therefore, the molecular diagnosis of Covid-19 can be optimized by combining RT-PCR and our pseudo-convolutional method to identify DNA sequences for SARS-CoV-2 with greater specificity and sensitivity.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Biología Computacional/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/genética , ADN Viral , Humanos , Aprendizaje Automático , Sensibilidad y Especificidad , Máquina de Vectores de Soporte , Virus/genéticaRESUMEN
With the arrival of coronavirus disease 2019 (COVID-19) in Brazil in February 2020, several preventive measures were taken by the population aiming to avoid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including the use of masks, social distancing, and frequent hand washing then, these measures may have contributed to preventing infection also by other respiratory viruses. Our goal was to determine the frequencies of Influenza A and B viruses (FLUAV/FLUBV), human mastadenovirus C (HAdV-C), Enterovirus 68 (EV-68), and rhinovirus (RV) besides SARS-CoV-2 among hospitalized patients suspect of COVID-19 with cases of acute respiratory disease syndrome (ARDS) in the period of March to December 2020 and to detect possible coinfections among them. Nucleic acid detection was performed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) in respiratory samples using naso-oropharyngeal swabs and bronchoalveolar lavage. A total of 418 samples of the 987 analyzed (42.3%) were positive for SARS-CoV-2, 16 (1.62%) samples were positive for FLUAV, no sample was positive for FLUBV or EV-68, 67 (6.78%) samples were positive for HAdV-C, 55 samples were positive for RV 1/2 (26.3%) and 37 for RV 2/2 (13.6%). Coinfections were also detected, including a triple coinfection with SARS-CoV-2, FLUAV, and HAdV-C. In the present work, a very low frequency of FLUV was reported among hospitalized patients with ARDS compared to the past years, probably due to preventive measures taken to avoid COVID-19 and the high influenza vaccination coverage in the region in which this study was performed.
Asunto(s)
Infecciones por Adenoviridae/epidemiología , COVID-19/epidemiología , Resfriado Común/epidemiología , Infecciones por Enterovirus/epidemiología , Gripe Humana/epidemiología , Distanciamiento Físico , Infecciones por Adenoviridae/prevención & control , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , COVID-19/prevención & control , Niño , Preescolar , Coinfección/epidemiología , Coinfección/virología , Resfriado Común/prevención & control , Enterovirus Humano D/genética , Enterovirus Humano D/aislamiento & purificación , Infecciones por Enterovirus/prevención & control , Femenino , Humanos , Lactante , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/prevención & control , Masculino , Máscaras , Mastadenovirus/genética , Mastadenovirus/aislamiento & purificación , Persona de Mediana Edad , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Adulto JovenRESUMEN
BACKGROUND: COVID-19 can be asymptomatic in a substantial proportion of patients. The assessment and management of these patients constitute a key element to stop dissemination. AIM: To describe the assessment and treatment of asymptomatic infection in patients with a confirmed diagnosis of COVID-19. METHODS: We searched five databases and search engines for preprints/preproofs, up to August 22, 2020. We included cohort, cross-sectional, and case series studies, reporting the assessment and management of asymptomatic individuals. We extracted data on total discharges with negative PCR, length of hospitalization, treatment, and number of patients who remained asymptomatic. A random-effects model with inverse variance method was used to calculate the pooled prevalence. RESULTS: 41 studies (nine cross-sectional studies, five retrospective studies and 27 reports/case series; 647 asymptomatic individuals), were included, of which 47% were male (233/501). The age of patients was between 1month and 73 years. In patients who became symptomatic, length of hospitalization mean was 13.6 days (SD 6.4). Studies used lopinavir/ritonavir, hydroxychloroquine plus ritonavir/lopinavir, hydroxychloroquine with and without azithromycin, ribavirin plus interferon and interferon alfa. The proportion of individuals who remained asymptomatic was 91% (463/588 patients; 95%CI: 78.3%-98.7%); and asymptomatic individuals discharged with negative PCR was 86% (102/124 individuals; 95%CI: 58.4%-100%). CONCLUSIONS: There is no standard treatment for asymptomatic COVID-19 individuals. There are no studies of adequate design to make this decision. It has been shown that most asymptomatic individuals who were followed have recovered, but this cannot be attributed to standard treatment.
Asunto(s)
Infecciones Asintomáticas/terapia , Tratamiento Farmacológico de COVID-19 , COVID-19/diagnóstico , Adolescente , Adulto , Anciano , Antibacterianos/uso terapéutico , Antivirales/uso terapéutico , Azitromicina/uso terapéutico , Prueba de Ácido Nucleico para COVID-19/métodos , Niño , Preescolar , Quimioterapia Combinada , Femenino , Hospitalización , Humanos , Hidroxicloroquina/uso terapéutico , Lactante , Lopinavir/uso terapéutico , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ritonavir/uso terapéutico , SARS-CoV-2/aislamiento & purificación , Adulto JovenRESUMEN
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the design and development of multiple reverse-transcription polymerase chain reaction kits aimed to facilitate the rapid scale-up of molecular testing for massive screening. We evaluated the diagnostic performance of nine commercial kits, which showed optimal performance and high discriminatory power. However, we observed differences in terms of sensitivity, specificity, and E gene Ct Values and discuss these results in light of the influence of SARS-CoV-2 genetic variability and its potential impact in current molecular diagnostic assays.
Asunto(s)
COVID-19/diagnóstico , Juego de Reactivos para Diagnóstico/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , COVID-19/virología , Prueba de COVID-19 , Colombia , Humanos , Técnicas de Diagnóstico Molecular , Sensibilidad y EspecificidadRESUMEN
Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.
Asunto(s)
Insectos Vectores/parasitología , Leishmania infantum/genética , Leishmania/aislamiento & purificación , Leishmaniasis Visceral/transmisión , Psychodidae/parasitología , Animales , Brasil , Expresión Génica , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida , Psychodidae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
BACKGROUND: The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES: To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS: Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS: Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS: Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.
Asunto(s)
Antígenos Virales/sangre , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Inmunológicas/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas no Estructurales Virales/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Dengue/sangre , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
Asunto(s)
COVID-19/diagnóstico , COVID-19/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Manejo de Especímenes/métodos , Adulto , Anciano , Servicio de Urgencia en Hospital , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
We conducted a multicenter clinical validity study of the Panbio coronavirus disease 2019 Antigen Rapid Test of nasopharyngeal samples in pediatric patients with coronavirus disease 2019-compatible symptoms of ≤5 days of evolution. Our study showed limited accuracy in nasopharyngeal antigen testing: overall sensitivity was 45.4%, and 99.8% of specificity, positive-predictive value was 92.5%.
Asunto(s)
Antígenos Virales/análisis , COVID-19/diagnóstico , ADN Viral/análisis , Nasofaringe/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , SARS-CoV-2/genética , Adolescente , COVID-19/virología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Pandemias , Reproducibilidad de los Resultados , SARS-CoV-2/inmunologíaRESUMEN
We described the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in stool samples from patients presenting only acute gastroenteritis (AGE) symptoms. From January to July 2020, 121 AGE stool samples were screened by quantitative reverse-transcription polymerase chain reaction. We detected SARS-CoV-2 in 27.5% of samples received during the epidemic period. No infectious viruses were observed in Vero E6 cells.
Asunto(s)
COVID-19/diagnóstico , COVID-19/virología , Gastroenteritis/virología , ARN Viral/aislamiento & purificación , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Adulto , Brasil/epidemiología , COVID-19/epidemiología , Prueba de COVID-19 , Heces/virología , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto JovenRESUMEN
BACKGROUND: Despite most cases not requiring hospital care, there are limited community-based clinical data on COVID-19. METHODS: The Corona São Caetano programme is a primary care initiative providing care to all residents with COVID-19 in São Caetano do Sul, Brazil. It was designed to capture standardised clinical data on community COVID-19 cases. After triage of potentially severe cases, consecutive patients presenting to a multimedia screening platform between 13 April and 13 May 2020 were tested at home with SARS-CoV-2 reverse transcriptase (RT) PCR; positive patients were followed up for 14 days with phone calls every 2 days. RT-PCR-negative patients were offered additional SARS-CoV-2 serology testing to establish their infection status. We describe the clinical, virological and natural history features of this prospective population-based cohort. FINDINGS: Of 2073 suspected COVID-19 cases, 1583 (76.4%) were tested by RT-PCR, of whom 444 (28.0%, 95% CI 25.9 to 30.3) were positive; 604/1136 (53%) RT-PCR-negative patients underwent serology, of whom 52 (8.6%) tested SARS-CoV-2 seropositive. The most common symptoms of confirmed COVID-19 were cough, fatigue, myalgia and headache; whereas self-reported fever (OR 3.0, 95% CI 2.4 to 3.9), anosmia (OR 3.3, 95% CI 2.6 to 4.4) and ageusia (OR 2.9, 95% CI 2.3 to 3.8) were most strongly associated with a positive COVID-19 diagnosis by RT-PCR or serology. RT-PCR cycle thresholds were lower in men, older patients, those with fever and arthralgia and closer to symptom onset. The rates of hospitalisation and death among 444 RT-PCR-positive cases were 6.7% and 0.7%, respectively, with older age and obesity more frequent in the hospitalised group. CONCLUSION: COVID-19 presents in a similar way to other mild community-acquired respiratory diseases, but the presence of fever, anosmia and ageusia can assist the specific diagnosis. Most patients recovered without requiring hospitalisation with a low fatality rate compared with other hospital-based studies.