ABSTRACT
La explosión de casos de COVID-19 resaltó el papel fundamental que desempeñan las pruebas de diagnóstico en la toma de decisiones médicas y de salud pública, para contener y mitigar la pandemia de SARS-CoV-2. Este estudio reporta la evaluación e implementación de diferentes tests para la detección molecular de SARS-CoV-2 en la región central de Argentina. Evaluamos 3 kits de RT-PCR en tiempo real (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection kit y WGene SARS-CoV-2 RT Detection), 2 métodos de extracción de ácidos nucleicos [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-minutes vs. 9-minutes], un reactivo pre-analítico (FlashPrep®) y 2 tests de amplificación isotérmica (Neokit Plus and ELA CHEMSTRIP®). El orden de rendimiento de los 3 kits de RT-PCR en tiempo real evaluados fue el siguiente: DisCoVery>GeneFinderTM>WGene. Los 2 métodos de extracción de RNA mostraron buenos y similares resultados;se seleccionó MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min debido a su rápido tiempo de procesamiento. El reactivo FlashPrep® mostró excelente resultado para realizar detección directa de RNA. Los ensayos de amplificación isotérmica mostraron valores de sensibilidad y especificidad aceptables (>80%), excepto en muestras con Ct>30. Nuestros resultados muestran kits de RT-PCR en tiempo real óptimos, como así también métodos moleculares alternativos para el diagnóstico de SARS-CoV-2 que resultan aceptables para su uso en contextos adversos, de descentralización y en diferentes escenarios epidemiológicos, para la detección rápida y precisa del SARS-CoV-2.
ABSTRACT
The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min], a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery>GeneFinderTM>WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct>30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be acceptable for their use in adverse contexts, decentralization, and different epidemiological scenarios, for rapid and accurate SARS-CoV-2 detection.
ABSTRACT
SARS-CoV-2 variants of concern (VOC) and interest (VOI) present mutations in reference to the original virus, being more transmissible. We implemented a rapid strategy for the screening of SARS-CoV-2 VOC/VOIs using real time RT-PCR and performed monitoring and surveillance of the variants in our region. Consecutive real-time RT-PCRs for detection of the relevant mutations/deletions present in the Spike protein in VOC/VOIs (TaqMan™ SARS-CoV-2 Mutation Panel, Applied Biosystems) were implemented. A total of 6,640 SARS-CoV-2 RNA samples (Cts < 30) from infected individuals in Central Argentina during 2021 were analyzed using different algorithms that were gradually adapted to the changing scenarios of local variant circulation. The strategy developed allowed the early detection and the identification of VOC/VOIs that circulated through the year, with a 100% of concordance with the WGS. The analyses of the samples showed introductions of VOCs Alpha and Gamma in February and March 2021, respectively. Gamma showed an exponential increase, with a peak of detection in July (72%), being responsible of the second wave of COVID19 in Argentina. Since VOC Delta entered into the region, it increased gradually, together with VOI Lambda, replacing VOC Gamma, until being the main variant (84.9%) on November. By December, these variants were replaced by the emergent VOC Omicron in a term of 2 weeks, producing the third wave. We report a useful tool for VOC/VOI detection, capable to quickly and cost-effectively monitor currently recognized variants in resource-limited settings, which allowed to track the recent expansion of Omicron in our region, and contributed to the implementation of public health measures to control the disease spread.
ABSTRACT
BACKGROUND: The current COVID-19 pandemic has overloaded the diagnostic capacity of laboratories by the gold standard method rRT-PCR. This disease has a high spread rate and almost a quarter of infected individuals never develop symptoms. In this scenario, active surveillance is crucial to stop the virus propagation. METHODS: Between July 2020 and April 2021, 11,580 oropharyngeal swab samples collected in closed and semi-closed institutions were processed for SARS-CoV-2 detection in pools, implementing this strategy for the first time in Córdoba, Argentina. Five-sample pools were constituted before nucleic acid extraction and amplification by rRT-PCR. Comparative analysis of cycle threshold (Ct) values from positive pools and individual samples along with a cost-benefit report of the whole performance of the results was performed. RESULTS: From 2,314 5-sample pools tested, 158 were classified as positive (6.8%), 2,024 as negative (87.5%), and 132 were categorized as indeterminate (5.7%). The Ct value shift due to sample dilution showed an increase in Ct of 2.6±1.53 cycles for N gene and 2.6±1.78 for ORF1ab gene. Overall, 290 pools were disassembled and 1,450 swabs were analyzed individually. This strategy allowed correctly identifying 99.8% of the samples as positive (7.6%) or negative (92.2%), avoiding the execution of 7,806 rRT-PCR reactions which represents a cost saving of 67.5%. CONCLUSION: This study demonstrates the feasibility of pooling samples to increase the number of tests performed, helping to maximize molecular diagnostic resources and reducing the work overload of specialized personnel during active surveillance of the COVID-19 pandemic.
Subject(s)
COVID-19 , Pandemics , COVID-19/diagnosis , COVID-19/epidemiology , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Specimen Handling/methods , Watchful WaitingABSTRACT
Monitoring wastewater for the traces of viruses allows effective surveillance of entire communities, including symptomatic and asymptomatic infected individuals, providing information on whether a specific pathogen is circulating in a population. In the context of the COVID-19 pandemic, 261 wastewater samples from six communities of the province of Córdoba, Argentina were analyzed. From mid-May 2020 to the end of August 2021, raw sewage samples were collected from the central network pipe that enters into the Wastewater Treatment Plants (WWTP) in Córdoba city and five communities in the Punilla Valley. SARS-CoV-2 was concentrated by using the polyethylene glycol-6000 precipitation method. Viral genomes were extracted from concentrated samples, and N- and E-SARS-CoV-2 genes were detected by using real time RT-PCR. Wastewater samples that resulted positive for SARS-CoV-2 genome detection were subjected to viral variants of concern (VOCs) identification by real time RT-PCR. Overall, just by using the identification of the N gene or E gene, the rates of viral genome detection were 43.4% (86/198) and 51.5% (102/198) respectively, and by using both methodologies (positivity criterion: detection of N and / or E gene), the detection rate was 71.2% (141/198). Thereby, the optimal strategy to study the SARS-CoV-2 genome in wastewater would be the use of the combined detection of both genes. Detection of SARS-CoV-2 variants in wastewater reflected their circulation in the community, showing no VOCs detection in the first COVID-19 wave and their co-circulation with Gamma, Alpha and Delta VOCs during 2021. Therefore, SARS-CoV-2 Wastewater Based Epidemiology (WBE) described the introduction, permanence and/or the co-circulation of viral variants in the community. In geographical areas with a stable population, SARS-CoV-2 WBE could be used as an early warning sign of new COVID-19 cases, whereas in localities with a low number of inhabitants and high tourist influx, WBE may only be useful to reflect the circulation of the virus in the community. Overall, the monitoring of SARS-CoV-2 in wastewater can become a silent sentinel of the trend of viral circulation in the community, providing supplementary information for clinical surveillance to support public health measures.
Subject(s)
COVID-19 , SARS-CoV-2 , Argentina/epidemiology , COVID-19/epidemiology , Humans , Pandemics , RNA, Viral , Wastewater , Wastewater-Based Epidemiological MonitoringABSTRACT
Although the nasopharyngeal swab (NPS) is considered the gold standard for the diagnosis of the SARS-CoV-2 infection, the Nasal Mid-Turbinate swab (NMTS) is often used due to its higher tolerance among patients. We compared the diagnostic performance of the NPS and the NMTS for the Panbio™ COVID-19 antigen-detecting rapid diagnostic test (Ag-RDT). Two hundred and forty-three individuals were swabbed three times by healthcare professionals: a NMTS and a NPS specimen for the Ag-RDT and an oropharyngeal swab for real time RT-PCR. Forty-nine participants were RNA-SARS-CoV-2 positive by real time RT-PCR: 45 and 40 were positive by the Ag-RDT with NPS and NMTS, respectively. The overall sensitivity and specificity were 91.8% (95% CI: 83.2-100.0) and 99.5% (95% CI: 98.2-100.0) for Ag-RDT with NPS, and 81.6% (95% CI: 69.8-93.5) and 100.0% (95% CI: 99.7-100.0) for the Ag-RDT with NMTS. The Cohen's kappa index was 0.92 (95% CI: 0.85-0.98). Among asymptomatic individuals, the Ag-RDT with both sampling techniques showed a high sensitivity [100.0% (95% CI: 95.5-100.0) with NPS; 90.9% (95% CI: 69.4-100.0) with NMTS], while the performance of the test decreased in samples with Ct≥ 30 and in patients tested after the first 7 days from symptom onset. Although the NMTS yielded a lower sensitivity compared to NPS, it might be considered a reliable alternative, as it presents greater adherence among patients, enabling scaling of antigen testing strategies, particularly in countries with under-resourced health systems.
Subject(s)
COVID-19 , Antigens, Viral , COVID-19/diagnosis , Humans , SARS-CoV-2 , Sensitivity and Specificity , TurbinatesABSTRACT
SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.