ABSTRACT
BackgroundThe ongoing COVID-19 pandemic caused by SARs-CoV-2 was transmitted person to person via droplet infections and fecal-oral transmission. This illustrates the probability of environmentally facilitated transmission, mainly the sewage. MethodWe used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. ResultsA total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID patients and COVID 19 patient quarantine center drainage strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. DiscussionThis study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as early warning system to monitor viral tracking and circulation in cities with lower COVID-19 disease burden or heavily populated areas where door-to-door tracing may not be possible. However, attention needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.
ABSTRACT
The pandemic SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has created a widespread panic across the globe especially in the developing countries like Pakistan. The lack of resources and technical staff are causing havoc challenges in the detection and prevention of this global outbreak. Therefore, a less expensive and massive screening of suspected individuals for COVID-19 is required. In this study, a user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated to suggest a potential RT-qPCR alternate for rapid testing of COVID-19 suspected individuals. A total of 12 COVID-19 negative and 72 COVID-19 suspected individuals were analyzed. Both RT-qPCR and RT-LAMP assays were performed for all the individuals using open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay. Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. Interestingly all samples (45) having Ct values less than 30 showed 100% sensitivity. However, samples with weaker Ct values (i.e., => 35) showed 54% concordance, suggesting potential false negatives or false positives in RT-LAMP or RT-qPCR results, respectively. Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as an alternative strategy for rapid COVID-19 testing. Hence, based on fast processing time, minimal risk of specimens transfer and utilizing available resources, LAMP based detection of COVID-19 is strongly advocated especially for developing countries.
