The COVID-19 pandemic has been evolving in Pakistan with the emergence of the United Kingdom, South African, and Brazilian variants. These variants of concern (VOC) are known for increased transmissibility and can also be responsible for avoiding immune responses. The gold standard to detect VOC is sequencing, however routine genomic surveillance in resource-limited countries like Pakistan is not always readily available.
The inadvertent detection of the B.1.1.7 (United Kingdom) VOC by a target failure due to the key deletion in spike Δ69-70 by commercially available PCR assay helps to understand target failures as an alternative approach to detect variants. In pursuit of VOC it was further discovered that a deletion in the ORF1a gene (ORF1a Δ3675-3677) is common in B.1.1.7, B.1.351 (South African), and P.1 (Brazilian) VOC. The Real-Time Quantitative PCR (RT-qPCR) assay can distinguish target failures and can discriminate SARS-CoV-2 VOC. The study uses positive samples archived in respective labs.
Samples were divided into two groups. Group I constitutes 261 positive samples out of total of 16,964 (1.53%) performed from August till September 2020, while group II consists of 3501 positive samples out of a total of 46,041 (7.60%) performed, from November 2020 till January 2021. The RT-qPCR analysis showed that no VOC was present in positive samples of group I. However, a staggering difference in results was noted in group II where the positivity ratio increased exponentially and the VOC started appearing in significant numbers (53.64%). This concludes that the third wave in Pakistan is due to the importation of SARS-CoV-2 variants.

Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded.
The three nucleotides closest to the 3′ end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein.
This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.

Canine Olfactory Detection of SARS-COV2-Infected Patients: A One Health Approach

The aim of the present study is to apply the canine olfactory sensitivity to detect COVID-19-positive axillary sweat samples as a One Health approach in Latin America. One hundred volunteers with COVID-like symptoms were invited to participate, and both axillary sweat samples for dog detection and nasopharynx/oropharynx swabs for qPCR were collected.
Two dogs, previously trained, detected 97.4% of the samples positive for COVID-19, including a false-negative qPCR-test, and the positive predictive value was 100% and the negative predictive value was 98.2%. Therefore, we can conclude that canine olfactory sensitivity can detect a person infected with COVID-19 through axillary sweat successfully and could be used as an alternative to screen them without invasive testing.
qpcr covid, SARS-CoV-2 UK, South African and Brazilian Variants in Karachi- Pakistan

Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater

Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology.
In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E).
Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%).
Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

Comprehensive investigation of SARS-CoV-2 fate in wastewater and finding the virus transfer and destruction route through conventional activated sludge and sequencing batch reactor

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA transmission route was thoroughly investigated in the hospital wastewater, sewage collection network, and wastewater treatment plants. Samples were taken on four occasions from December 2020 to April 2021. The performance of two different wastewater treatment processes of sequencing batch reactor (SBR) and conventional activated sludge (CAS) was studied for virus destruction. For this purpose, liquid phase, solid phase and bioaerosol samples were taken from different units of the investigated wastewater treatment plants (WWTPs).
The results revealed that all untreated hospital wastewater samples were positive for SARS-CoV-2 RNA. The virus detection frequency increased when the number of hospitalized cases increased. Detection of viral RNA in the wastewater collection system exhibited higher load of virus in the generated wastewater in areas with poor socioeconomic conditions. Virus detection in the emitted bioaerosols in WWTPs showed that bioaerosols released from CAS with surface aeration contains SARS-CoV-2 RNA posing a potential threat to the working staff of the WWTPs. However, no viral RNA was detected in the bioaerosols of the SBR with diffused aeration system.

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Investigation of SARS-CoV-2 RNA in WWTPs showed high affinity of the virus to be accumulated in biosolids rather than transporting via liquid phase. Following the fate of virus in sludge revealed that it is completely destructed in anaerobic sludge treatment process. Therefore, based on the results of the present study, it can be concluded that receiving water resources could not be contaminated with virus, if the wastewater treatment processes work properly.

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