Genomic surveillance of SARS-CoV-2 reveals emergence of Omicron BA.2 in Islamabad, Pakistan

The Omicron variant of SARS-CoV-2 has rapidly replace previous variants of SARS CoV2 around the globe and is now a major variant of concern. The genomic surveillance of Omicron variant also reveals spread of its subvariant BA.2 which has differing transmissibility in comparison to its other subvariant BA.1. BA.1 and BA.2 harbors different mutational profile. One of the important change in both the subvariants is the presence of 69-70 deletion in BA.1 and absence of this deletion in BA.2. This deletion can be used as tool for the detection of omicron sub variants using real time PCR. In the current study we have used the TaqPath COVID-19 PCR kit for the detection of 69-70 deletion followed by genotyping using SNPsig(R) SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK) that target K417N, E484K, and P681R mutations. The samples with the amplification of spike gene and K417N were termed as probable BA.2 cases. A subset of samples (n=13) were further subjected to whole genome sequencing. The results showed all the 13 samples were of BA.2. Hence, this assay can be used as a cost effective method for the detection of omicron BA.2 variant using real time PCR in resource limiting settings. Moreover, the detection of BA.2 with highly transmissible mutations in Islamabad, Pakistan may potentially increase the number of positive cases. In that scenario, there has to be stringent genomic surveillance to understand the circulating lineages in the country.

Genomic Characterization of Sars-Cov-2 from Islamabad Pakistan by Rapid Nanopore Sequencing

Since the start of COVID-19 pandemic, Pakistan has experienced four waves of pandemic. The fourth wave ended in October, 2021 while the fifth wave of pandemic starts in January, 2022. The data regarding the circulating strains after the fourth wave of pandemic from Pakistan is not available. The current study explore the genomic diversity of SARS-CoV-2 after fourth wave and before fifth wave of pandemic through whole genome sequencing. The results showed the circulation of different strains of SARS-CoV-2 during November-December, 2021. We have Omicron BA.1 (n=4), Lineage A (n=2) and delta AY.27 (n=1) variants of SARS-CoV-2 in the population of Islamabad. All the isolates harbors characteristics mutations of omicron and delta variant in the genome. The lineage A isolate harbors a nine amino acid (68-76) and a ten amino acid (679-688) deletion in the genome. The circulation of omicron in the population before the fifth wave of pandemic and subsequent upsurges of COVID-19 positive cases in Pakistan highlights the importance of genomic surveillance.

Detection and upsurge of SARS-CoV-2 Omicron variant in Islamabad Pakistan

The SARS-CoV-2 omicron variant was first detected in South Africa in November, 2021 and has rapidly spread to more than 90 countries. The emergence of Omicron variant demands for enhanced genomic surveillance to track the mutation profile and spread of virus. In the current study, we have sequenced 15 whole-genome sequences of SARS-CoV-2 Omicron variant from Islamabad region of Pakistan. Among the 15 isolates, 66% were from Islamabad whereas 33% of cases had international travel history of United Kingdom, Maldives, South Africa, and Oman. The detection of Omicron in local community and in travelers highlights the need for rigorous screening at national level and at entry points in order to contain the spread of variant.

Genomic surveillance reveals the emergence of SARS-CoV-2 Lineage A from Islamabad Pakistan

The lineage A of SARS-CoV-2 has been around the world since the start of the pandemic. In Pakistan the last case of lineage A was reported in April, 2021 since then no case has been reported. In November, 2021 during routine genomic surveillance at National Institute of Health we have found 07 cases of lineage A from Islamabad, Pakistan. The study reports two novel deletions in the spike glycoprotein. One 09 amino acid deletion (68-76 a.a) is found in the S1 subunit while another 10 amino acid deletion (679-688 a.a) observed at the junction of S1/S2 referred as furin cleavage site. The removal of furin cleavage site may result in impaired virus replication thus decreasing its pathogenesis. The actual impact of these two deletions on the virus replication and disease dynamics needs to be studied in detail. Moreover, the enhanced genomic surveillance will be required to track the spread of this lineage in other parts of the country.

Genomic diversity of SARS-CoV-2 in Pakistan during fourth wave of pandemic

The emergence of different variants of concern of SARS-CoV-2 has resulted in upsurges of COVID positive cases around the globe. Pakistan is also experiencing fourth wave of COVID-19 with increasing number of positive cases. In order to understand the genomic diversity of circulating SARS-CoV-2 strains during fourth wave of pandemic in Pakistan, the current study was designed. The samples from 89 COVID-19 positive patients were subjected to whole genome sequencing using GeneStudio S5. The results showed that 99% (n=88) of isolates belonged to delta variant and only one isolate belonged to alpha variant. Among delta variant cases 26.1% (n=23) isolates were showing B.1.617.2 while 74% of isolates showing AY.4 lineage. Islamabad was found to be the most affected city with 54% (n=48) of cases, followed by Karachi (28%, n=25), and Rawalpindi (10%, n=9). AY.4 has slight difference in mutation profile compared to B.1.617.2. E156del, G142D and V26I mutations in spike and T181I in NSP6 were present in B.1.617.2 but not in AY.4. Interestingly, A446V mutation in NSP4 has been only observed in AY.4. The current study highlights the circulation of primarily delta variant (B.1.617.2 and AY.4) during fourth wave of pandemic in Pakistan.

Detection and whole-genome sequencing of SARS-CoV-2 B.1.617.2 and B.1.351 variants of concern from Pakistan during the COVID-19 third wave

The viral lineages reflecting variants of concern have emerged worldwide and among them B.1.1.7 (Alpha), B.1351 (Beta) and B.1.617.2 (Delta) variants are the most significant ones and merit close monitoring. In Pakistan, very limited information is available on the circulation of these variants and only the alpha variant has been reported as the main circulating lineage. The objective of this study was to detect and explore the genomic diversity of B.1.351 and B.1.617.2 during the third wave in the indigenous population. During the current study, a total of 2274 samples were tested on real-time PCR for the presence of SARS-CoV-2 from May 14 to May 31, 2021, and among these, 17% were spike positive, whereas 83% of samples had the spike gene target failure (SGTF). From these spike positive samples, 22 samples were processed for whole-genome sequencing. Among VOCs, 45.5% (n=10) belonged to B.1.351 followed by B.1.617.2, 36% (n=8). The delta variant cases were reported mostly from Islamabad (n = 5; 63%) followed by Peshawar and Azad Kashmir (n = 1; 13% each). Beta variant cases originated from Islamabad (n=5; 56%), Peshawar (n=2; 22%), Azad Kashmir and Rawalpindi (n=1; 11% each). The mutation profile of delta variant isolates reported significant mutations, L452R, T478K, P681R, and D950N. The beta variant isolates reported characteristic mutations, D215G, K417N, E484K, N501Y, and A701V. Notably, a characteristic mutation, E484Q was also found in delta variant, B.1.617.2. Our current findings suggest detection of these VOCs from the local population and warrants large-scale genomic surveillance in the country. In addition, it provides timely information to the health authorities to take appropriate actions.

Genomic epidemiology of SARS-CoV-2 in Pakistan

Pakistan has been severely affected by the COVID-19 pandemic. To investigate the initial introductions and transmissions of the SARS-CoV-2 in the country, we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected before June 1, 2020. We identified a total of 347 variants, 29 of which were over-represented in Pakistan. Meanwhile, we found over one thousand intra-host single-nucleotide variants. Several of them occurred concurrently, indicating possible interactions among them. Some of the hypermutable positions were not observed in the polymorphism data, suggesting strong purifying selections. The genomic epidemiology revealed five distinctive spreading clusters. The largest cluster consisted of 74 viruses which were derived from different geographic locations and formed a deep hierarchical structure, indicating an extensive and persistent nation-wide transmission of the virus that was probably contributed by a signature mutation of this cluster. Twenty-eight putative international introductions were identified, several of which were consistent with the epidemiological investigations. No progenies of any of these 150 viruses have been found outside of Pakistan, most likely due to the nonphmarcological intervention to control the virus. This study has inferred the introductions and transmissions of SARS-CoV-2 in Pakistan, which could provide a guidance for an effective strategy for disease control.

An upsurge of SARS CoV-2 B.1.1.7 Variant in Pakistan

The emergence of a more transmissible variant of SARS-CoV-2 (B1.1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported from several countries, only two cases have been identified through whole-genome sequencing from Pakistan. We used a two-step strategy for the detection of B.1.1.7 with initial screening through ThermoFisher TaqPath SARS-CoV-2 kit followed by partial sequencing of Spike (S) gene of samples having spike gene target failure (SGTF) on real-time PCR. From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for the presence of SARS-CoV-2 using TaqPath kit and 70.4% (n=1,867) showed amplification of all the 3 genes (S, N, and ORF). Notably, 29.6% (n=783) samples had the spike gene target failure (SGTF). The SGTF cases were detected at a low frequency during the first three weeks of January (n=10, n=13, and n=1 respectively) however, the cases started to increase in the last week. During February, 726 (93%) cases of SGTF was reported with a peak (n=345) found during the 3rd week. Based on the partial sequencing of spike gene of SGTF samples (n=15), 93% (n=14) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. Our findings highlight the high prevalence of B.1.1.7 in Pakistan and warrant large scale genomic surveillance and strengthening of laboratory network in the country.

Laboratory based surveillance of SARS-CoV-2 in Pakistan

COVID-19 cases are alarmingly increasing in Pakistan since May 2020. Laboratory based surveillance system has been in place since the start of the pandemic. The genomic surveillance of SARS-CoV-2 strains isolated locally has been conducted based on partial ORF1b. The sequences were classified to show the phylogenetic correlation and showed 100% homology with those detected in neighboring countries India and China. The rapid increase in cases has led to development of robust strategies to enhance the laboratory testing capacity. We are currently meeting the country requirement to diagnose the virus in the community. Nonetheless, factors like recent ease in lockdown measures has led to massive rise in number of cases in few weeks time.

Detection of SARS-Coronavirus-2 in wastewater, using the existing environmental surveillance network: An epidemiological gateway to an early warning for COVID-19 in communities.

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.

Development Optimization and Validation of RT-LAMP based COVID-19 Facility in Pakistan

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.