Breakthrough cases of Omicron and Delta variants of SARS-CoV-2 during the fifth wave in Pakistan.

COVID-19 pandemic has severely affected Pakistan with 1,557,134 cases as of August 4, 2022. However, the data regarding breakthrough infections in Pakistan is scant. Hence, the objective was to analyze SARS-CoV-2 breakthrough infections with respect to vaccines and variants during the fifth wave in Pakistan. Therefore, the Department of Virology (NIH, Pakistan) genotyped 2,467 randomly selected individuals between November 2021 and February 2022 using the SNPsig® SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK). P681R and K417N mutations were used to distinguish delta and omicron. Data on the patient's age, gender, date of collection, variant, and vaccination status were analyzed using Statistical Package for Social Sciences (SPSS) software. Among 2,467 genotyped samples, Omicron was detected in 58.6% (n = 1445), Delta in 40.4% (n = 998) and undetermined/wildtype variant in 24 samples. The vaccination status of omicron-positive patients showed (49.7%; n = 718/1445) and Delta-positive patients (39.67%; n = 396/998) to be fully vaccinated. Of note, a high percentage 85% of breakthrough cases (n = 947) were identified among fully vaccinated individuals (n = 1114). Among them, 85.9% (n = 617/718) belonged to omicron and 83.3% (n = 330/396) to delta. Moreover, 76.7% (n = 855) of vaccinated individuals (n = 1114) received Sinopharm (n = 432) and Sinovac (n = 423) vaccines. The majority of breakthrough subjects who contracted Omicron were vaccinated with Sinopharm (93.0%, n = 256) and delta with Cansino (100%, n = 44). Individuals vaccinated with Sinovac showed the most frequent breakthrough cases for both Omicron and Delta variant between the 4th and 6th months (n = 278) after primary vaccination as compared to the 7th to 9th months (n = 24) category. While in case of Sinopharm, maximum breakthrough cases occurred between 7th to 9th months (n = 234) as compared to the 4th to 6th months (n = 120) after primary vaccination. Omicron and Delta breakthrough cases in men (n = 364 and 193) are more frequently seen than women (n = 253 and 138) respectively and breakthrough majority cases (n = 392) occurred in individuals aged 18-33 years. Breakthrough cases limiting monitoring in Pakistan impose a substantial constraint on policymakers' ability to take timely effective decisions. Since the current study consists of only a 2,467-genotyped sample, comprehensive data should be analyzed.

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

The emergence of different variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in upsurges of coronavirus disease 2019 (COVID-19) cases around the globe. Pakistan faced the fourth wave of COVID-19 from July to August 2021 with 314,786 cases. To understand the genomic diversity of circulating SARS-CoV-2 strains during the fourth wave of the pandemic in Pakistan, this study was conducted. The samples from 140 COVID-19-positive patients were subjected to whole-genome sequencing using the iSeq Sequencer by Illumina. The results showed that 97% (n = 136) of isolates belonged to the delta variant while three isolates belonged to alpha and only one isolate belonged to the beta variant. Among delta variant cases, 20.5% (n = 28) isolates were showing B.1.617.2 while 23.5% (n = 25), 17.59% (n = 19), 14.81% (n = 16), and 13.89% (n = 15) of isolates were showing AY.108, AY.43 AY.127, and AY.125 lineages, respectively. Islamabad was found to be the most affected city with 65% (n = 89) of delta variant cases, followed by Karachi (17%, n = 23), and Rawalpindi (10%, n = 14). Apart from the characteristic spike mutations (T19R, L452R, T478K, P681R, and D950N) of the delta variant, the sublineages exhibited other spike mutations as E156del, G142D, T95I, A222V, G446V, K529N, N532S, Q613H, and V483A. The phylogenetic analysis revealed the introductions from Singapore, the United Kingdom, and Germany. This study highlights the circulation of delta variants (B.1.617.2 and sublineages) during the fourth wave of pandemic in Pakistan.

Genomic surveillance reveals the detection of SARS-CoV-2 delta, beta, and gamma VOCs during the third wave in Pakistan.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide and gained significant importance due to their high transmissibility and global spread, thus meriting close monitoring. In Pakistan, limited information is available on circulation of these variants as the alpha variant has been reported the main circulating lineage. The current study was designed to detect and explore the genomic diversity of SARS-CoV-2 lineages circulating during the third wave of the pandemic in the indigenous population. From May 01 to June 09, 2021, a total of 16 689 samples were tested using TaqPath™ COVID-19 kit for the presence of SARS-CoV-2. Overall, 2562 samples (15.4%) were COVID-19 positive. Out of these positive samples, 2124 (12.7%) did not show the spike gene amplification (spike gene target failure ([SGTF]), whereas 438 (2.6%) showed spike gene amplification (non-SGTF). A subset (n = 58/438) of non-SGTF samples were randomly selected for whole-genome sequencing. Among VOCs, 45% (n = 26/58) were delta, 46% (n = 27/58) were beta, and one was gamma variant. The delta variant cases were reported mainly from Islamabad (n = 15; 58%) followed by Rawalpindi and Azad Kashmir (n = 1; 4% each). Beta variant cases originated mainly from Karachi (n = 8; 30%) and Islamabad (n = 11; 41%) and the gamma variant case was reported in a traveler from Italy. The delta, beta, and gamma variants possessed lineage-specific spike mutations. Notably, two rare mutations (E484Q and L5F) were found in the delta variant. Furthermore, in the beta variant, two significant rare non-synonymous spike mutations (A879S and K444R) were also reported. High prevalence of beta and delta variants in local population may increase the number of cases in the near future and provides an early warning to national health authorities to take timely decisions and devise suitable interventions to contain a possible fourth wave.

Genomic Epidemiology of SARS-CoV-2 in Pakistan.

COVID-19 has swept globally and Pakistan is no exception. To investigate the initial introductions and transmissions of the SARS-CoV-2 in Pakistan, we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected from March 16 to June 1, 2020. We identified a total of 347 mutated positions, 31 of which were over-represented in Pakistan. Meanwhile, we found over 1000 intra-host single-nucleotide variants (iSNVs). Several of them occurred concurrently, indicating possible interactions among them or coevolution. Some of the high-frequency iSNVs in Pakistan were not observed in the global population, 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 of Pakistan and formed a deep hierarchical structure, indicating an extensive and persistent nation-wide transmission of the virus that was probably attributed to a signature mutation (G8371T in ORF1ab) of this cluster. Furthermore, 28 putative international introductions were identified, several of which are consistent with the epidemiological investigations. In all, this study has inferred the possible pathways of introductions and transmissions of SARS-CoV-2 in Pakistan, which could aid ongoing and future viral surveillance and COVID-19 control.

Genetic and evolutionary analysis of SARS-CoV-2 circulating in the region surrounding Islamabad, Pakistan.

Genomic epidemiology of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has provided global epidemiological insight into the COVID-19 pandemic since it began. Sequencing of the virus has been performed at scale, with many countries depositing data into open access repositories to enable in-depth global phylogenetic analysis. To contribute to these efforts, we established an Oxford Nanopore Technologies (ONT) sequencing capability at the National Institutes of Health (NIH), Pakistan. This study highlights multiple SARS-CoV-2 lineages co-circulating during the peak of a second COVID-19 wave in Pakistan (Nov 2020-Feb 2021), with virus origins traced to the United States of America and Saudi Arabia. Ten SARS-CoV-2 positive samples were used for ONT library preparation. Sequence and phylogenetic analysis determined that the patients were infected with lineage B.1.1.250, originally identified in the United Kingdom and Bangladesh during March and April of 2020, and in circulation until the time of this study in Europe, USA and Australia. Lineage B.1.261 was originally identified in Saudi Arabia with widespread local dissemination in Pakistan. One sample clustered with the parental B.1 lineage and the other with lineage B.6 originally from Singapore. In the future, monitoring the evolutionary dynamics of circulating lineages in Pakistan will enable improved tracing of the viral spread, changing trends of their expansion trajectories, persistence, changes in their demographic dynamics, and provide guidance for better implementation of control measures.

Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission.

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We 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. A 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 19 patients and quarantine center 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. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is 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.

Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account.

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 region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

Whole-genome sequencing of SARS-CoV-2 reveals the detection of G614 variant in Pakistan.

Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.