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Background: The coronavirus pandemic has substantially influenced the transmission pattern of other respiratory viruses. However, screening and detecting other respiratory pathogens was unheeded during this time to combat the COVID-19 pandemic. High virulence and re assortment factors intensify the importance of influenza virus surveillance for effective disease management. Therefore, the present surveillance study was designed to determine the influenza positivity rate from 2008–24. This study will provide integral support in depicting a panoramic representation of two respiratory-pandemic periods, 2010–11 and 2019–2021, for influenza and COVID-19 pandemics, respectively. Methods: An inferential cross-sectional study was conducted from 2008 to 2024 by collecting influenza surveillance data from twelve sentinel sites in Pakistan. Clinical and demographic data was recorded at sample collection time. Specimens were collected through nasopharyngeal/throat swabs and stored in viral transport medium (VTM) at the sentinel site laboratory at 2‐4 °C. Viral RNA was isolated from the samples using KingFisher TM Flex Purification System and MagMAX™ Viral/Pathogen Nucleic Acid Extraction Kit. Results: Within 16 years, 78118 samples were tested for influenza, of which 7999 (10.2 %) appeared positive. The positivity rate appeared very low in recent years, with only a 3.5 % positivity rate observed in 2020. Influenza A strain H1N1pdm09 seemed to be the prominent strain (n = 3407, 42.6 %), followed by influenza B (n = 2125, 26.6 %). Conclusion: The positivity of influenza samples was 10.2 % and recorded in patients where typical clinical representation of influenza was absent. Fewer samples were reported during the coronavirus pandemic, which might be because influenza screening was hindered and overlooked to combat the SARS-CoV-2 virus, and the patient threshold was very high for COVID-19 virus screening.

Introduction: Amid coronavirus disease 2019 (COVID‐19) pandemic, accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is critical for diagnosis management and breaking down transmission chains. We designed a national external quality assessment panel (EQAP) for SARS‐CoV‐2 molecular detection comprising working laboratories nationwide. Methods: A molecular diagnostic EQA panel that consists of five samples for SARS CoV‐2 testing was distributed to 141 public and private sector laboratories across country. These samples contain different concentrations of SARS‐CoV‐2 to evaluate the sensitivity of commercial kits available. Results: Sensitivity among public and private sector laboratories was variable, particularly lower SARS‐CoV‐2 concentrations significantly increased the risk of false‐negative tests, whereas Ct values of accurately tested SARS‐CoV‐2 specimens increased as concentration decreased. These findings highlighted that performance of used commercial kits was not significantly correlated to various extraction or PCR methods. Conclusion: This study highlights the need for a national external quality assessment panel (EQAP) in the country to improve the quality of the healthcare system while ensuring the accuracy and reliability of results. Furthermore, EQAPs can help laboratories meet accreditation and regulatory requirements. However, continued participation in EQAP is recommended for quality enhancement of laboratories. [ABSTRACT FROM AUTHOR]

Background: The first case of influenza A(H1N1)pdm09 was detected in Pakistan in June 2009. Since then, it has continued to circulate causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in influenza A(H1N1)pdm09 viruses from 2009 to 2016 and their relevance to the current vaccine viruses. Methods: Respiratory specimens (throat or nasopharyngeal swabs) were collected from patients with influenza-like illness and severe acute respiratory illness. Samples were processed following the protocol of the US Centers for Disease Control and Prevention. Sequencing and phylogenetic analysis of Haemagglutinin and neuraminidase genes were carried out on representative isolates of Pakistan viruses. Results: Between January 2009 and February 2016, out of 16,024 samples analysed, 1950 (12%) were positive for influenza A. During the pandemic period (2009–2010), influenza A(H1N1)pdm09 was the dominant strain with 366 out of 808 (45%) total influenza positive cases. In the post-pandemic period (2011–2016), a total of 1078 out of 1911 (56%) cases were positive for influenza A(H1N1)pdm09 with co-circulation of different influenza A subtypes. The Pakistan A(H1N1)pdm09 viruses belonged to two genetic clades: clade 7 in the pandemic period, and clade 7 (2011) and clade 6B (2015) in the post-pandemic period. Sequence analysis of genes coding for surface glycoprotein’s of Haemagglutinin and neuraminidase had a high degree of sequence similarity with corresponding genes of regional viruses circulating in South-East Asia. Conclusion: Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Key substitutions in the receptor binding site and a few changes indicative of virulence were also detected in the post-pandemic strains. Continued monitoring of the viruses is essential for early identification of potential variants of high virulence and their relevance to current vaccine strains. Keywords: Influenza A(H1N1)Pdm09, Pandemic, Evolutionary analysis, Sequence analysis, Phylogeny, Pakistan

Background: Data on the viral etiology of acute lower respiratory infections are scarce in Pakistan. Human respiratory syncytial virus (RSV) is an important cause of morbidity in children but no effective vaccine or antiviral therapy is currently available. As vaccines are expected to become available in the future, it is important to understand the epidemiology of locally prevalent RSV subtypes. This study aimed to define the molecular epidemiology of RSV (A and B) genotypes in Pakistani children under 5 years. Methods: World Health Organization case definitions for influenza-like illness (ILI) and severe acute respiratory illness (SARI) were used for case selection. Children under 5 years who presented with ILI or SARI at tertiary care hospitals from all provinces/regions, including the eight influenza sentinel sites, during October–April each year between 2010 and 2013 were enrolled. Demographic and clinical data of the children were recorded and nasopharyngeal/throat swabs taken for analysis. All samples were tested for RSV A and B using real-time polymerase chain reaction for non-influenza respiratory viruses. Specific oligonucleotide primers for RSV A and B were used for subtyping and sequencing of the G protein, followed by phylogenetic analysis. Results: A total of 1941 samples were included. RSV was detected in 472 (24%) children, with RSV A detected in 367 (78%) and RSV B in 105 (22%). The G protein of all RSV A strains clustered in the NA1/GA2 genotype while RSV B strains carried the signature 60 nucleotide duplication and were assigned to three BA genotypes: BA-9, BA-10 and the new BA-13 genotype. Conclusions: This study highlights the importance of RSV as a viral etiologic agent of acute respiratory infections in children in Pakistan, and the diversity of RSV viruses. Continued molecular surveillance for early detection of prevalent and newly emerging genotypes is needed to understand the epidemiology of RSV infections in Pakistan. Keywords: Respiratory syncytial virus, Human, Child, Molecular epidemiology, Genotype, Pakistan

During December 2016–May 2017, an outbreak of chikungunya virus infection occurred across Pakistan. The East/Central/South African genotype was predominant. This study provides baseline data on the virus strain and emphasizes the need for active surveillance and implementation of preventive interventions to contain future outbreaks.

Chikungunya is a vectorborne viral disease that causes large outbreaks, mainly in tropical and subtropical countries (1). The term chikungunya is derived from a word in the Makonde language (spoken [...]

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.

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.

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.

Chikungunya virus (CHIKV) is considered a public health problem due to its rapid spread and high morbidity. In 2016-2017 an outbreak of CHIKV was occurred in Pakistan but the data regarding the genomic diversity of CHIKV was not reported. Hence, the current study aimed to determine the genetic diversity of CHIKVs in Pakistan. A cross sectional study was carried out using sera of infected CHIKV patients (n = 1549) during the outbreak in Pakistan (2016-2018). Nucleotide sequencing of non-structural genes of CHIKV from eight isolates were performed followed by phylogenetic analysis using Bayesian method. Phylogenetic analysis suggested that the Pakistani CHIKV strains belonged to Indian Ocean Lineage (IOL) of genotype ECSA and C1.3a clade. Furthermore, the Pakistani isolates showed several key mutations (nsP2-H130Y, nsP2-E145D, nsP4-S55N and nsP4- R85G) corresponding to mutations reported in 2016 Indian strains of CHIKV. The molecular analysis revealed high evolutionary potential of CHIKV strains as well as better understanding of enhanced virulence and pathogenesis of this outbreak. The study highlights the need to continue surveillance in order to understand viral diversity over time and to devise preventive measures to limit diseases transmission in the region.

'Background:' Influenza is a common respiratory disease in Pakistan. However, the absence of a robust surveillance system makes it difficult to estimate the burden of disease. 'Purpose:' We conducted this study to identify key strengths and weaknesses of the laboratory-based influenza surveillance system in Pakistan and to make recommendations for improvement. 'Methods:' We conducted an evaluative descriptive study of the national laboratory-based influenza surveillance system from April to July 2017. We conducted this assessment using the updated guidelines for evaluating public health surveillance systems issued by CDC in 2011. 'Findings: 'The system was found to be simple and easy to operate, but with little flexibility to integrate with other pathogens and diseases. Data quality was good, given that 80% of observed forms were completed. Timeliness was good, as it takes only 24-48 hours from sample collection to report a submission to the central level. Acceptability was good, since both private and public sector hospitals and labs are involved. Sensitivity was 62% and positive predictive value (PPV) was 37.2%. The representativeness of lab based influenza surveillance system was poor, since it is a sentinel surveillance system with specific, strategically placed reporting sites. 'Conclusions:' The system meets its objectives. Sustainability and stability of the system needs to be improved by allocation of public funds. Coverage of the system should be expanded to improve representativeness. Regular capacity building with staff at reporting sites will ensure continued quality of reporting.

BackgroundInfluenza is known to have a specific pattern of seasonality the reasons for which are yet to be fully ascertained. Temperate zones show influenza epidemic during the winter months. The tropical and subtropical regions show more diverse influenza outbreak patterns. This study explores the seasonality of influenza activity and predicts influenza peak based on historical surveillance time series data in Islamabad and Multan, Pakistan.MethodsThis is a descriptive study of routinely collected monthly influenza sentinel surveillance data and meteorological data from 2012-16 in two sentinel sites of Pakistan: Islamabad (North) and Multan (Central).ResultsMean number of cases of influenza and levels of precipitation were higher in Islamabad compared to Multan. Mean temperature and humidity levels were similar in both the cities. The number of influenza cases rose with decrease in precipitation and temperature in Islamabad during 2012-16, although the same cannot be said about humidity. The relationship between meteorological parameters and influenza incidence was not pronounced in case of Multan. The forecasted values in both the cities showed a significant peak during the month of January.ConclusionThe influenza surveillance system gave a better understanding of the disease trend and could accurately forecast influenza activity in Pakistan.