Evolutionary analysis of seasonal influenza A viruses in Pakistan 2020-2023.

INTRODUCTION: Influenza A viruses cause global health concerns due to their high amino acid substitution rates. They are linked to yearly seasonal epidemics and occasional pandemics. This study focused on sequencing influenza A virus strains in Pakistan. MATERIALS AND METHODS: We analyzed the genetic characteristics of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Pakistan from January 2020 to January 2023. Whole genome sequences from influenza A (n = 126) virus isolates were amplified and sequenced by the Oxford Nanopore (MinION) platform. RESULTS: The HA genes of influenza A(H1N1)pdm09 underwent amino acid substitutions at positions K54Q, A186T, Q189E, E224A, R259K, and K308R in sequenced samples. The HA genes of influenza A(H3N2) had amino acid substitutions at G53D, E83K, D104G, I140M, S205F, A212T, and K276R in the sequenced samples. Furthermore, the HA gene sequences of influenza A(H1N1)pdm09 in this study belonged to subclade 6B.1A.5a.2a. Similarly, the HA gene sequences of influenza A(H3N2) were classified under six subclades (3C.3a.1 and 3C.2a1b.2a [2, 2a.1, 2b, 2c, and 2a.3b]). Notably, amino acid substitutions in other gene segments of influenza A(H1N1)pdm09 and A(H3N2) were also found. CONCLUSION: These findings indicate influenza A(H1N1)pdm09 and A(H3N2) viruses co-circulated during the 2020-2023 influenza season in Pakistan. Continued surveillance is crucial for real-time monitoring of possible high-virulence variation and their relevance to existing vaccine strains.

Estimation of seasonal influenza disease burden using sentinel site data in Pakistan 2017-2019: A cross-sectional study.

Background: The influenza A(H1N1)pdm09 pandemic highlighted the need for reliable disease burden estimation from low- and middle-income countries like Pakistan. We designed retrospective age-stratified estimation of influenza-related severe acute respiratory infections (SARIs) incidence in Islamabad Pakistan 2017-2019. Materials and Methods: The catchment area was mapped on SARI data from one designated influenza sentinel site and other healthcare facilities in the Islamabad region. The incidence rate was calculated as per 100,000 for each age group with 95% confidence interval. Results: The catchment population for the sentinel site was 0.7 million against the total denominator of 1.015 million, and incidence rates were adjusted. During January 2017 to December 2019, among 13,905 hospitalizations, 6715 (48%) patients were enrolled; 1208 of these (18%) were positive for influenza. During 2017, influenza A/H3 dominated with 52% detections followed by A(H1N1)pdm09 (35%) and influenza B (13%). Furthermore, elderly 65+ years age group had highest hospitalizations and influenza positive. The incidence rates of all cause respiratory and influenza-related SARI were highest among children >5 years; highest incidence was found in 0 to 11 month/year group with 424/100,000 cases and lowest in 5-15 years 56/100,000. The estimated average annual influenza-associated hospitalization percentage was 29.3% during the study period. Conclusion: Influenza accounts for a significant proportion of respiratory morbidity and hospitalization. These estimates would enable governments for evidence-based decisions and priority allocation of health resources. It is necessary to test for other respiratory pathogens for more clear disease burden estimation.

Prevalence Of Human Adenovirus In Paediatric Patients Presenting With Acute Respiratory Symptoms At Different Hospitals Of Pakistan.

BACKGROUND: Human Adenovirus (HAdV) is one of the most common causes of infection in children. HAdV commonly affects respiratory system, however can also involve other parts of the body like nervous system, eyes and urinary tract. The virus usually causes a mild infection of the lower and upper respiratory tract. Objective of the study was to find the prevalence of HAdV in paediatric patients presenting with Influenza like symptoms and severe acute respiratory illness across Pakistan. METHODS: This cross-sectional study was conducted at the National Institute of Health, Islamabad. Respiratory swabs were collected from 389 children with age less than five years from 14 hospitals in different regions of Pakistan from October 1, 2017 to September 30, 2018. Patients' demographics, signs and symptoms were recorded through a predesigned proforma while Real-time polymerase chain reaction (RT-PCR) was performed for respiratory samples. RESULTS: Out of all 389 samples, HAdV was found in 25 (6.4%) cases. The proportion of HAdV obtained was greater in females 18 (4.6%) than male 7 (1.8%). The influenza-like illness in children attending outpatient department had a higher prevalence of HAdV 13 (3.3%) compared to admitted children 12 (3.1%). Similarly, patients from one to 6 months of age had higher positive outcome than older children. Majority of positive patients were from Islamabad (2.0%) followed by Gilgit (1.8%), Azad Jammu Kashmir (1.0%), Multan (0.5%), and Karachi (0.5%). The most frequent signs and symptoms were cough, fever, sore throat, nasal congestion and shortness of breath. CONCLUSIONS: The present study concludes that HAdV infection is common in Pakistan especially in female patients aged 1-6 months. It's crucial to improve the diagnosis of HAdV infections in our country to prevent complications associated with the virus. Furthermore, genetic analysis may help find different genotypes of HAdV circulating in Pakistan.

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.

Chikungunya virus: Molecular epidemiology of nonstructural proteins in Pakistan.

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.

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.

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.

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.

Epidemiology of Chikungunya virus isolates 2016-2018 in Pakistan.

The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it remerged in India and Indian Ocean regions in 2005-2006. The purpose of this study was to evaluate the genetic diversity and evolutionary changes in CHIKV from 2016 to 2018 in Pakistan. Blood specimens were collected and processed following the Centers for Disease Control and Prevention Trioplex Protocol. Sequencing and phylogenetic analysis of complete coding sequence of representative isolates from the CHIKV outbreak was carried out during December 2016 to July 2018, a total of 1549 samples were received, out of which 50% (n = 774) were found positive for CHIKV RNA. Mean age of chikungunya positive patients was 31.8 ± 15.7 years and most affected were between 21 and 40 years of age. The Pakistan CHIKV strains clustered with the Indian Ocean sublineage of East/Central/South African with cocirculation of some variants In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Key substitutions in the neutralizing epitopes site and a few changes indicative of adaptive to other insect cells were also detected in Pakistani strains. This study provides the emerging trend of CHIKV in the country for early identification of potential variants of high virulence and preventive measures for vector borne disease especially in the endemic areas.

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.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based diagnosis: A potential alternative to quantitative real-time PCR based detection of the novel SARS-COV-2 virus.

The sudden outbreak of the novel Coronavirus infectious disease (COVID-19) resulted in significant challenges to global health systems. One of the primary challenges is rapid, reliable, and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus among the suspected COVID-19-infected individuals. At present, quantitative real-time PCR (qRT-PCR) is a widely used diagnostic method. However, it requires expensive instruments and expertise in the interpretation of results. These constraints reflect the significant need for the development of alternative diagnostic options. This study will validate the use and efficiency of the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay as a potential alternative for the detection of COVID-19. For this purpose, a cohort of 297 suspected COVID-19 patients was tested using both the RT-LAMP assay and the conventional RT-PCR method. For the RT-LAMP assay, three genes (orf-1ab, N, and S) were identified as the target sites for the detection of COVID-19. Based on a comparative assessment, 117 out of 124 positive COVID-19 cases were observed using the RT-LAMP technique with an overall 91.45% sensitivity. Interestingly, where a consensus on 163 individuals free of SARS-Cov-2 was observed, RT-LAMP specificity was 90%. Based on these findings, the robustness of the technique, and the reduced dependency on expensive instrumentation, RT-LAMP-based COVID-19 detection is strongly recommended as a potential alternative assay.

Epidemiology of Influenza among patients with influenza-like illness and severe acute respiratory illness in Pakistan: A 10-year surveillance study 2008-17.

In Pakistan, the burden of influenza was largely unknown, as no formal surveillance system was in place. In 2008, an influenza surveillance system was set up in eight sentinel sites. This study describes the epidemiology of influenza virus using a 10-year surveillance data from 2008 to 2017. Nasopharyngeal/throat swabs were collected from patients with influenza-like illness (ILI) and severe acute respiratory illness (SARI) along with relevant epidemiological information. The samples were tested using real-time reverse transcriptase-polymerase chain reaction for the detection and characterization of influenza viruses. A total of 17 209 samples were tested for influenza, out of which 3552 (20.6%) were positive; 2151/11 239 (19.1%) were patients with ILI, whereas 1401/5970 (23.5%) were patients with SARI. Influenza A/H1N1pdm09 was the predominant strain with 40.6% (n = 1442) followed by influenza B (936, 26.4%). Influenza A/H1N1pdm09 was predominant among the children (5-14 years) and adults (15-64 years). Influenza B strain was predominantly found in the elderly age group (≥ 65 years) accounting for 48% of cases followed by children (2-4 years) accounting for 37% of cases. This 10-year surveillance data provides evidence of influenza activity in the country throughout the year with seasonal winter peaks. The results could be used to strengthen the epidemic preparedness and response plan.

Emergence of Chikungunya Virus, Pakistan, 2016-2017.

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.

Evolutionary analysis of influenza A(H1N1)pdm09 during the pandemic and post-pandemic period in Pakistan.

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.

Molecular characterization of circulating respiratory syncytial virus genotypes in Pakistani children, 2010-2013.

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.

Seasonality of influenza and its association with meteorological parameters in two cities of Pakistan: A time series analysis.

BACKGROUND: Influenza 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. METHODS: This 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). RESULTS: Mean 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. CONCLUSION: The influenza surveillance system gave a better understanding of the disease trend and could accurately forecast influenza activity in Pakistan.