Surveillance To Track Progress Toward Polio Eradication - Worldwide, 2022-2023.

The reliable and timely detection of poliovirus cases through surveillance for acute flaccid paralysis (AFP), supplemented by environmental surveillance of sewage samples, is a critical component of the polio eradication program. Since 1988, the number of polio cases caused by wild poliovirus (WPV) has declined by >99.9%, and eradication of WPV serotypes 2 and 3 has been certified; only serotype 1 (WPV1) continues to circulate, and transmission remains endemic in Afghanistan and Pakistan. This surveillance update evaluated indicators from AFP surveillance, environmental surveillance for polioviruses, and Global Polio Laboratory Network performance data provided by 28 priority countries for the program during 2022-2023. No WPV1 cases have been detected outside of Afghanistan and Pakistan since August 2022, when an importation into Malawi and Mozambique resulted in an outbreak during 2021-2022. During 2022-2023, among 28 priority countries, 20 (71.4%) met national AFP surveillance indicator targets, and the number of environmental surveillance sites increased. However, low national rates of reported AFP cases in priority countries in 2023 might have resulted from surveillance reporting lags; substantial national and subnational AFP surveillance gaps persist. Maintaining high-quality surveillance is critical to achieving the goal of global polio eradication. Monitoring surveillance indicators is important to identifying gaps and guiding surveillance-strengthening activities, particularly in countries at high risk for poliovirus circulation.

Surveillance To Track Progress Toward Poliomyelitis Eradication - Worldwide, 2021-2022.

Since the Global Polio Eradication Initiative (GPEI) was established in 1988, the number of wild poliovirus (WPV) cases has declined by >99.9%, and WPV serotypes 2 and 3 have been declared eradicated (1). By the end of 2022, WPV type 1 (WPV1) transmission remained endemic only in Afghanistan and Pakistan (2,3). However, during 2021-2022, Malawi and Mozambique reported nine WPV1 cases that were genetically linked to Pakistan (4,5), and circulating vaccine-derived poliovirus (cVDPV) outbreaks were detected in 42 countries (6). cVDPVs are oral poliovirus vaccine-derived viruses that can emerge after prolonged circulation in populations with low immunity allowing reversion to neurovirulence and can cause paralysis. Polioviruses are detected primarily through surveillance for acute flaccid paralysis (AFP), and poliovirus is confirmed through stool specimen testing. Environmental surveillance, the systematic sampling of sewage and testing for the presence of poliovirus, supplements AFP surveillance. Both surveillance systems were affected by the COVID-19 pandemic's effects on public health activities during 2020 (7,8) but improved in 2021 (9). This report updates previous reports (7,9) to describe surveillance performance during 2021-2022 in 34 priority countries.* In 2022, a total of 26 (76.5%) priority countries met the two key AFP surveillance performance indicator targets nationally compared with 24 (70.6%) countries in 2021; however, substantial gaps remain in subnational areas. Environmental surveillance expanded to 725 sites in priority countries, a 31.1% increase from the 553 sites reported in 2021. High-quality surveillance is critical to rapidly detect poliovirus transmission and enable prompt poliovirus outbreak response to stop circulation. Frequent monitoring of surveillance guides improvements to achieve progress toward polio eradication.

Molecular detection and genetic characterization of human metapneumovirus strains circulating in Islamabad, Pakistan.

Lower respiratory illness is one of the leading causes of death among children in low- and high-income countries. Human metapneumovirus (hMPV) is a key contributor to respiratory illnesses commonly reported among children and causes serious clinical complications ranging from mild respiratory infections to severe lower respiratory tract anomalies mainly in the form of bronchiolitis and pneumonia. However, due to the lack of a national surveillance system, the clinical significance of hMPV remains obscure in the Pakistani population. This study was conducted to screen throat swabs samples collected from 127 children reported with respiratory symptoms at a tertiary care hospital in Islamabad. Out of 127, 21 (16.5%) samples were positive for hMPV with its genotype distribution as A2a (10%), A2b (20%), B1 (10%), and B2 (60%). Phylogenetic analysis showed that the hMPV viruses were closely related to those reported from neighboring countries including India and China. This work will contribute to a better understanding of this virus, its diagnosis, and the handling of patients in clinical setups. Further studies at a large-scale are warranted for a better understanding of the disease burden and epidemiology of hMPV in Pakistan.

Isolation and molecular characterization of Adenovirus in suspected acute flaccid paralysis patients: A preliminary report from Pakistan.

Human adenoviruses (HAdVs) usually cause asymptomatic or mild infection, but infrequently, they are responsible for various severe syndromes including neurological disorders. Various research studies have investigated the association of HAdVs with acute flaccid paralysis (AFP). The purpose of this study was to investigate the genetic diversity of HAdVs and their association with AFP. Stool samples from patients ≤ 12 years of age with suspected AFP were collected from all over Pakistan within the framework of poliovirus surveillance. Poliovirus- and enterovirus-negative samples were screened for HAdVs. For virus isolation, the human epithelial cell line HEp-2c was used, culture-positive samples were screened by nested PCR assay, and partial hexon gene sequences were used for genotype identification. Out of 172 samples, 94 were positive by virus isolation, 89 were positive by PCR, and 32 isolates were genotyped successfully. Phylogenetic analysis showed that the HAdVs belonged to species A (HAdV-A12 and A31), B (HAdV-B3 and B7), C (HAdV-C1 and C6), D (HAdV-D19 and D93), and F (HAdV-F41), showing 99-100% nucleotide sequence identity and 98.3-100% amino acid sequence identity). Most of these genotypes have been reported previously in AFP cases, but this is the first report of the detection of HAdV-D93 in stool samples from AFP cases. The detection of a significant fraction of the HAdVs genotypes indicates that these genetically distinct genotypes are circulating in Pakistan and suggests their possible role in the pathogenesis of AFP.

Spatial analysis of genetic clusters and epidemiologic factors related to wild poliovirus type 1 persistence in Afghanistan and Pakistan.

Following the certification of the World Health Organization Region of Africa as free of serotype 1 wild poliovirus (WPV1) in 2020, Afghanistan and Pakistan represent the last remaining WPV1 reservoirs. As efforts continue in these countries to progress to eradication, there is an opportunity for a deeper understanding of the spatiotemporal characteristics and epidemiological risk factors associated with continual WPV1 circulation in the region. Using poliovirus surveillance data from 2017-2019, we used pairwise comparisons of VP1 nucleotide sequences to illustrate the spatiotemporal WPV1 dispersal to identify key sources and destinations of potentially infected, highly mobile populations. We then predicted the odds of WPV1 detection at the district level using a generalized linear model with structural indicators of health, security, environment, and population demographics. We identified evidence of widespread population mobility based on WPV1 dispersal within and between the countries, and evidence indicating five districts in Afghanistan (Arghandab, Batikot, Bermel, Muhamandara and Nawzad) and four districts in Pakistan (Charsada, Dera Ismail Khan, Killa Abdullah and Khyber) act as cross-border WPV1 circulation reservoirs. We found that the probability of detecting WPV1 in a district increases with each armed conflict event (OR = 1·024, +- 0·008), level of food insecurity (OR = 1·531, +-0·179), and mean degrees Celsius during the months of greatest precipitation (OR = 1·079, +- 0·019). Our results highlight the multidisciplinary complexities contributing to the continued transmission of WPV1 in Afghanistan and Pakistan. We discuss the implications of our results, stressing the value of coordination during this final chapter of the wild polio virus eradication initiative.

Conserved Antigenic Structure of Contemporary Wild Poliovirus Type 1 Strains Endemic in Pakistan.

BACKGROUND: Elimination of poliovirus in Pakistan and Afghanistan is challenged by notions against the role of oral poliovirus vaccine (OPV) in eradicating contemporary wild poliovirus (WPV) strains. METHODS: A total of 1055 WPV type 1 (WPV1) strains isolated between 2013 and 2018 were categorized into 68 antigenic groups and tested for neutralization by OPV-derived antibodies. Molecular docking was conducted to determine neutralization efficiency of antibodies against WPV. The clinical significance of WPV1 variants was assessed to ascertain their role in patient outcomes. RESULTS: We found that 88% of WPV1 strains isolated from paralytic children belonged to a single antigenic lineage identical to the WPV1 strain detected in 1993. WPV1 antigenic variants were effectively neutralized by OPV-derived antibodies, with geometric mean titers comparable to the neutralization titers found for 3 strains in OPV (OPV1-3, 7.96-9.149 [95% confidence interval, 6.864-10.171]; WPV1 strains, 7.542-8.786 [6.493-9.869]). Docking examination underscored a strong antigen-antibody interaction despite variations within the viral protein 1 epitopes. There was no significant association (P = .78) with clinical prognosis among patients infected with antigenically diverse WPV1 strains and patient outcomes, including death. CONCLUSIONS: Our findings substantiate the robustness of OPV for neutralizing the contemporary WPV1 strains endemic in Pakistan and Afghanistan. Vaccination coverage must be augmented to achieve early eradication.

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.

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.

Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing.

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

Genetic Epidemiology Reveals 3 Chronic Reservoir Areas With Recurrent Population Mobility Challenging Poliovirus Eradication in Pakistan.

BACKGROUND: Pakistan is among 3 countries endemic for wild poliovirus type 1 (WPV1) circulation that are still struggling for eradication of poliomyelitis. Active clinical and environmental surveillance with meticulous laboratory investigations provide insights into poliovirus transmission patterns and genomic diversity to inform decisions for strategic operations required to achieve eradication. METHODS: We analyzed epidemiological and virological data to comprehend the current epidemiological status of WPV1 in Pakistan during 2015-2017. Stool specimens of patients with acute flaccid paralysis (AFP) and sewage samples collected from 60 environmental sites were tested. Viral culturing, intratypic differentiation by real-time polymerase chain reaction, and nucleic acid sequencing of the VP1 region of the poliovirus genome to determine genetic relatedness among WPV1 strains were applied. RESULTS: Poliovirus isolates were grouped into 11 distinct clusters, which had ≥95% nucleotide homology in the VP1 coding region. Most of the poliovirus burden was shared by 3 major reservoirs: Karachi, Peshawar, and Quetta block (64.2% in 2015, 75.4% in 2016, and 76.7% in 2017). CONCLUSIONS: Environmental surveillance reveals importations and pockets of unimmunized children that dictate intensive target mop-up campaigns to contain poliovirus transmission. A decrease in the number of orphan isolates reflects effective combination of AFP and environmental surveillance in Pakistan. The genetic data reflect sustained transmission within reservoir areas, further expanded by periodic importations to areas of high immunity reflected by immediate termination of imported viruses. Improved immunization coverage with high-quality surveillance is vital for global certification of polio eradication.

Assessing the sensitivity of the polio environmental surveillance system.

BACKGROUND: The polio environmental surveillance (ES) system has been an incredible tool for advancing polio eradication efforts because of its ability to highlight the spatial and temporal extent of poliovirus circulation. While ES often outperforms, or is more sensitive than AFP surveillance, the sensitivity of the ES system has not been well characterized. Fundamental uncertainty of ES site sensitivity makes it difficult to interpret results from ES, particularly negative results. METHODS AND FINDINGS: To study ES sensitivity, we used data from Afghanistan and Pakistan to examine the probability that each ES site detected the Sabin 1, 2, or 3 components of the oral polio vaccine (OPV) as a function of virus prevalence within the same district (estimated from AFP data). Accounting for virus prevalence is essential for estimating site sensitivity because Sabin detection rates should vary with prevalence-high immediately after supplemental immunization activities (SIAs), but low in subsequent months. We found that most ES sites in Pakistan and Afghanistan are highly sensitive for detecting poliovirus relative to AFP surveillance in the same districts. For example, even when Sabin poliovirus is at low prevalence of ~0.5-3% in AFP surveillance, most ES sites have ~34-50% probability of detecting Sabin. However, there was considerable variation in ES site sensitivity and we flagged several sites for re-evaluation based on low sensitivity rankings and low wild polio virus detection rates. In these areas, adding new sites or modifying collection methods in current sites could improve sensitivity of environmental surveillance. CONCLUSIONS: Relating ES detections to virus prevalence significantly improved our ability to evaluate site sensitivity compared to evaluations based solely on ES detection rates. To extend our approach to new sites and regions, we provide a preliminary framework for relating ES and AFP detection rates, and descriptions of how detection rates might relate to SIAs and natural seasonality.

Environmental Surveillance Reveals Complex Enterovirus Circulation Patterns in Human Populations.

BACKGROUND: Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples. METHODS: We used a next-generation sequencing approach analyzing reverse transcriptase polymerase chain reaction products synthesized directly from sewage concentrates. RESULTS: We determined whole-capsid genome sequences of multiple enterovirus strains from all 4 A to D species present in environmental samples from the United Kingdom, Senegal, and Pakistan. CONCLUSIONS: Our results indicate complex enterovirus circulation patterns in human populations with differences in serotype composition between samples and evidence of sustained and widespread circulation of many enterovirus serotypes. Our analyses revealed known and divergent enterovirus strains, some of public health relevance and genetically linked to clinical isolates. Enteroviruses identified in sewage included vaccine-derived poliovirus and enterovirus D-68 stains, new enterovirus A71 and coxsackievirus A16 genogroups indigenous to Pakistan, and many strains from rarely reported serotypes. We show how this approach can be used for the early detection of emerging pathogens and to improve our understanding of enterovirus circulation in humans.

Progress Toward Poliomyelitis Eradication - Pakistan, January 2017-September 2018.

Among the three wild poliovirus (WPV) serotypes, only WPV type 1 (WPV1) has been reported in polio cases or detected from environmental surveillance globally since 2012. Pakistan remains one of only three countries worldwide (the others are Afghanistan and Nigeria) that has never had interrupted WPV1 transmission. This report documents Pakistan's activities and progress toward polio eradication during January 2017-September 2018 and updates previous reports (1,2). In 2017, Pakistan reported eight WPV1 cases, a 60% decrease from 20 cases in 2016. As of September 18, 2018, four cases had been reported, compared with five cases at that time in 2017. Nonetheless, in 2018, WPV1 continues to be isolated regularly from environmental surveillance sites, primarily in the core reservoir areas of Karachi, Quetta, and Peshawar, signifying persistent transmission. Strategies to increase childhood immunity have included an intense schedule of supplemental immunization activities (SIAs), expanding and refining deployment of community-based vaccination implemented by community health workers recruited from the local community in reservoir areas, and strategic placement of permanent transit points where vaccination is provided to mobile populations. Interruption of WPV1 transmission will require further programmatic improvements throughout the country with a focus on specific underperforming subdistricts in reservoir areas.

Evaluation of the bag-mediated filtration system as a novel tool for poliovirus environmental surveillance: Results from a comparative field study in Pakistan.

Poliovirus (PV) environmental surveillance (ES) plays an important role in the global eradication program and is crucial for monitoring silent PV circulation especially as clinical cases decrease. This study compared ES results using the novel bag-mediated filtration system (BMFS) with the current two-phase separation method. From February to November 2016, BMFS and two-phase samples were collected concurrently from twelve sites in Pakistan (n = 117). Detection was higher in BMFS than two-phase samples for each Sabin-like (SL) PV serotype (p<0.001) and wild PV type 1 (WPV1) (p = 0.065). Seventeen sampling events were positive for WPV1, with eight discordant in favor of BMFS and two in favor of two-phase. A vaccine-derived PV type 2 was detected in one BMFS sample but not the matched two-phase. After the removal of SL PV type 2 (SL2) from the oral polio vaccine in April 2016, BMFS samples detected SL2 more frequently than two-phase (p = 0.016), with the last detection by either method occurring June 12, 2016. More frequent PV detection in BMFS compared to two-phase samples is likely due to the greater effective volume assayed (1620 mL vs. 150 mL). This study demonstrated that the BMFS achieves enhanced ES for all PV serotypes in an endemic country.

High prevalence of G3 rotavirus in hospitalized children in Rawalpindi, Pakistan during 2014.

Rotavirus A species (RVA) is the leading cause of severe diarrhea among children in both developed and developing countries. Among different RVA G types, humans are most commonly infected with G1, G2, G3, G4 and G9. During 2003-2004, G3 rotavirus termed as "new variant G3" emerged in Japan that later disseminated to multiple countries across the world. Although G3 rotaviruses are now commonly detected globally, they have been rarely reported from Pakistan. We investigated the genetic diversity of G3 strains responsible RVA gastroenteritis in children hospitalized in Rawalpindi, Pakistan during 2014. G3P[8] (18.3%; n = 24) was detected as the most common genotype causing majority of infections in children less than 06 months. Phylogenetic analysis of Pakistani G3 strains showed high amino acid similarity to "new variant G3" and G3 strains reported from China, Russia, USA, Japan, Belgium and Hungary during 2007-2012. Pakistani G3 strains belonged to lineage 3 within sub-lineage 3d, containing an extra N-linked glycosylation site compared to the G3 strain of RotaTeqTM. To our knowledge, this is the first report on the molecular epidemiology of G3 rotavirus strains from Pakistan and calls for immediate response measures to introduce RV vaccine in the routine immunization program of the country on priority.

Population sensitivity of acute flaccid paralysis and environmental surveillance for serotype 1 poliovirus in Pakistan: an observational study.

BACKGROUND: To support poliomyelitis eradication in Pakistan, environmental surveillance (ES) of wastewater has been expanded alongside surveillance for acute flaccid paralysis (AFP). ES is a relatively new method of surveillance, and the population sensitivity of detecting poliovirus within endemic settings requires estimation. METHODS: Data for wild serotype 1 poliovirus from AFP and ES from January 2011 to September 2015 from 14 districts in Pakistan were analysed using a multi-state model framework. This framework was used to estimate the sensitivity of poliovirus detection from each surveillance source and parameters such as the duration of infection within a community. RESULTS: The location and timing of poliomyelitis cases showed spatial and temporal variability. The sensitivity of AFP surveillance to detect serotype 1 poliovirus infection in a district and its neighbours per month was on average 30.0% (95% CI 24.8-35.8) and increased with the incidence of poliomyelitis cases. The average population sensitivity of a single environmental sample was 59.4% (95% CI 55.4-63.0), with significant variation in site-specific estimates (median varied from 33.3-79.2%). The combined population sensitivity of environmental and AFP surveillance in a given month was on average 98.1% (95% CI 97.2-98.7), assuming four samples per month for each site. CONCLUSIONS: ES can be a highly sensitive supplement to AFP surveillance in areas with converging sewage systems. As ES for poliovirus is expanded, it will be important to identify factors associated with variation in site sensitivity, leading to improved site selection and surveillance system performance.

Epidemiological and molecular investigation of a measles outbreak in Punjab, Pakistan, 2013-2015.

Despite the availability of an effective vaccine, the measles virus continues to cause significant morbidity and mortality in children worldwide. Molecular characterization of wild-type measles strains is an invaluable component of epidemiological studies or surveillance systems that provides important information pertinent to outbreak linkages and transmission pathways. Serum samples and throat swabs were collected from suspected measles cases from the Punjab province of Pakistan (2013-2015) and further tested for measles immunoglobulin M (IgM) through enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction for molecular characterization. Among the total of 5415 blood samples, 59% tested positive for measles IgM. Males had a higher infection rate (55%) than females (45%), and the highest frequency of positive cases (63%) was found in the age group of 0 to 5 years. Partial sequencing of the nucleoprotein gene showed that 27 strains belonged to the B3 genotype, whereas 2 viruses were identified as D4. On phylogenetic analysis, Pakistani B3 strains were found to be closely related to previously reported indigenous strains and those from neighboring countries of Iran and Qatar. This is the first report on the detection of the measles B3 genotype from Punjab, Pakistan. The current study shows a high burden of measles infections in Punjab province owing to poor routine immunization coverage in major cities. It is imperative that national health authorities adopt strategic steps on an urgent basis for improvement of routine immunization coverage. Molecular epidemiology of the measles viruses circulating in different parts of the country can provide useful data to manage future outbreaks.

Identification of measles virus genotype B3 associated with outbreaks in Islamabad, Pakistan, 2013-2015.

BACKGROUND: Measles virus infection remains a significant cause of childhood mortality and morbidity despite continued global efforts and the availability of a safe and effective vaccine. Molecular analysis of indigenous measles viruses could provide critical information on outbreak linkages and transmission pathways that can aid the implementation of appropriate control programs in Pakistan. METHODS: Blood samples and throat swabs were collected from subjects suspected with measles in Islamabad, Pakistan from 2013 to 2015. Serum samples were tested for the presence of measles immunoglobulin M (IgM) antibodies using enzyme-linked immunosorbent assay (ELISA) while throat swabs were used for the isolation (Vero/SLAM cell line) and subsequent characterization and phylogenetic analysis of measles strains. RESULTS: Of 373 blood samples, 66% tested positive for measles IgM. Male subjects were more often infected (58%) than female (42%) with the highest frequency of positive cases (63%) in the 0-5-years age group. Among the positive cases, only 13% had received one or two doses of the measles vaccine, while 87% were unvaccinated. Of 80 throat swabs, 29 (36%) showed a measles virus-specific cytopathic effect (CPE) and were characterized as genotype B3 through partial sequencing of the nucleoprotein (N) gene. Phylogenetic analysis revealed the Pakistani B3 strains to be closely related to strains from neighboring countries (Iran and Afghanistan) as well as with B3 viruses from the USA, Germany, and the UK. CONCLUSIONS: The study results showed that despite the availability of an effective vaccine, the burden of measles infections is very high in Pakistan due to poor routine immunization coverage even in major cities, including the capital city of Islamabad. It is imperative that national health authorities take urgent strategic steps to improve routine immunization and implement adequate molecular identification methods to tackle future measles outbreaks.