Building up a genomic surveillance platform for SARS-CoV-2 in the middle of a pandemic: a true North-South collaboration.

Next-generation sequencing technology has revolutionised pathogen surveillance over the last two decades. However, the benefits are not equitably distributed, with developing countries lagging far behind in acquiring the required technology and analytical capacity. Recent declines in the cost associated with sequencing-equipment and running consumables have created an opportunity for broader adoption. During the COVID-19 pandemic, rapid diagnostics development and DNA sequencing revolutionised the ability to diagnose and sequence SARS-CoV-2 rapidly. Socioeconomic inequalities substantially impact the ability to sequence SARS-CoV-2 strains and undermine a developing country's pandemic preparedness. Low- and middle-income countries face additional challenges in establishing, maintaining and expanding genomic surveillance. We present our experience of establishing a genomic surveillance system at the Aga Khan University, Karachi, Pakistan. Despite being at a leading health sciences research institute in the country, we encountered significant challenges. These were related to collecting standardised contextual data for SARS-CoV-2 samples, procuring sequencing reagents and consumables, and challenges with library preparation, sequencing and submission of high-quality SARS-CoV-2 genomes. Several technical roadblocks ensued during the implementation of the genomic surveillance framework, which were resolved in collaboration with our partners. High-quality genome sequences were then deposited on open-access platforms per the best practices. Subsequently, these efforts culminated in deploying Pakistan's first SARS-CoV-2 phyllo surveillance map as a Nextstrain build. Our experience offers lessons for the successful development of Genomic Surveillance Infrastructure in resource-limited settings struck by a pandemic.

Co-carriage of Staphylococcus aureus and Streptococcus pneumoniae among children younger than 2 years of age in a rural population in Pakistan.

Objectives: Staphylococcus aureus and Streptococcus pneumoniae are common colonizers of the human nasopharynx. In this study, we describe S. aureus nasopharyngeal carriage and evaluate its association with S. pneumoniae carriage post-10-valent pneumococcal conjugate vaccine (PCV10) introduction in Pakistan. Methods: A serial cross-sectional study was undertaken from 2014 to 2018, children <2 years were randomly selected, and nasopharyngeal swabs were collected using standard WHO guidelines. S. aureus and S. pneumoniae isolates were identified using standard methods and tested for antimicrobial susceptibility by the standard Kirby-Bauer disk-diffusion method as per Clinical & Laboratory Standards Institute (CLSI) recommendations. Regression analysis was used to determine predictors associated with S. aureus carriage. Results: We enrolled 3140 children. S. aureus carriage prevalence was 5.6% (176/3140), and 50.1% (81/176) of the isolates were methicillin-resistant S. aureus (MRSA). S. aureus carriage was higher in the absence of pneumococcus compared to isolates in which pneumococcus was present (7.5% vs 5.0%). S. aureus carriage was negatively associated with pneumococcal carriage, being in 3rd and 4th year of enrollment, and vaccination with two and three PCV10 doses, in addition, fast breathing, ≥2 outpatients visits, and rainy season were positively associated. The following resistance rates were observed: 98.9% for penicillin, 74.4% for fusidic acid, and 23.3% for gentamicin, 10.2% for erythromycin, and 8.5% for cotrimoxazole. All isolates were susceptible to amikacin. Conclusions: Overall S. aureus carriage prevalence was low, PCV10 vaccine was protective against the carriage. The proportion of MRSA carriage and antimicrobial resistance was high in this community warranting continuous monitoring for invasive infections.

Pneumococcal Carriage in Infants Post-PCV10 Introduction in Pakistan: Results from Serial Cross-Sectional Surveys.

The 10-valent pneumococcal vaccine was introduced in Pakistan's Expanded Program on Immunization (EPI) in 2013 as a 3 + 0 schedule without catchup. We conducted three annual cross-sectional surveys from 2014−2016 to measure vaccine-type (VT) carriage in infants from a rural part of Pakistan. Nasopharyngeal specimens were collected by random sampling of infants from two union councils of Matiari. Samples were then transported to the Infectious Disease Research Laboratory (IDRL) at the Aga Khan University within 6−8 h of collection. Serotypes were established using sequential multiplex PCR. Of the 665 children enrolled across three surveys, 547 were culture-positive for pneumococcus. VT carriage decreased from 21.8% in 2014 to 12.7% in 2016 (p-value for trend <0.001). Those who were not vaccinated or partially vaccinated were found to be at higher risk of carrying a VT serotype ((aOR 2.53, 95% CI 1.39, 4.63 for non-vaccinated) and (aOR 3.35, 95% CI 1.82, 6.16 for partially vaccinated)). On the other hand, being enrolled in the most recent survey was negatively associated with VT carriage (aOR 0.51, 95% CI 0.28, 0.93). We found that PCV10 was effective in decreasing the carriage of vaccine-type serotypes in Pakistani infants.

Serotype-specific effectiveness against pneumococcal carriage and serotype replacement after ten-valent Pneumococcal Conjugate Vaccine (PCV10) introduction in Pakistan.

OBJECTIVE: Pakistan was one of the first South-Asian countries to introduce the ten-valent pneumococcal conjugate vaccine (PCV10) at the national level, using a 3+0 schedule without catchup, in 2013. METHODS: From 2014-18, fifteen children <2 years old were recruited every week in Matiari, Sindh, and nasopharyngeal swabs were collected. The samples were cultured, and pneumococcus was further serotyped through multiplex PCR at the Aga Khan University Hospital as per the method described by the Centers for Disease Control and Prevention, USA. RESULTS: Pneumococcus was detected in 2370/3140 (75%) children. Vaccine type (VT) and non-vaccine type (NVT) serotypes were carried by 379 and 1990 children. There was a significant decline in VT carriage (by 40.3%, p-value <0.001), whereas overall NVT carriage remained the same. The prevalence of VT serotypes 6B, 9V/9A, and 19F showed a significant decline by 58.8%, 79.3%, and 56%, respectively. The prevalence of NVT serotypes 19A, 21, and 10A increased by 70%, 33.3%, and 65.6%, respectively, whereas serotypes 13 and 9N/9L decreased by 53.4% and 51.8%, respectively. Serotype-specific vaccine effectiveness estimates that reached statistical significance were for 9V/9A (VE = 65.0, 95% CI 26.0-83.5%), 19F (VE = 55.3, 95% CI 15.5-76.4%) and for the vaccine related serotype 6A (VE = 28.4, 95% CI 0.9-48.2%). CONCLUSION: The emergence of NVT serotypes, primarily 19A replacing VT serotypes in this rural community, necessitates continuous monitoring of serotypes in the carriage and invasive disease to evaluate the utility of existing vaccine formulations.

Impact of enteropathogens on faltering growth in a resource-limited setting.

Introduction: Environmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy. Methods: A community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3-6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens. Results: Giardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO). Conclusion: Protozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

Usability and Acceptability of a Mobile App for Behavior Change and to Improve Immunization Coverage among Children in Pakistan: A Mixed-Methods Study.

BACKGROUND: Pakistan's immunization uptake rates are still significantly lower than anticipated despite several initiatives. Lack of awareness, forgetting about vaccination schedule, and vaccine misconception/misinformation are a few of the major drivers that mitigate the rates of immunization. The current COVID-19 pandemic emphasizes the importance of immunization. The significant reductions in regular childhood vaccination during pandemic have increased the risk of outbreaks of vaccine-preventable diseases. Concerns among parents over possibly exposing their children to COVID-19 during child visits may have contributed to the reported declines. Innovative and cost-effective mHealth interventions must be implemented in order to address the problem of inadequate immunization rates. In addition, it is also critical to understand the end user needs in order to reflect on the highly relevant essence of the customized healthcare experience. OBJECTIVE: The aim of this study was to learn about caregivers' attitudes toward the usability and acceptability of behavior-change smartphone applications (mobile phones) for improving immunization coverage in Pakistan. METHODS: A mixed-method design was employed for this study. The study was conducted at Aga Khan University, Hospital. Parents visiting the Community Health Center for 6-week vaccination of their children were recruited. The study was conducted in two stages. Stage 1 consisted of qualitative interviews that grasped the parent's attitudes and challenges to immunization, as well as their acceptability and accessibility of the smartphone-based behavior-change application to increase vaccine uptake. Stage 1 was followed by stage 2, in which data were collected through a questionnaire designed by using data from qualitative interviews. RESULTS: The majority of participants agreed that immunization serves an important role in protecting their child from illnesses that cause morbidity and mortality. Almost all of them emphasized the importance of using a pre-appointment method at vaccination center in order to reduce the waiting time. Furthermore, participants were also interested in AI-based behavior modification applications related to immunization. They also wanted to have applications in their native language for better understanding and communication of related information. In our study, approximately 95.2 percent of participants agreed to accept SMS immunization updates, which was also reasonably high. Lastly, the majority of them identified forgetfulness as a significant contributor to regular immunization. CONCLUSION: To enhance the uptake of childhood vaccines, overall vaccination rates, and overcome barriers related to vaccination coverage, cost-effective and user-friendly mHealth AI-based smart phone applications are required to raise awareness regarding the continuation of vaccination service and the importance of timely vaccination. Parents' experiences and attitudes must be considered while designing and evaluating the efficacy of mHealth-based interventions.

Methods for estimating the direct and indirect effect of 10 valent pneumococcal vaccine on nasopharyngeal carriage in children under 2 years in Matiari, Pakistan.

Pneumonia is the leading cause of morbidity and mortality in children worldwide. The ten valent pneumococcal vaccine (PCV10) was introduced in Pakistan's Expanded Program on Immunization (EPI) in 2012 as a 3 + 0 schedule without catchup immunization. Nasopharyngeal carriage is taken as a surrogate marker to measure the impact of pneumococcal vaccine on populations. Carriage surveys are necessary to monitor the persistence of Vaccine Type (VT) serotypes, the emergence of Non-Vaccine Type (NVT) serotypes, and their role in both transmission and disease. This article describes various troubleshooting measures which we undertook to adopt the protocol to our setting. We also used an innovative approach to describe various epidemiological parameters of vaccine effectiveness against carriage. It is important to publish these methods to allow for valid regional and temporal comparisons of results in different settings. Thus, in this article, we describe the following methods for isolating upper airway pneumococcal carriage:•Methods for the collection, transport, and storage of nasopharyngeal samples.•Methods for identification and serotyping of pneumococci.•Methods for estimation of the direct and indirect effects of pneumococcal vaccines on nasopharyngeal carriage.