Understanding factors associated with protective immunity against emerging viral infections is crucial for global health. Pakistan reported its first COVID-19 case on 26 February 2020, but experienced relatively low COVID-19-related morbidity and mortality between 2020 and 2022. The underlying reasons for this remain unclear, and our research aims to shed light on this crucial issue. We conducted a serial population-based serosurvey over 16 months (rounds 1-4, July 2020 to November 2021) across households in urban (Karachi) and rural (Matiari) Sindh, sampling 1100 households and 3900 individuals. We measured antibodies in sera and tested a subset of respiratory samples for COVID-19 using polymerase chain reaction (PCR) and antigen tests, also measuring haemoglobin (Hb), C-reactive protein (CRP), vitamin D, and zinc in round 1. Participants showed 23% (95% confidence interval (CI) = 21.9-24.5) antibody seroprevalence in round 1, increasing across rounds 2-4 to 29% (95% CI = 27.4-30.6), 49% (95% CI = 47.2-50.9), and 79% (95% CI = 77.4-80.8), respectively. Urban residents had 2.6 times (95% CI = 1.9-3.6) higher odds of seropositivity than rural residents. Seropositivity did not differ between genders. Individuals aged 20-49 years had 7.5 (95% CI = 4.6-12.4) times higher odds of seropositivity compared to children aged 0-4 years. Most participants had no symptoms associated with COVID-19, with no reported mortality. Vitamin D deficiency was linked to seroprevalence. COVID-19 was confirmed in 1.8% of individuals tested via RT-PCR and antigen tests. The data suggests a steady increase in humoral immunity in Pakistan, likely due to increased transmission and associated asymptomatic disease. Overall, this reflects the longitudinal trend of protection against severe acute respiratory syndrome coronavirus 2, leading to the relatively low morbidity and mortality observed in the population.

Introduction Undernutrition during pregnancy is linked to adverse pregnancy and birth outcomes and has downstream effects on the growth and development of children. The gut microbiome has a profound influence on the nutritional status of the host. This phenomenon is understudied in settings with a high prevalence of undernutrition, and further investigation is warranted to better understand such interactions. Methods and analysis This is a prospective, longitudinal observational study to investigate the relationship between prokaryotic and eukaryotic microbes in the gut and their association with maternal body mass index (BMI), gestational weight gain, and birth and infant outcomes among young mothers (17–24 years) in Matiari District, Pakistan. We aim to enrol 400 pregnant women with low and normal BMIs at the time of recruitment (<16 weeks of gestation). To determine the weight gain during pregnancy, maternal weight is measured in the first and third trimesters. Gut microbiome dynamics (bacterial and eukaryotic) will be assessed using 16S and 18S rDNA surveys applied to the maternal stool samples. Birth outcomes include birth weight, small for gestational age, large for gestational age, preterm birth and mortality. Infant growth and nutritional parameters include WHO z-scores for weight, length and head circumference at birth through infancy. To determine the impact of the maternal microbiome, including exposure to pathogens and parasites on the development of the infant microbiome, we will analyse maternal and infant microbiome composition, micronutrients in serum using metallomics (eg, zinc, magnesium and selenium) and macronutrients in the stool. Metatranscriptomics metabolomics and markers of inflammation will be selectively deployed on stool samples to see the variations in dietary intake and maternal nutritional status. We will also use animal models to explore the bacterial and eukaryotic components of the microbiome. Ethics and dissemination The study is approved by the National Bioethics Committee (NBC) in Pakistan, the Ethics Review Committee (ERC) at Aga Khan University and the Research Ethics Board (REB) at the Hospital for Sick Children, and findings will be published in peer-reviewed journals. Trial registration number NCT05108675.

Background: Zinc deficiency poses significant health risks, particularly in low-income settings. This study aims to evaluate the impact of agronomically zinc biofortified (fermented and non-fermented) and post-harvest wheat flour flatbread on zinc status and metabolic health in adolescents and adult women in rural Pakistan. Methods: A four-arm triple-blind randomized controlled trial will be conducted in a rural district of Pakistan. Participants (adolescents aged 10–19 and adult women aged 20–40) will be assigned to receive fermented or unfermented high zinc agronomically biofortified wheat flour flatbread, post-harvest zinc-fortified wheat flour flatbread, or low zinc conventional whole wheat flour flatbread. The meal would be served once a day, six days a week for six months. The study aims to enroll 1000 participants and will be analyzed based on the intention-to-treat principle. The trial is registered with number NCT06092515. Outcomes: Primary outcomes will include serum zinc concentration and metabolic markers, while secondary outcomes include anthropometric measurements, blood pressure, and dietary intake. Conclusion: This trial will provide valuable insights into the efficacy of agronomically zinc biofortified wheat flour in improving zinc status and metabolic health. Findings may inform public health strategies to combat zinc deficiency in resource-limited settings.

Background According to the World Health Organization (WHO), worldwide anemia prevalence in children of 6–59 months is 40% [1]. Though iron deficiency is recognized as a significant cause of anemia, vitamin B₁₂ (B₁₂) and folate deficiencies are also important causes. The previous Pakistan National Nutrition Survey (NNS) 2011 showed a high prevalence among women of reproductive age (WRA), which necessitated further investigation in follow-up surveys among children. Methods We performed a secondary analysis of the National Nutrition Survey (NNS) 2018–2019. Blood samples were obtained from 31,828 children (< 5 years), with a subsample analyzed for B₁₂ (n=2693) and folate (n=6690) deficiency. B₁₂ was assessed by electrochemiluminescence immunoassay, and folate by CDC’s microbiologic assay. Logistic regressions were applied to determine factors associated with deficiency. Results 35.1%, while 24.8% of children showed folate and B₁₂ deficiency, respectively. Higher odds of folate and B₁₂ deficiency were observed in Khyber Pakhtunkhwa, OR = 1.424 (95% CI 1.157, 1.752), and newly merged districts of Khyber Pakhtunkhwa, OR = 11.257 (95% CI 2.291, 55.314), respectively. Additionally, mother’s education was associated with B₁₂ deficiency i.e. uneducated OR = 2.017, (95% CI 1.229, 3.308), primary OR = 2.361 (95% CI 1.359, 4.103) and middle OR = 2.108 (95% CI 1.144, 3.882) while underweight was associated with folate deficiency only, OR = 1.181 (95% CI 1.016, 1.371). Conclusion This analysis highlights a considerable burden of folate (35.1%) and vitamin B₁₂ (24.8%) deficiencies among Pakistani children under five years of age, with regional disparities. Socioeconomic and nutritional factors, particularly maternal education and child undernutrition, further influence the risk of deficiency. These results emphasize the need for region-specific nutrition interventions, maternal education programs, and strengthened food fortification strategies.

Background: Understanding factors associated with protective immunity against emerging viral infections is crucial for global health. Pakistan reported its first COVID-19 case on February 26, 2020, but compared to other countries, it experienced relatively low COVID-19-related morbidity and mortality between 2020 and 2022. The underlying reasons remain unclear. Our research aims to shed light on this crucial issue. Methods: We conducted serial household surveys over 16 months (Rounds 1-4, July 2020 - November 2021) in urban (Karachi) and rural (Matiari) Sindh, sampling 1100 households and 3900 individuals. Our comprehensive approach included measuring antibodies in sera, testing a subset of respiratory samples for COVID-19 using RT-PCR and antigen tests, and measuring Hemoglobin, CRP, Vitamin D, and zinc in Round 1. Results Participants showed 23% (95% CI 21.9-24.5) antibody seroprevalence in round 1, increasing across rounds to 29% (95% CI 27.4-30.6), 49% (95% CI 47.2-50.9) and 79% (95% CI 77.4-80.8). Urban residents had 2.6 times (95% CI 1.9-3.6) higher odds of seropositivity than rural residents. Seropositivity did not differ between genders. Individuals aged 20-49 had 7.5 (95% CI 4.6-12.4) times higher odds of seropositivity compared to children aged 0-4. Most participants were asymptomatic, with no reported mortality. Vitamin D deficiency was linked to seroprevalence. COVID-19 was confirmed in 1.8% of individuals via RT-PCR and antigen tests. Conclusions: The data suggests a steady increase in humoral immunity in Pakistan, likely due to increased transmission and potential cross-protective immunity, which may be associated with asymptomatic disease, leading to the reduction of morbidity and mortality observed.

Background: Anemia is a major public health issue, particularly among women of reproductive age (WRA) in low- and middle-income countries (LMICs). Pakistan’s National Nutrition Survey (NNS) 2011 showed a high prevalence of vitamin B₁₂ (B₁₂) and folate deficiency among WRA, necessitating further investigation in subsequent surveys. Methods: Blood samples from 31,828 WRA (15–49 years old) were collected using a stratified multi-stage sampling technique in NNS-2018. We conducted a secondary analysis using population-weighted logistic regression to assess the association of potential factors with B₁₂ and folate deficiency. B₁₂ (n = 4442) and folate (n = 12,662) samples were measured using an electrochemiluminescence immunoassay and a Centers for Disease Control and Prevention, USA (CDC)-approved microbiologic assay, respectively. Results: Folate deficiency was present in 44.7% WRA, and 20.2% had B₁₂ deficiency. Provincial distribution was associated with folate deficiency, i.e., Sindh (OR = 1.140, 95% CI 1.018, 1.285), Baluchistan (OR = 1.237, 95% CI 1.052, 1.453), and Islamabad (OR = 1.524, 95% CI 1.109, 2.092), while B₁₂ deficiency was prevalent in Islamabad (OR = 1.673, 95% CI 1.122, 2.497), Gilgit Baltistan (OR = 2.472, 95% CI 1.197, 5.106), and newly merged districts of KPK (OR = 1.584, 95% CI 0.977, 2.570). Rural residence (OR = 1.407, 95% CI 1.125, 1.760), obesity (OR = 1.649, 95% CI 1.282, 2.122), and overweight (OR = 1.560, 95% CI 1.262, 1.928) were associated with B₁₂ deficiency. Conclusions: Our results show regional and demographic differences in the prevalence of folate and B₁₂ deficiencies among WRA. This underscores the need for targeted nutritional interventions and further longitudinal studies to identify potentially associated factors.