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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.

IntroductionClimate change has led to extreme heat events, disproportionately affecting vulnerable populations. Heat stress during pregnancy is linked to adverse health outcomes, yet the biological mechanisms remain poorly understood. This research study aims to investigate the effect of environmental heat on maternal, fetal and infant health and examine the biological pathways linking heat stress to adverse pregnancy outcomes.Methods and analysisThis prospective cohort study will recruit 6000 pregnant women from three districts in Sindh, Pakistan. Eligible participants ≥18 years old, will have a minimum of five scheduled visits from <14 weeks’ gestation and will be followed up to 12 months postpartum. Primary outcomes include low birth weight and small vulnerable newborns (SVN); secondary outcomes include preterm birth, small for gestational age (SGA), miscarriage, stillbirth, and composite maternal and neonatal morbidity and mortality. Fetal ultrasound scans with Doppler assessments will be performed at each visit to measure fetal growth, uteroplacental and fetoplacental circulation. Each woman’s heat exposure will be measured using wearable sensors and heat strain biomarkers. In a subset of 1000 women, maternal heart rate, skin temperature sleep patterns and physical activity will be monitored throughout pregnancy using wearable devices. Time-varying, distributed lag and non-linear models will examine associations between heat stress indices and pregnancy outcomes.Ethics and disseminationThe study has received ethical approval from the Aga Khan University (AKU) (Ref: 26249) and the Pakistan National Bioethics Committee (Ref: 1065/23/1736). Written informed consent will be obtained from all participants before enrolment. Referral pathways to healthcare facilities will be established to ensure timely management of pregnancy complications. Findings will be disseminated through peer-reviewed publications, scientific conferences, and engagement with policymakers and public health stakeholders to inform climate-resilient maternal health strategies. Results will also be shared with participants and communities through meetings and informal sessions to raise awareness and support evidence-based heat adaptation.Trial registration numberNCT01234567.

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. 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. Primary outcomes will include serum zinc concentration and metabolic markers, while secondary outcomes include anthropometric measurements, blood pressure, and dietary intake. 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.

IntroductionUndernutrition 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 analysisThis 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 disseminationThe 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.

BackgroundVitamin D deficiency during pregnancy is a public health problem in Pakistan and is prevalent among most women of reproductive age in the country. Vitamin D supplementation during pregnancy is suggested to prevent adverse pregnancy outcomes and vitamin D deficiency in both the mother and her newborn.MethodsWe conducted a double-blinded, randomised controlled trial in Karachi, Pakistan to evaluate the effect of different doses of vitamin D supplementation during pregnancy on biochemical markers (serum 25(OH)D, calcium, phosphorus and alkaline phosphatase) in women and neonates, and on pregnancy and birth outcomes (gestational diabetes, pre-eclampsia, low birth weight, preterm births and stillbirths).ResultsPregnant women (N=350) in their first trimester were recruited and randomised to three treatment groups of vitamin D supplementation: 4000 IU/day (group A, n=120), 2000 IU/day (group B, n=115) or 400 IU/day (group C, n=115). Women and their newborn in group A had the lowest vitamin D deficiency at endline (endline: 75.9%; neonatal: 64.9%), followed by group B (endline: 84.9%; neonatal: 73.7%) and then the control group (endline: 90.2%; neonatal: 91.8%). Vitamin D deficiency was significantly lower in group A than in group C (p=0.006) among women at endline and lower in both groups A and B than in the control group (p=0.001) in neonates. Within groups, serum 25(OH)D was significantly higher between baseline and endline in group A and between maternal baseline and neonatal levels in groups A and B. Participant serum 25(OH)D levels at the end of the trial were positively correlated with those in intervention group A (4000 IU/day) (β=4.16, 95% CI 1.6 to 6.7, p=0.002), with food group consumption (β=0.95, 95% CI 0.01 to 1.89, p=0.047) and with baseline levels of serum 25(OH)D (β=0.43, 95% CI 0.29 to 0.58, p<0.0001).ConclusionThe evidence provided in our study indicates that vitamin D supplementation of 4000 IU/day was more effective in reducing vitamin D deficiency among pregnant women and in improving serum 25(OH)D levels in mothers and their neonates compared with 2000 IU/day and 400 IU/day. Trial registration number NCT02215213.

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.

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 B[sub.12] (B[sub.12]) 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 B[sub.12] and folate deficiency. B[sub.12] (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 B[sub.12] 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 B[sub.12] 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 B[sub.12] deficiency. Conclusions: Our results show regional and demographic differences in the prevalence of folate and B[sub.12] deficiencies among WRA. This underscores the need for targeted nutritional interventions and further longitudinal studies to identify potentially associated factors.

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. 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. 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.

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 B (B ) and folate deficiency among WRA, necessitating further investigation in subsequent surveys. 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 B and folate deficiency. B ( = 4442) and folate ( = 12,662) samples were measured using an electrochemiluminescence immunoassay and a Centers for Disease Control and Prevention, USA (CDC)-approved microbiologic assay, respectively. Folate deficiency was present in 44.7% WRA, and 20.2% had B 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 B 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 B deficiency. Our results show regional and demographic differences in the prevalence of folate and B deficiencies among WRA. This underscores the need for targeted nutritional interventions and further longitudinal studies to identify potentially associated factors.