High levels of mycotoxin contamination have been reported in various food commodities in Pakistan, however, there has been no exposure assessment study using multiple mycotoxins’ biomarkers. This study aimed to simultaneously assess the exposure to the five major mycotoxins: aflatoxin B₁ (AFB₁), deoxynivalenol (DON), fumonisin B₁ (FB₁), ochratoxin A (OTA) and zearalenone (ZEN) in a Pakistani population using an integrated approach of human biomonitoring. Human urine samples (n = 292) were analyzed by a super-sensitive liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. Rice and wheat were also collected and analyzed for mycotoxins by the LC-MS/MS method. Food consumption data were collected using a 24 h recall method. A high prevalence of urinary AFM₁ (66%, mean ± SD 20.8 ± 41.3 pg/mL) and OTA (99%, 134.7 ± 312.0 pg/mL) were found, whilst urinary DON, FB₁ and ZEN levels were low. The probable daily intake (PDI) derived from the urinary biomarkers revealed that 89% of the participants had exposure to OTA exceeding the established tolerable daily intake (TDI = 17 ng/kg bw/day). The average PDI of AFB₁ for the studied population was 43 ng/kg bw/day, with rice as the main source of AFB₁ exposure. In summary, exposure to AFB₁ and OTA are of health concern and require further management.

There are limited data on exposure to mycotoxins in Pakistan. Here, we measured exposure to deoxynivalenol (DON), a common contaminant of wheat, and aflatoxin B₁ (AFB₁), a known contaminant of rice, using biomarkers of exposure. Wheat (n = 195) and rice (n = 62) samples were analyzed for AFB₁ and DON levels, and the corresponding urinary biomarkers were analyzed in urine samples from a rural population (n = 264, aged 4–80 years, male 58%) using ultra-sensitive liquid chromatography–tandem mass spectrometry. AFB₁ was detected in 66% of rice (5.04 ± 11.94 µg/kg) and 3% of wheat samples. AFM₁ (hydroxylated form of AFB₁) was detected in 69% of urine samples, mean 0.023 ± 0.048 ng/mL and DON was detected in 20% of urine samples, mean 0.170 ± 0.129 ng/mL. The maximum probable daily intake for DON derived from the urinary biomarker was 59.8 ng/kg b.w./day, which is below the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives’ tolerable daily intake (1000 ng/kg b.w./day). However, for aflatoxin, the derived margin of exposure (MoE) of (13.2) was well below the safe MoE (10,000) suggested by the European Food Safety Authority. The calculated aflatoxin-associated cancer risk of 0.514/10⁵ individuals/year suggests that measures should be taken to reduce the AFB₁ contamination in food, particularly rice, in Pakistan.

Aflatoxins are liver carcinogens and are common contaminants in unpackaged peanut (UPP) oil. However, the health risks associated with consuming aflatoxins in UPP oil remain unclear. In this study, aflatoxin contamination in 143 UPP oil samples from Guangdong Province were assessed via liquid chromatography–tandem mass spectrometry (LC-MS). We also recruited 168 human subjects, who consumed this oil, to measure their liver functions and lipid metabolism status. Aflatoxin B1 (AFB1) was detected in 79.72% of the UPP oil samples, with levels ranging from 0.02 to 174.13 μg/kg. The average daily human intake of AFB1 from UPP oil was 3.14 ng/kg·bw/day; therefore, the incidence of liver cancer, caused by intake of 1 ng/kg·bw/day AFB1, was estimated to be 5.32 cases out of every 100,000 persons per year. Meanwhile, Hepatitis B virus (HBV) infection and AFB1 exposure exerted a synergistic effect to cause liver dysfunction. In addition, the triglycerides (TG) abnormal rate was statistically significant when using AFB1 to estimate daily intake (EDI) quartile spacing grouping (p = 0.011). In conclusion, high aflatoxin exposure may exacerbate the harmful effects of HBV infection on liver function. Contamination of UPP oil with aflatoxins in Guangdong urgently requires more attention, and public health management of the consumer population is urgently required.

Background: Chronic exposure to aflatoxins is associated with liver cancer, impaired child growth, and compromised immune function. The Childhood Acute Illness and Nutrition (CHAIN) Network cohort was established to identify risk factors for mortality in acutely ill children admitted to nine hospitals in four African and two South Asian countries. We examined the role of aflatoxin exposure in inpatient and post-discharge mortality. Methods: In a nested case-cohort from the CHAIN cohort, we compared aflatoxin exposure at admission and discharge with death or survival in hospital (n=755) or up to 180-days post-discharge (n=585) and with community participants (CP, n=222). Children were stratified into non-wasting, medium-wasting and severe-wasting groups based on mid-upper arm circumference. Serum samples were analysed for an aflatoxin exposure biomarker, the aflatoxin-albumin adduct (AF-alb) using ELISA. Findings: Overall, 56% of hospitalised participants tested positive for AF-alb at admission. The AF-alb level was higher in deceased (geometric mean and 95% CI (GM and 95% CI) 5.9 (4.9 to 7.1)) than in survivors (4.2 (3.8 to 4.7)) and CP (3.7 (3.1 to 4.3)) pg/mg alb. AF-alb concentration was higher at admission (4.7, (4.2 to 5.1)) than at discharge (3.7, (3.3 to 4.1)) and in the CP group (3.7, (3.1 to 4.3)) pg/mg alb (p<0.01) and in African vs Asian children (7.4 (6.5 to 8.5) vs 1.9 (1.8 to 2.1)) (p<0.001). Adjusted logistical regression showed no significant association between AF-alb levels and mortality, but after separating the nutrition strata, AF-alb was significantly associated with mortality (highest vs lowest quartile group OR=4.84, p=0.014) in non-wasted children. Interpretation: Moderate to severe malnutrition is a more important risk factor for mortality than aflatoxin in acutely ill children, but aflatoxin exposure may contribute to mortality in non-wasted children. Controlling aflatoxin exposure should be integrated into clinical and public health interventions to reduce mortality in areas with high levels of exposure. Funding: Bill and Melinda Gates Foundation (OPP1131320 & INV-003225).

Introduction: Many acutely ill children in low- and middle-income settings have a high risk of mortality both during and after hospitalisation despite guideline-based care. Understanding the biological mechanisms underpinning mortality may suggest optimal pathways to target for interventions to further reduce mortality. The Childhood Acute Illness and Nutrition (CHAIN) Network (www.chainnnetwork.org) Nested Case-Cohort Study (CNCC) aims to investigate biological mechanisms leading to inpatient and post-discharge mortality through an integrated multi-omic approach. Methods and analysis; The CNCC comprises a subset of participants from the CHAIN cohort (1278/3101 hospitalised participants, including 350 children who died and 658 survivors, and 270/1140 well community children of similar age and household location) from nine sites in six countries across sub-Saharan Africa and South Asia. Systemic proteome, metabolome, lipidome, lipopolysaccharides, haemoglobin variants, toxins, pathogens, intestinal microbiome and biomarkers of enteropathy will be determined. Computational systems biology analysis will include machine learning and multivariate predictive modelling with stacked generalization approaches accounting for the different characteristics of each biological modality. This systems approach is anticipated to yield mechanistic insights, show interactions and behaviours of the components of biological entities, and help develop interventions to reduce mortality among acutely ill children. Ethics and dissemination. The CHAIN Network cohort and CNCC was approved by institutional review boards of all partner sites. Results will be published in open access, peer reviewed scientific journals and presented to academic and policy stakeholders. Data will be made publicly available, including uploading to recognised omics databases. Trial registration NCT03208725.