Molecular detection of Cyclospora cayetanensis in fresh produce and irrigation water in peri- urban settings: a One Health cross-sectional study

Background Cyclospora cayetanensis is recognized as an important foodborne parasite worldwide, with fresh produce and contaminated irrigation water serving as major transmission vehicles. In South Asia, environmental surveillance data for this pathogen remain limited, hindering the development of evidence-based food safety measures. We investigated the occurrence of C. cayetanensis in fresh produce and irrigation water across peri-urban areas of Khyber Pakhtunkhwa, Pakistan, and assessed environmental and farm-level factors associated with contamination. Methods A cross-sectional study was conducted in Peshawar and Kohat districts from April to September 2025. A total of 420 samples were collected, including 300 fresh produce samples (six commonly consumed vegetables and herbs) and 120 irrigation water samples from canal, tube-well, and mixed sources. Samples were processed using concentration techniques, and detection was performed by nested PCR targeting the 18S rRNA gene. Structured field questionnaires were used to capture farm-level practices, and logistic regression was applied to identify risk factors. Results C. cayetanensis DNA was detected in 6.0% (18/300) of produce samples and 12.5% (15/120) of irrigation water samples (p = 0.028). Contamination was significantly higher in canal water (20.0%) compared to tube-well sources (5.0%) (OR 4.75; 95% CI: 1.01–22.3). Leafy vegetables and herbs showed higher contamination rates than smooth-surfaced produce (p = 0.009). In multivariable analysis, canal irrigation (aOR 3.41; p = 0.031), proximity to drainage channels within 50 meters (aOR 3.98; p = 0.007), and use of untreated surface water for rinsing (aOR 2.91; p = 0.045) were independently associated with contamination. Conclusions This study provides the first molecular evidence of C. cayetanensis contamination at the produce–water interface in peri-urban Khyber Pakhtunkhwa, Pakistan. Surface irrigation systems and inadequate water management practices emerged as critical risk factors. By combining molecular detection with environmental and farm-level assessments under a One Health approach, our findings provide practical guidance for targeted food safety interventions in settings where environmental surveillance has historically been sparse.