Interlaboratory comparison of molecular detection of Cyclospora cayetanensis in routine diagnostic laboratories

Although molecular detection of Cyclospora cayetanensis is increasingly incorporated into multiplex gastrointestinal diagnostic workflows, dedicated external quality assessment (EQA) programmes for Cyclospora remain limited. We performed a nationwide interlaboratory comparison to evaluate routine molecular detection of C. cayetanensis across decentralized ISO15189-accredited laboratories in the Netherlands.

An anonymized 11-sample panel containing unpreserved stool specimens and eNAT-preserved stool suspensions was distributed to laboratories in the Netherlands (n = 8) and Curaçao (n = 1). Laboratories analysed samples using their routine diagnostic workflows. Workflow characteristics, DNA extraction procedures, molecular platforms, and interpretation criteria were collected by a structured questionnaire. Because detection of Cryptosporidium spp. is usually performed within the same workflow and present similar extraction challenges, Cryptosporidium-positive samples were included as internal workflow controls. Aggregated retrospective laboratory data were collected to explore surveillance feasibility and to gain insight in seasonality of C. cayetanensis and Cryptosporidium spp.

Seven laboratories performed molecular detection of C. cayetanensis, while all nine performed detection of Cryptosporidium spp. Complete qualitative agreement was observed for Cryptosporidium spp. detection. For C. cayetanensis, overall qualitative agreement was high, although one false-positive and three false-negative results occurred among core samples. Detection of a low-positive educational sample varied substantially between laboratories, indicating reduced reproducibility near assay-specific limits of detection. This study shows that eNAT-preserved stool suspensions remained stable under ambient transport conditions. Retrospective laboratory data showed low-level year-round C. cayetanensis detection and a seasonal increase in Cryptosporidium spp. positivity during late summer and autumn.

Routine molecular detection of C. cayetanensis demonstrated good overall interlaboratory agreement across decentralized diagnostic laboratories. However, reproducibility decreased for low-positive samples, highlighting the importance of assay validation, external quality assessment, and cautious interpretation of weak positive C. cayetanensis PCR results.