Clinical evaluation of recombinant antigen ELISAs and broad-range helminth PCRs for the diagnosis of human paragonimiasis in Gabon

Abstract Background Paragonimiasis is a neglected food-borne trematode infection endemic to parts of Africa, Asia, and the Americas acquired by raw/undercooked freshwater crab consumption. Pulmonary manifestations mimic pulmonary tuberculosis (TB). Diagnosis relies on microscopy, with low sensitivity. Development of molecular assays is constrained by an absence of genomic data for African Paragonimus species. Serological testing depends on crude worm antigen, resulting in restricted availability and reproducibility. Methods This study was nested within a prospective cohort of adults with presumed TB in Lambaréné, Gabon. The reference standard for paragonimiasis consisted of sputum and stool microscopy. Molecular index tests included PCRs (ITS2, cox1) on sputum and stool, followed by sequencing. Serological index tests consisted of recombinant ELISAs using GST-tagged Myo-1, MDP, and Egg antigens. Diagnostic accuracy was evaluated using positive control sera (three individuals with confirmed P. westermani infection). Stool, urine and blood were examined for potentially serologically cross-reactive parasites. Results Among 103 participants (enrolled September 2024–January 2025), no Paragonimus eggs were detected. PCR assays amplified only non- Paragonimus sequences, including other helminths. Control sera (confirmed P. westermani ) showed marked reactivity, particularly toward Myo-1 and Egg antigen, background reactivity in participants was generally low. Without confirmed cases of African paragonimiasis, we could only calculate analytical sensitivity for P. westermani -sera. Exploratory cut-offs resulted in tentative diagnostic sensitivity between 17 and 100%. Exploratory cut-offs yielded specificities of 76–100%, assuming that no infections were missed by the reference standard. No relevant cross-reactivity with other infections was observed. Conclusions In this first real-life application of recombinant antigen-based ELISAs for the serodiagnosis of paragonimiasis, our results showed promise, particularly for Myo-1 and Egg antigen. These results require further validation in larger and diverse populations including cases with African Paragonimus species to better define the epidemiology and burden of paragonimiasis in Gabon and beyond.