Seroprevalence and optimized quantitative PCR detection of Entamoeba histolytica using self-collected rectal swabs among men who have sex with men in southern Taiwan: a cross-sectional study

Background Amebiasis, caused by Entamoeba histolytica , has emerged as a sexually transmitted enteric infection among men who have sex with men (MSM) and people with HIV (PWH) in developed countries. While previous surveillance studies in Taiwan among PWH showed ongoing transmission, investigation on E. histolytica infection among non-HIV MSM using newer diagnostic methods and ELISA-IgG seroprevalence surveys is lacking. Methods From March 2024 to February 2025, we conducted a cross-sectional study during an LGBTQ + Pride event and at a tertiary medical center in southern Taiwan. MSM participants were recruited during anonymous HIV and syphilis testing, or during follow-up visits for HIV pre-exposure prophylaxis or antiretroviral therapy. We collected serum samples for E. histolytica ELISA IgG and self-collected rectal swabs for molecular diagnosis. DNA extraction was performed using an optimized protocol. A novel quantitative PCR assay utilizing specific primers and a double-quencher probe was developed to enhance detection specificity and reduce background noise. Results Among 139 non-HIV MSM participants, 11 individuals tested positive for E. histolytica IgG, yielding a seroprevalence of 7.9%. Molecular testing by rectal swab quantitative PCR identified two positive cases (1.4%). Of the two positive cases, one developed diarrhea just before examination, while the other remained asymptomatic. The novel PCR primer probe set detected E. histolytica DNA from rectal swab samples with high specificity. Conclusions The present study investigated E. histolytica infection among non-HIV MSM in Taiwan using ELISA IgG and novel molecular diagnostic methods. The seroprevalence of 7.9% suggests ongoing transmission in this population. The optimized self-collected rectal swab protocol combined with the novel PCR assay provides a practical surveillance tool. Further investigation and targeted prevention strategies are warranted.