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Effective diagnostic methods are critical, yet traditional microscopy, though widely used for its simplicity, lacks the sensitivity and specificity of modern techniques like real-time Polymerase Chain Reaction (qPCR), making the latter a more effective tool for monitoring and assessing the burden of intestinal protozoa diseases. In this study, we implemented two duplex qPCR assays to detect <jats:italic>Entamoeba dispar<\/jats:italic>\u2009+\u2009<jats:italic>Entamoeba histolytica<\/jats:italic> and <jats:italic>Cryptosporidium<\/jats:italic> spp.\u2009+\u2009<jats:italic>Chilomastix mesnili<\/jats:italic>, along with singleplex assays for <jats:italic>Giardia duodenalis<\/jats:italic> and <jats:italic>Blastocystis<\/jats:italic> spp., using a 10 \u00b5L reaction volume. This marks the first molecular detection of <jats:italic>Chilomastix mesnili<\/jats:italic> by qPCR, enhancing diagnostic precision. Using these, we analyzed stool samples from 70 patients on Pemba Island, Tanzania, before and 54 samples after treatment with 20, 25, or 30\u00a0mg of emodepside or placebo, aiming to assess protozoa prevalence for this region and emodepside\u2019s potential antiprotozoal effects. Our qPCR reliably detected protozoa in 74.4% of samples, with <jats:italic>Entamoeba histolytica<\/jats:italic> and <jats:italic>Entamoeba dispar<\/jats:italic> in 31.4% of cases. Notably, one-third of these infections were caused by <jats:italic>Entamoeba histolytica<\/jats:italic>. No significant reduction in protozoa was observed after emodepside treatment compared to placebo. The study highlights the utility of qPCR in providing species-level differentiation and improving the speed and cost-effectiveness of testing. The high prevalence of protozoa in this region underscores the need for continued monitoring and control efforts, though emodepside was not effective against protozoa infections.\n<\/jats:p>","DOI":"10.1007\/s00436-025-08483-3","type":"journal-article","created":{"date-parts":[[2025,4,8]],"date-time":"2025-04-08T18:54:29Z","timestamp":1744138469000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Implementation of real-time PCR assays for diagnosing intestinal protozoa infections"],"prefix":"10.1007","volume":"124","author":[{"given":"Christian N.","family":"Lotz","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8586-4937","authenticated-orcid":false,"given":"Pierre H. 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