Diagnostic Accuracy of ARK Checker® C/G – DyLight® 488: Simultaneous Detection of Giardia and Cryptosporidium by Fluorescent Antibody Microscopy

Background Giardiasis and cryptosporidiosis are often misdiagnosed by stool ova and parasite test (classical O&P). Multiplex PCR and rapid antigen immunochromatography (rapid-IC) could offer high diagnostic accuracy; however, their cost and restricted coverage, especially for parasites, limit routine use. This study evaluated the efficacy of adapted fluorescent antibody microscopy using ARK Checker® C/G – DyLight® 488 (FAM-TEST). The reagent is a liquid-form conjugated antibody preparation that directly reacts with Giardia cysts and Cryptosporidium oocysts. Methods Stool samples were incubated with DyLight-488–labeled antibodies as indicated in our original protocol and examined by fluorescence microscopy. Diagnostic accuracy of FAM-TEST was assessed with multiplex PCR results as reference and compared with that of rapid-IC. Samples with discordant results among these methods were additionally tested by conventional PCR with Sanger sequencing to verify infection status. Results A total of 694 stool samples were submitted for microbiological examination. FAM-TEST identified Giardia and/or Cryptosporidium in 35 samples. In addition, 49 FAM-TEST- negative samples from a randomly selected month were included as negative controls. For Giardia , all FAM-TEST results were identical to those of rapid-IC, including three false negatives [88.0% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 95.2% negative predictive value (NPV)]. For Cryptosporidium , FAM-TEST showed 93.8% sensitivity, 100% specificity, 100% PPV, and 98.6% NPV, which were comparable with those of rapid-IC. Notably, nested PCR for Giardia produced negative results for three samples considered to yield FAM-TEST false negatives, raising another possibility of multiplex PCR false positives or extremely low pathogen burden, whereas conventional PCR for Cryptosporidium showed results fully consistent with those of multiplex PCR. Conclusion Concurrent use of FAM-TEST with classical O&P offers a cost-effective, practical diagnostic approach for enteric parasites, which especially strengthens diagnostic accuracy for Giardiasis/Cryptosporidiosis.