Improving Practical Diagnostic Measure for Enteric Parasites: Development of Fluorescent Antibody Microscopy (FAM) for Giardiasis and Cryptosporidiosis

BackgroundGiardiasis 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 accuracy; however, its cost and restricted coverage, especially for parasites, limit their use. This study evaluated the efficacy of newly developed "fluorescent antibody microscopy (FAM)" for these protozoa. Methodology/Principal FindingesDyLight 488-conjugated antibodies against Giardia cysts and Cryptosporidium oocysts were used for FAM examination. Diagnostic accuracy was assessed by reference to multiplex PCR results and compared with that of rapid-IC. FAM identified Giardia and/or Cryptosporidium in 35 of 694 stool samples tested. Also, FAM-negative stools examined in a randomly chosen month (49 samples) were selected as negative control. For Giardia, all FAM results were identical to those of rapid-IC, including three false negatives [100% positive predictive value (PPV), and 95.2% negative predictive value (NPV)]. For Cryptosporidium, FAM showed 100% PPV, and 98.6% NPV, which were comparable with those of rapid-IC. Conventional PCR for Giardia sequences produced negative results for three samples considered to yield FAM false negative, raising another possibility of multiplex PCR false positives. Conclusions/SignificanceConcurrent use of FAM with classical O&P, which strengthens diagnostic accuracy for Giardiasis/Cryptosporidiosis, is an option for cost-effective, practical diagnosis of enteric parasites. Author summaryGiardiasis and Cryptosporidiosis are often misdiagnosed through traditional stool tests. Although multiplex-PCR and rapid antigen tests (rapid-IC) offer high accuracy, their cost and limited parasite coverage restrict their use. To overcome these challenges, we developed a new diagnostic method named "Fluorescent Antibody Microscopy (FAM)." This study evaluated the diagnostic accuracy of FAM by using multiplex-PCR as a reference standard and compared it with rapid-IC. For Giardia, FAMs results were identical to rapid-IC, including three false negatives (100% PPV and 95.2% NPV). Similarly, for Cryptosporidium, FAM showed high diagnostic accuracy (100% PPV and 98.6% NPV), comparable to rapid-IC. Furthermore, in some cases, the concomitant use of bright-field microscopic examination occasionally identified enteric parasites, which were not covered by multiplex-PCR. Our results highlight that morphological parasitical identification with FAM may provide a cost-effective diagnostic strategy for enteric parasites, offering high accuracy for Giardia and Cryptosporidium.