Background: Microscopy is the conventional method for the identification of gastrointestinal parasitic pathogens in fecal specimens; however, it presents numerous challenges, including high technical expertise burden, multiple staining procedures, and prolonged turnaround time. Molecular methods provide higher throughput and potentially higher sensitivity and specificity.
Methods: We validated a commercial, automated DNA extraction platform and multiplex parasitic real-time PCR panel (Seegene Allplex
TM GI-Parasite Assay) detecting six protozoal pathogens:
Blastocystis hominis (Bh),
Cryptosporidium spp.,
Cyclospora cayetanensis (Cc),
Dientamoeba fragilis (Df),
Entamoeba histolytica (Eh), and
Giardia lamblia (Gl) in unpreserved fecal specimens submitted for diagnostic parasitology. Microscopy was the reference standard for all organisms, with stool ELISA as an additional reference assay for Eh.
Results: Among 461 unpreserved fecal specimens, sensitivity, specificity, positive predictive and negative predictive values of the enteric multiplex for fresh specimens were as follows: 93%, 98.3%, 85.1%, 99.3% for Bh; 100% for all measures in
Cryptosporidium and Cc; 100%, 99.3%, 88.5%, 100% for Df; 33.3%, 100%, 100%, 99.6% for Eh; and 100%, 98.9%, 68.8%, 100% for Gl, respectively. With the addition of 17 frozen specimens, the sensitivity for Eh increased to 75%. On a per-batch basis, the molecular platform reduced pre-analytical and analytical testing turnaround time by 7 h.
Conclusions: The enteric multiplex platform provides a useful diagnostic tool for clinically relevant enteric protozoa, including
Cryptosporidium spp.,
Cyclospora cayetanensis,
Dientamoeba fragilis, and
Giardia lamblia. Further evaluation of the assay is required for
Entamoeba histolytica prior to clinical use; however, given the widespread availability of confirmatory serology and stool antigen testing for
E. histolytica, such performance limitations are of lesser concern.
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