Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa

<b>Background</b>: 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. <b>Methods</b>: 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: <i>Blastocystis hominis</i> (Bh), <i>Cryptosporidium</i> spp., <i>Cyclospora cayetanensis</i> (Cc), <i>Dientamoeba fragilis</i> (Df), <i>Entamoeba histolytica</i> (Eh), and <i>Giardia lamblia</i> (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. <b>Results</b>: 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 <i>Cryptosporidium</i> 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. <b>Conclusions</b>: The enteric multiplex platform provides a useful diagnostic tool for clinically relevant enteric protozoa, including <i>Cryptosporidium</i> spp., <i>Cyclospora cayetanensis</i>, <i>Dientamoeba fragilis</i>, and <i>Giardia lamblia</i>. Further evaluation of the assay is required for <i>Entamoeba histolytica</i> prior to clinical use; however, given the widespread availability of confirmatory serology and stool antigen testing for <i>E. histolytica</i>, such performance limitations are of lesser concern.