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. Mo...
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MDPI AG
2025-03-01
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| author | Rachel Lau Jason Kwan Kimberley Marks-Beaubrun Ruben Cudiamat Min Qun Ellen Chen Krista Orejana Filip Ralevski Andrea K. Boggild |
| author_facet | Rachel Lau Jason Kwan Kimberley Marks-Beaubrun Ruben Cudiamat Min Qun Ellen Chen Krista Orejana Filip Ralevski Andrea K. Boggild |
| author_sort | Rachel Lau |
| collection | DOAJ |
| description | <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<sup>TM</sup> 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. |
| format | Article |
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| institution | Kabale University |
| issn | 2673-947X |
| language | English |
| publishDate | 2025-03-01 |
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| spelling | doaj-art-44b9c613bfdb43148adf6ea1e942e1882025-08-20T03:43:27ZengMDPI AGHygiene2673-947X2025-03-0151810.3390/hygiene5010008Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric ProtozoaRachel Lau0Jason Kwan1Kimberley Marks-Beaubrun2Ruben Cudiamat3Min Qun Ellen Chen4Krista Orejana5Filip Ralevski6Andrea K. Boggild7Public Health Ontario Laboratories, Public Health Ontario, Toronto, ON M5G 1M1, CanadaFaculty of Medicine, University of British Columbia, Vancouver, BC V6T 1Z3, CanadaDepartment of Psychology, University of Toronto, Toronto, ON M5S 2E5, CanadaPublic Health Ontario Laboratories, Public Health Ontario, Toronto, ON M5G 1M1, CanadaPublic Health Ontario Laboratories, Public Health Ontario, Toronto, ON M5G 1M1, CanadaPublic Health Ontario Laboratories, Public Health Ontario, Toronto, ON M5G 1M1, CanadaPublic Health Ontario Laboratories, Public Health Ontario, Toronto, ON M5G 1M1, CanadaDepartment of Medicine, University of Toronto, Toronto, ON M5S 3H2, Canada<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<sup>TM</sup> 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.https://www.mdpi.com/2673-947X/5/1/8diagnosticsgastrointestinal protozoaperformance characteristicsPCRvalidation |
| spellingShingle | Rachel Lau Jason Kwan Kimberley Marks-Beaubrun Ruben Cudiamat Min Qun Ellen Chen Krista Orejana Filip Ralevski Andrea K. Boggild Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa Hygiene diagnostics gastrointestinal protozoa performance characteristics PCR validation |
| title | Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa |
| title_full | Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa |
| title_fullStr | Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa |
| title_full_unstemmed | Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa |
| title_short | Validation of an Automated High-Throughput Multiplex Real-Time PCR Assay for Detection of Enteric Protozoa |
| title_sort | validation of an automated high throughput multiplex real time pcr assay for detection of enteric protozoa |
| topic | diagnostics gastrointestinal protozoa performance characteristics PCR validation |
| url | https://www.mdpi.com/2673-947X/5/1/8 |
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