Thomas Köller1, Andreas Hahn1, Enkhtsetseg Altangerel2, Jaco J Verweij3, Olfert Landt4, Simone Kann5, Denise Dekker6, Jürgen May6, Ulrike Loderstädt6, Andreas Podbielski1, Hagen Frickmann7. 1. Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Rostock, Germany. 2. Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, Hamburg, Germany. 3. Laboratory for Medical Microbiology and Immunology, Elisabeth-Tweesteden Hospital, Tilburg, The Netherlands. 4. TIB MOLBIOL, Berlin, Germany. 5. Medical Mission Institute, Würzburg, Germany. 6. Bernhard Nocht Institute for Tropical Medicine Hamburg, Hamburg, Germany. 7. Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Rostock, Germany; Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, Hamburg, Germany. Electronic address: Frickmann@bni-hamburg.de.
Abstract
INTRODUCTION: A test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a gold standard. Three different commercial kits were included in the comparison, with a range of 3-15 different PCR targets, while 14 targets were covered by in-house testing, so not all 16 target pathogens were covered by all assays. METHODS: Residual materials from nucleic acid extractions of stool samples with very high likelihood of being colonized or infected by at least one enteric parasite species or microsporidia were tested. In all, 500 DNA samples were analyzed, but due to limited sample volume, only 250 of the 500 samples were tested per assay. Each sample was assessed with the qPCR platforms being compared and cycle threshold (Ct) values were included in a descriptive comparison. RESULTS: Depending on the assay applied, qPCR detected per 250 tested samples Giardia duodenalis (184-205), Blastocystis spp. (174-183), Trichuris trichiura (118-120), Ascaris lumbricoides (79-96), Necator americanus (78-106), Hymenolepis nana (40-42), Cryptosporidium spp. (27-36), Dientamoeba fragilis (26-28), Schistosoma spp. (13-23), Enterobius vermicularis (8-14), Entamoeba histolytica (7-16), Strongyloides stercoralis (6-38), Cyclospora spp. (6-13), Taenia spp. (1-4), microsporidia (1-5), and Ancylostoma spp. (1-2). Inter-assay agreement kappa was almost perfect (0.81-1) for Dientamoeba fragilis, Hymenolepis nana, Cryptosporidium spp., and Ascaris lumbricoides, substantial (0.61-0.8) for Necator americanus, Blastocystis spp., Ancylostoma spp., Giardia duodenalis, Schistosoma spp., Trichuris trichiura, and Enterobius vermicularis, moderate (0.41-0.6) for Entamoeba histolytica, fair (0.21-0.4) for microsporidia, slight (0-0.2) for Cyclospora spp. and Strongyloides stercoralis, and poor (<0) for Taenia spp. CONCLUSIONS: Varying inter-assay agreement makes interpretation of microsporidia and parasite PCR in stool samples challenging. Intra-assay agreement had been controlled during the developing of the assays. Future studies, e.g., with optimized nucleic acid procedures and including microscopically characterized samples, are advisable.
INTRODUCTION: A test comparison of in-house and commercial real-time PCR (qPCR) kits for the detection of human parasites and microsporidia in stool samples was conducted without a gold standard. Three different commercial kits were included in the comparison, with a range of 3-15 different PCR targets, while 14 targets were covered by in-house testing, so not all 16 target pathogens were covered by all assays. METHODS: Residual materials from nucleic acid extractions of stool samples with very high likelihood of being colonized or infected by at least one enteric parasite species or microsporidia were tested. In all, 500 DNA samples were analyzed, but due to limited sample volume, only 250 of the 500 samples were tested per assay. Each sample was assessed with the qPCR platforms being compared and cycle threshold (Ct) values were included in a descriptive comparison. RESULTS: Depending on the assay applied, qPCR detected per 250 tested samples Giardia duodenalis (184-205), Blastocystis spp. (174-183), Trichuris trichiura (118-120), Ascaris lumbricoides (79-96), Necator americanus (78-106), Hymenolepis nana (40-42), Cryptosporidium spp. (27-36), Dientamoeba fragilis (26-28), Schistosoma spp. (13-23), Enterobius vermicularis (8-14), Entamoeba histolytica (7-16), Strongyloides stercoralis (6-38), Cyclospora spp. (6-13), Taenia spp. (1-4), microsporidia (1-5), and Ancylostoma spp. (1-2). Inter-assay agreement kappa was almost perfect (0.81-1) for Dientamoeba fragilis, Hymenolepis nana, Cryptosporidium spp., and Ascaris lumbricoides, substantial (0.61-0.8) for Necator americanus, Blastocystis spp., Ancylostoma spp., Giardia duodenalis, Schistosoma spp., Trichuris trichiura, and Enterobius vermicularis, moderate (0.41-0.6) for Entamoeba histolytica, fair (0.21-0.4) for microsporidia, slight (0-0.2) for Cyclospora spp. and Strongyloides stercoralis, and poor (<0) for Taenia spp. CONCLUSIONS: Varying inter-assay agreement makes interpretation of microsporidia and parasite PCR in stool samples challenging. Intra-assay agreement had been controlled during the developing of the assays. Future studies, e.g., with optimized nucleic acid procedures and including microscopically characterized samples, are advisable.
Authors: Felix Weinreich; Andreas Hahn; Kirsten Alexandra Eberhardt; Torsten Feldt; Fred Stephen Sarfo; Veronica Di Cristanziano; Hagen Frickmann; Ulrike Loderstädt Journal: Pathogens Date: 2022-01-26
Authors: Konstantin Tanida; Andreas Hahn; Kirsten Alexandra Eberhardt; Egbert Tannich; Olfert Landt; Simone Kann; Torsten Feldt; Fred Stephen Sarfo; Veronica Di Cristanziano; Hagen Frickmann; Ulrike Loderstädt Journal: Pathogens Date: 2021-05-26