Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples
Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays ampl...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | Diagnostics |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2075-4418/12/4/1007 |
| _version_ | 1797436096086278144 |
|---|---|
| author | Felix Weinreich Andreas Hahn Kirsten Alexandra Eberhardt Simone Kann Thomas Köller Philipp Warnke Susann Dupke Denise Dekker Jürgen May Hagen Frickmann Ulrike Loderstädt |
| author_facet | Felix Weinreich Andreas Hahn Kirsten Alexandra Eberhardt Simone Kann Thomas Köller Philipp Warnke Susann Dupke Denise Dekker Jürgen May Hagen Frickmann Ulrike Loderstädt |
| author_sort | Felix Weinreich |
| collection | DOAJ |
| description | Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint. |
| first_indexed | 2024-03-09T10:56:46Z |
| format | Article |
| id | doaj.art-ebf7d387ab224944bf6aabeec1a54247 |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-09T10:56:46Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-ebf7d387ab224944bf6aabeec1a542472023-12-01T01:35:40ZengMDPI AGDiagnostics2075-44182022-04-01124100710.3390/diagnostics12041007Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool SamplesFelix Weinreich0Andreas Hahn1Kirsten Alexandra Eberhardt2Simone Kann3Thomas Köller4Philipp Warnke5Susann Dupke6Denise Dekker7Jürgen May8Hagen Frickmann9Ulrike Loderstädt10Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, 20359 Hamburg, GermanyInstitute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, GermanyDepartment of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine Hamburg & I. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20359 Hamburg, GermanyMedical Mission Institute, 97074 Würzburg, GermanyInstitute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, GermanyInstitute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, GermanyCentre for Biological Threats and Special Pathogens/Highly Pathogenic Microorganisms, Robert Koch Institute, 13353 Berlin, GermanyInfectious Disease Epidemiology Department, Bernhard Nocht Institute for Tropical Medicine Hamburg, 20259 Hamburg, GermanyInfectious Disease Epidemiology Department, Bernhard Nocht Institute for Tropical Medicine Hamburg, 20259 Hamburg, GermanyDepartment of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, 20359 Hamburg, GermanyDepartment of Hospital Hygiene & Infectious Diseases, University Medicine Göttingen, 37075 Göttingen, GermanyPrior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.https://www.mdpi.com/2075-4418/12/4/1007test evaluationreal-time PCRparasitebacteriaenteric pathogengastrointestinal infection |
| spellingShingle | Felix Weinreich Andreas Hahn Kirsten Alexandra Eberhardt Simone Kann Thomas Köller Philipp Warnke Susann Dupke Denise Dekker Jürgen May Hagen Frickmann Ulrike Loderstädt Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples Diagnostics test evaluation real-time PCR parasite bacteria enteric pathogen gastrointestinal infection |
| title | Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples |
| title_full | Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples |
| title_fullStr | Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples |
| title_full_unstemmed | Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples |
| title_short | Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples |
| title_sort | multicentric evaluation of seegene allplex real time pcr assays targeting 28 bacterial microsporidal and parasitic nucleic acid sequences in human stool samples |
| topic | test evaluation real-time PCR parasite bacteria enteric pathogen gastrointestinal infection |
| url | https://www.mdpi.com/2075-4418/12/4/1007 |
| work_keys_str_mv | AT felixweinreich multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT andreashahn multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT kirstenalexandraeberhardt multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT simonekann multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT thomaskoller multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT philippwarnke multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT susanndupke multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT denisedekker multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT jurgenmay multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT hagenfrickmann multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples AT ulrikeloderstadt multicentricevaluationofseegeneallplexrealtimepcrassaystargeting28bacterialmicrosporidalandparasiticnucleicacidsequencesinhumanstoolsamples |