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Article: Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

TitleImproving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay
Authors
Keywordsagar gel electrophoresis
Ancylostoma duodenale
Anisakis simplex
Ascaris lumbricoides
cerebrospinal fluid
Issue Date2019
PublisherBMJ Publishing Group. The Journal's web site is located at http://jcp.bmjjournals.com/
Citation
Journal of Clinical Pathology, 2019, v. 72 n. 7, p. 487-492 How to Cite?
AbstractAIMS: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay. METHODS: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested. RESULTS: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples. CONCLUSIONS: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.
Persistent Identifierhttp://hdl.handle.net/10722/273430
ISSN
2017 Impact Factor: 2.894
2015 SCImago Journal Rankings: 1.260

 

DC FieldValueLanguage
dc.contributor.authorWong, SSY-
dc.contributor.authorPOON, RWS-
dc.contributor.authorTo, KKW-
dc.contributor.authorChan, JFW-
dc.contributor.authorLU, G-
dc.contributor.authorXING, F-
dc.contributor.authorCHENG, VCC-
dc.contributor.authorYuen, KY-
dc.date.accessioned2019-08-06T09:28:48Z-
dc.date.available2019-08-06T09:28:48Z-
dc.date.issued2019-
dc.identifier.citationJournal of Clinical Pathology, 2019, v. 72 n. 7, p. 487-492-
dc.identifier.issn0021-9746-
dc.identifier.urihttp://hdl.handle.net/10722/273430-
dc.description.abstractAIMS: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay. METHODS: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested. RESULTS: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples. CONCLUSIONS: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.-
dc.languageeng-
dc.publisherBMJ Publishing Group. The Journal's web site is located at http://jcp.bmjjournals.com/-
dc.relation.ispartofJournal of Clinical Pathology-
dc.rightsJournal of Clinical Pathology. Copyright © BMJ Publishing Group.-
dc.rightsThis article has been accepted for publication in [Journal, Year] following peer review, and the Version of Record can be accessed online at [insert full DOI eg. http://dx.doi.org/10.1136/xxxxx]. [© Authors (or their employer(s)) OR © BMJ Publishing Group Ltd ( for assignments of BMJ Case Reports)] <year>-
dc.subjectagar gel electrophoresis-
dc.subjectAncylostoma duodenale-
dc.subjectAnisakis simplex-
dc.subjectAscaris lumbricoides-
dc.subjectcerebrospinal fluid-
dc.titleImproving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay-
dc.typeArticle-
dc.identifier.emailWong, SSY: samsonsy@hku.hk-
dc.identifier.emailTo, KKW: kelvinto@hku.hk-
dc.identifier.emailChan, JFW: jfwchan@hku.hk-
dc.identifier.emailYuen, KY: kyyuen@hkucc.hku.hk-
dc.identifier.authorityWong, SSY=rp00395-
dc.identifier.authorityTo, KKW=rp01384-
dc.identifier.authorityChan, JFW=rp01736-
dc.identifier.authorityYuen, KY=rp00366-
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1136/jclinpath-2018-205590-
dc.identifier.pmid30952829-
dc.identifier.scopuseid_2-s2.0-85063990813-
dc.identifier.hkuros300602-
dc.identifier.volume72-
dc.identifier.issue7-
dc.identifier.spage487-
dc.identifier.epage492-
dc.publisher.placeUnited Kingdom-

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