Aims 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.
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Handling editor Dr Tony Mazzulli.
Contributors SSYW is responsible for the identification of parasites in the corresponding institution, acquisition of data, and prepared and approved the final version of the manuscript. RWSP is responsible for all aspects of molecular identification of the parasites. KKWT, JFWC and VCCC are responsible for acquisition of data and review of the manuscript. GL and FFX are responsible for acquisition of data and identification of parasites in the corresponding institutions. K-YY is responsible for initiation and supervision of the study and critical evaluation of the manusript.
Funding The study was partly funded by the High Level Hospital-Summit Program in Guangdong, China; donations from the Shaw Foundation Hong Kong, Dr Richard Yu and Carol Yu, Michael Seak-Kan Tong, Hui Ming, Hui Hoy, Chow Sin Lan Charity Fund Limited and Chan Yin Chuen Memorial Charitable Foundation.
Competing interests None declared.
Patient consent for publication Not required
Provenance and peer review Not commissioned; externally peer reviewed.