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

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 ( gene) and 33 nematode ( 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 gene failed to identify 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 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.