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

Abstract

AimsHelminth 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.MethodsWe 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.ResultsThe 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.ConclusionsThe 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.

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

“…A number of genus-and species-specific in-house helminth real-time PCR approaches [28,42,43] supplemented by some commercial assays [28] were applied. Among the in-house assays were both previously described [29,30] and newly introduced group-specific helminth real-time PCRs targeting nematodes, trematodes, and cestodes. With both microscopically positive and negative samples (details are shown in Tables 4 and 5 and Appendix A), these group-specific real-time PCRs showed imperfect sensitivity and specificity, predominantly with sensitivity issues.…”

“…mansoni complex [42] and S. haematobium complex [43], which were originally designed for application with blood samples, were used as detailed previously [42,43] with DNA from stool. Group-specific helminth real-time PCRs targeting nematodes, trematodes, and cestodes were applied as described [29,30] as simplex assays. In addition, groupspecific assays for nematodes, trematodes, and cestodes targeting the 18S-rRNA-gene were run (details on the group-specific assays are provided in the Tables 1 and 2).…”

“…Multiple genus-and species-specific PCRs and real-time PCRs [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] have been introduced for helminths. A cox-gene-based group-specific real-time PCR approach targeting nematodes and cestodes has been described [29], as has a 28S-rRNA-gene-based approach targeting the trematode group [30].…”

Recent advances in nucleic acid-based methods for detection of helminth infections and the perspective of biosensors for future development

“…In the Philippines, a study indicated that the multiplex PCR could simultaneously detect two cestode parasites T. saginata and T. solium alongside A. lumbricoides and hookworms in stool samples (Gordon et al, 2015). Moreover, a recent study indicated that a multiplex single-tube PCR assay can specifically discriminate trematode, cestode and nematode species by targeting the 28S rRNA gene (trematode), COX1 gene (cestode) and COX1 gene (nematode) (Wong et al, 2019). By incorporating Luminex beads or fluorescent dye, the multiplex PCR can be modified as qPCR to improve its sensitivity and specificity.…”

Capillaria hepatica (syn. Calodium hepaticum) as a Cause of Asymptomatic Liver Mass

“…12 For example, Wong et al developed a multiplex test for various helminths in several biologic media, and Mattiucci et al described molecular identification of Anisakis pegreffii in intestinal granulomas. 13,14 We are not aware of published molecular diagnosis tools for capillariasis in humans.…”