Development and Validation of a Real-Time PCR for the Detection of Haycocknema perplexum

Haycocknema perplexum is a rare and emerging cause of parasitic myositis. Detection and surveillance are of growing importance given the increasing degree of climate conditions conducive to transmission. In this study, we developed a real-time PCR method for direct detection of two genomic regions of Haycocknema perplexum. The real-time PCR assays, SSU-PCR and COX1-PCR targeted the small subunit of nuclear ribosomal RNA and cytochrome oxidase-1 genomic regions, respectively. The performance of the assays was assessed using a panel of H. perplexum samples, both fresh frozen and formalin fixed paraffin embedded tissue (FFPE) (n = 22, derived from eight patients) and non-H. perplexum (n = 8) tissue biopsy specimens. Both H. perplexum assays showed 83% sensitivity and 100% specificity, with negative and positive predictive values of 100% and 93% respectively. The results indicate that the LOD was 10-5 dilution (Ct value 40.2) for COX1-PCR, and 10-3 dilution (Ct value 39.6) for SSU-PCR, making the latter a more sensitive assay for detecting lower concentrations of organism in the patient biopsy. The sensitivity of fresh frozen samples was superior to FFPE samples. All but 1 sample was negative following treatment. Feasibility of real-time PCR detection of H. perplexum directly from tissue biopsies has been demonstrated for diagnosis, possible test of cure and could enhance transmission surveillance.