Development and validation of a real-time polymerase chain reaction 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 polymerase chain reaction (PCR) method for direct detection of two genomic regions of H. perplexum. The real-time PCR assays, SSU-PCR and COX-1-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 (FFPE) tissue (n=22, derived from eight patients) and tissue biopsy specimens not derived from H. perplexum (n=8). Both H. perplexum assays showed 92% and 84% sensitivity for SSU and COX-1 targets, respectively, 100% specificity, and negative and positive predictive values of 100% and 93%, respectively. The results indicate that the limit of detection was 10-5 dilution [cycle threshold (Ct) value 40.2] for COX-1-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 one 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.