A novel multiplex real-time polymerase chain reaction for the molecular diagnosis of metacestode infections in human patients

Abstract

Objectives: The diagnosis of larval cestodiases in humans primarily depends on using imaging techniques in combination with serological tests. However, in case of atypical imaging results, negative serology results due to immunosuppression, or infection with rare taeniid species, traditional diagnostic tools may not provide a definitive species-level diagnosis. We aimed to validate a rapid, reliable, and cost-effective single-step real-time PCR method that can identify and differentiate larval cestodiases from biopsy material.

Methods: We validated a real-time PCR technique able to distinguish Echinococcus multilocularis, E. granulosus sensu lato (s.l.), and Taenia spp. from biopsy or cytology material in a single-step analysis. Further Sanger sequencing of E. granulosus s.l. and Taenia spp. amplicons enables differentiation of various Echinococcus and Taenia species. The assay was validated on (a) a reference sample collection of 69 clinical and veterinary cases confirmed by imaging, serology, and morphological analysis, (b) 38 routine human patient samples confirmed for aforementioned pathogens by a conventional end-point PCR, and (c) 127 samples from patients with suspected echinococcosis that were submitted to our laboratory for diagnostic analysis.

Results: Compared to a conventional reference end-point PCR approach, the quadruplex real-time PCR exhibited a lower limit of detection in a serial dilution with 5-log dilutions for all three targets (2 log for E. multilocularis, 1 log for E. granulosus s.s., and 1 log for T. saginata). We were able to detect DNA from E. multilocularis, E. granulosus s.l. (E. granulosus s.s., E. canadensis, E. ortleppi, and E. felidis), a wide range of Taenia spp., as well as from non-echinococcal metacestodes such as Hydatigera taeniaformis, Hymenolepis spp., Versteria sp., and Spirometra erinaceieuropaei.

Discussion: We suggest that the presented real-time PCR method is a suitable tool to be routinely used in a clinical microbiology laboratory to rapidly detect and identify larval cestodiases in human tissue.

Keywords: Echinococcus granulosus sensu lato; Echinococcus multilocularis; Real-time polymerase chain reaction; Taeniaspp.