A novel multiplex real-time PCR for the molecular diagnosis of metacestode infections in human patients.

Oberli, Alexander; Furrer, Lavinia; Skoko, Lena; Müller, Norbert; Gottstein, Bruno; Bittel, Pascal (2023). A novel multiplex real-time PCR for the molecular diagnosis of metacestode infections in human patients. (In Press). Clinical microbiology and infection Elsevier 10.1016/j.cmi.2023.07.032

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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 E. 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 (i) a reference sample collection of 69 clinical and veterinary cases confirmed by imaging, serology and morphological analysis, (ii) 38 routine human patient samples confirmed for aforementioned pathogens by a conventional end-point PCR and (iii) 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. but also non-echinococcal metacestodes such as Hydatigera taeniaformis, Hymenolepis spp., Versteria sp. and Spirometra erinaceieuropaei.

CONCLUSIONS

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.

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