SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.