Abstract
While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.
Highlights
Species of the Schistosoma japonicum complex with human pathogenic potential comprise S. mekongi and the closely related S. malayensis next to S. japonicum sensu stricto [1,2]
Due to the lack of sequence information in public databases corresponding to S. japonicum retrotransposons SjR2 [17] and SjCHGCS19 [19] in S. mekongi and S. malayenis, the genomes of the latter two species were partially sequenced
As there is considerable sequence variability of the different retrotransposon copies even within the same species, various primer combinations were introduced into multi-primer PCR assays in order to increase sensitivity when applied to DNA extracted from helminth material instead of plasmids which are usually used as positive controls
Summary
Species of the Schistosoma japonicum complex with human pathogenic potential comprise S. mekongi and the closely related S. malayensis next to S. japonicum sensu stricto [1,2]. For the African Schistosoma species, highly sensitive real-time PCRs targeting the multicopy sequences Sm1-7 for S. mansoni complex as well as Dra for S. haematobium complex in human serum have been developed [2,7,8,9,10] and well evaluated using sera from travel returnees [11,12] and individuals in endemic areas [13,14]. These assays provide highly sensitive tools for screening and facilitate the identification of both species complexes in parallel in human serum samples as a one-tube multiplex approach [12,14]. Its application for the diagnosis of early infections prior to seroconversion and prior to the excretion of eggs, as well as for the discrimination of active infections from old serological responses to infections that have been successfully cured long ago, is considered as well established [12,13,14,15,16]
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