A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a ‘One Health’ context

Abstract

Studying the epidemiology of schistosomiasis—the most prevalent gastropod-borne human disease and an economic burden for the livestock industry—relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.

Development of a Molecular Snail Xenomonitoring Assay to Detect Schistosoma haematobium and Schistosoma bovis Infections in their Bulinus Snail Hosts

“…Here, we describe the development and application of a molecular xenomonitoring pipeline for the detection and differentiation of S. haematobium and S. bovis patent and non-patent infections in Bulinus freshwater snails, using three previously developed primers [27,31]. The MIX assay screens for Schistosoma and other trematode species, while also incorporating an internal control, in this case, gastropod DNA, an important feature for any molecular diagnostic assay.…”

“…Alignments of the Schistosoma-specific ITS primers (ITS2_Schisto_F and ITS2_Schisto_R [27]) showed 100% and 90% (2 mismatches) homology to S. haematobium and S. bovis, respectively, with no cross-reactivity to the Bulinus reference rDNA data. When paired with their opposing universal primers (ITS2_Schisto_F + ETTS1 or ITS2_Schisto_R + ETTS2), the amplicon sizes of 538 and 835 bp were predicted, respectively, for Schistosoma.…”

“…However, several assays have been developed for detecting trematode species in freshwater snails, including Fasciola spp. [22,[37][38][39][40][41][42][43][44], other wildlife trematode species [45], and medically important schistosome species-S. japonicum [46,47], S. mansoni [24,27,28,[48][49][50][51][52][53][54], and S. haematobium [23,26,27,30,52,54,55]. The first developed assay for the molecular detection of S. haematobium DNA in Bulinus employs the highly repetitive Dra1 target, and this has been the marker of choice for studies investigating S. haematobium infections in snails due to its high sensitivity [55].…”

“…Molecular xenomonitoring is a DNA-based method that has been developed to monitor the transmission of several vector-borne diseases, including trypanosomiasis [18,19], filariasis and malaria [20], helminthiases [21], and fascioliasis [22], including to some extent schistosomiasis [23][24][25][26][27][28][29]. Screening snails provides evidence on the extent of environmental contamination (i.e., schistosome miracidia penetrating snails), as well as environmental infection risk (i.e., schistosome sporocysts and cercariae developing inside the (pre-patent) snails, eventually emerging from the (patent) snail.…”

“…In the current study, we adapted available universal [31] and Schistosoma-specific [27] internal transcribed spacer (ITS) rDNA primers to design a three primer multiplex assay and tested this as a simple, robust, and rapid xenomonitoring PCR assay to enable the large-scale screening of Bulinus snails for Schistosoma infections (S. haematobium and S. bovis). We used a conventional PCR-based approach focused on simplicity, ease of data interpretation, sensitivity, and specificity, with a primary aim to provide a xenomonitoring tool for monitoring S. haematobium transmission in endemic settings.…”