Application of a Genus-Specific LAMP Assay for Schistosome Species to Detect Schistosoma haematobium x Schistosoma bovis Hybrids

Abstract

Schistosomiasis is a disease of great medical and veterinary importance in tropical and subtropical regions caused by different species of parasitic flatworms of the genus Schistosoma. The emergence of natural hybrids of schistosomes indicate the risk of possible infection to humans and their zoonotic potential, specifically for Schistosoma haematobium and S. bovis. Hybrid schistosomes have the potential to replace existing species, generate new resistances, pathologies and extending host ranges. Hybrids may also confuse the serological, molecular and parasitological diagnosis. Currently, LAMP technology based on detection of nucleic acids is used for detection of many agents, including schistosomes. Here, we evaluate our previously developed species-specific LAMP assays for S. haematobium, S. mansoni, S. bovis and also the genus-specific LAMP for the simultaneous detection of several Schistosoma species against both DNA from pure and, for the first time, S. haematobium x S. bovis hybrids. Proper operation was evaluated with DNA from hybrid schistosomes and with human urine samples artificially contaminated with parasites’ DNA. LAMP was performed with and without prior DNA extraction. The genus-specific LAMP properly amplified pure Schistosoma species and different S. haematobium-S. bovis hybrids with different sensitivity. The Schistosoma spp.-LAMP method is potentially adaptable for field diagnosis and disease surveillance in schistosomiasis endemic areas where human infections by schistosome hybrids are increasingly common.

Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis

“…Despite the tremendous efforts to prevent and control schistosomiasis, the helminthic disease still causes large morbidity and mortality in developing countries and beyond as seen by the occurrence of inter-species and/or inter-lineages schistosomal hybrids in Europe [3]. Efforts surely are impacted by misdiagnoses and underdiagnoses of infective subjects constituting sources of ongoing parasite transmission due to limitations of conventional diagnostics [2,7,[14][15][16]; their performance is influenced by disease status including atypical disease presentation [3,69], endemicity/co-endemicity levels, and chemotherapeutic treatment [1,17] A limitation of all investigations warranting future large-scale high-throughput research is the rather small sample size reaching a few hundred participants at the maximum [27]. Cross-reaction with other Schistosoma species and co-endemic helminths, protozoa, bacteria and viruses was rarely seen throughout.…”

“…To address the growing concerns of cross-species hybridization leading to large diversity of novel inter-species and/or inter-lineages, LAMP was investigated to detect hybrids originating from S. haematobium and the phylogenetic closely-related livestock species S. bovis [3,69]. Urine specimens from healthy donors were spiked with DNA of hybrids originating from Côte d'Ivoire and Corsica/France; hybrids were confirmed previously based on mitochondrial cytochrome c oxidase (COX) and nuclear ribosomal ITS profiles [3].…”

“…bovis x S. haematobium profiles. Hybrids likely retrieved from an initial crossing between male S. haematobium and female S. bovis leading to the introgression of S. bovis mitochondrial DNA into the S. haematobium genome [69].…”

Natural Intra- and Interclade Human Hybrid Schistosomes in Africa with Considerations on Prevention through Vaccination

“…Compared to free-living animals, parasites are often hidden and have limited morphological characters or other identifiable phenotypes, thus the frequency, and ecological, evolutionary importance of parasite hybridization is largely underestimated. However, schistosomes are known to hybridize and natural hybrids are more and more frequently found disorienting baseline laboratory and clinical diagnoses; see Figure 2 for likely natural schistosomal hybridization events across Africa [ 46 ]. The probability of introgressive hybridization with production of viable progeny or parthenogenesis depends on the phylogenetic distance of Schistosoma species, thus species of low distance within the same monophyletic clade are more likely to exchange their gene pools than species of high distance [ 5 , 44 , 47 ].…”

“…Therefore, articles included in this investigation were limited to hybrid confirmation by mitochondrial cytochrome c oxidase (COX) and/or nuclear ribosomal internal transcribed spacer (ITS), both commonly used in phylogeny and phylogeography, and due to assumed poor sensitivity of other confirmatory tests; see Figure 3 for more details [ 58 , 59 , 60 ]. ITS, as a genus-specific marker, distinguishes hybrids and their backcrossed progeny as parental sequences retain for generations before getting homogenized by concerted evolution; COX is a haploid-inherited mitochondrial marker for species-specific identification evolving more rapidly than nuclear genes [ 2 , 39 , 44 , 46 , 56 , 61 ]. Hybrids are detected either when the biparentally inherited ITS marker shows heterozygous pattern at specific mutation points and/or if ITS and COX markers give discordant results in species assignation.…”