A RAPID DIAGNOSTIC PCR ASSAY FOR THE DETECTION OF SCHISTOSOMA MANSONI IN THEIR SNAIL VECTORS

Schistosoma mansoni is 1 of the causative agents for intestinal schistosomiasis, a neglected tropical disease responsible for severe morbidity in regions of sub-Saharan Africa and South America. Humans become infected when contacting freshwater that contains cercariae released by snail intermediate hosts of the genus Biomphalaria. There is a critical need to accurately determine infection status of snails in endemic settings (World Health Organization, 2022), and likewise, accurate determination is important to laboratory experiments (Lu et al., 2022). Incorrect assessment of snail infection status could confound interpretation, thereby hindering biologic discovery of factors that dictate snail susceptibility to schistosome infection and potential downstream development of schistosome transmission interventions.

Detecting schistosome infections within snails typically relies on microscopy to detect the emergence of cercariae; however, the development time of schistosomes to reach patency is variable and can take up to 30 wk in some cases (Tavalire et al., 2016). Thus, many infections may be missed following routine microscopy simply because the parasites have not fully developed (Fig. 1A). In addition, increased mortality is sometimes observed in snails challenged with schistosomes whether they become infected or not, resulting in reduced sample sizes that lower power to estimate infection rates (Anderson and Crombie, 1984; Blair and Webster, 2007). One alternative is the early dissection of snails for the detection of sporocysts; however, this method is cumbersome and requires specialized skills, and even skilled microscopists may miss larval parasites several weeks postexposure (Lu et al., 2016). Thus, a reliable tool that assesses snail infection status at early time points of infection could reduce experimental time and increase power and accuracy.

For diagnosis of schistosome infection in snail vectors, several PCR-, LAMP- and RPA-based methods have been developed that use single schistosome DNA markers (Hanelt et al., 1997; Hamburger et al., 2004, 2013; Abbasi et al., 2010, 2017; Fernández-Soto et al., 2014; Lu et al., 2016; Gandasegui et al., 2016, 2018; Casotti et al., 2020; Mesquita et al., 2021, 2024). Although these methods have proven useful, they lack an internal control, meaning that no DNA amplification would be interpreted as schistosome negative but could be caused by a procedural error in sample processing or the presence of PCR inhibitors, which commonly occurs with snails (Adema, 2021). A multiplexed PCR assay containing both snail and Schistosoma-specific DNA markers can overcome this (Schols et al., 2019; Pennance et al., 2020; Archer et al., 2024), as can high-throughput amplicon sequencing (Hammoud et al., 2022). However, the requirement of conducting multiple PCRs or additional sequencing can be cumbersome, particularly with a limited set of snails and parasites. In addition, universal primers can be problematic due to nonspecific amplification of other microorganisms found in or on snails.

The main aim of this study was to develop a rapid diagnostic PCR assay for detecting S. mansoni infections in Biomphalaria sudanica and Biomphalaria glabrata as soon as the first week of infection (Fig. 1A). This test aimed to offer higher sensitivity and faster assessment of snail infections postexposure compared with traditional microscopy methods, while incorporating an internal PCR control and easily interpretable results (Fig. 1B).

Snail-targeted primers were manually designed using mitochondrial genomes for B. sudanica (NC_038060) and B. glabrata (AY380531.1 and AY380567.1; DeJong et al., 2004; Zhang et al., 2018). The designed Biomphalaria mitochondrial ND4 gene primers specifically target B. sudanica (Bsud_ND4_F: 5′-CCTGAACGTTTACAAGCAGG-3′, Bsud_ND4_R: 5′-AGTCTTCGACTACGAACTTT-3′) and B. glabrata (Bglab_ND4_F: 5′-CCTGAGCGATTGCAAGCAGG-3′, Bglab_ND4_R: 5′-AGTCTTCGCCTGCGGACTTT-3′) and produce a conserved amplicon size across species (596 base pairs [bp]). To optimize the multiplex capacity of the snail primers with the S. mansoni ND5 targeted primers (Lu et al., 2016), 62 B. sudanica snails of 4–6 mm in shell diameter (F1 offspring of snails isolated from Kisumu, Kenya) were exposed to 8 S. mansoni miracidia that were obtained from pooled fecal samples of 5 infected local school children. Eight weeks postexposure, 6 snails that shed cercariae, along with the remaining 56 negative snails (no cercariae observed), were then gently crushed and individually preserved in 100% molecular grade ethanol and stored at −80 C until DNA extraction. The DNA extraction followed a modified protocol for the Qiagen blood and tissue kit (Qiagen, Germantown, Maryland) in that tissue was lysed overnight at 37 C and elution of DNA was with a volume of 110 μl of elution buffer.

For the rapid diagnostic PCR assay, the Thermo Scientific™ Phire™ Green Hot Start II PCR Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts) was adopted, which uses short extension times and results in improved amplicon yields compared with other polymerases. A negative control sample (water) and a positive control sample (DNA extracted from a lab-reared B. sudanica snail that was spiked with S. mansoni DNA) were included in every PCR.

Each PCR was performed in 20-μl reactions: 10 μl of 2× Phire Green HS Master Mix; 1.5 μl of each snail and schistosome targeted primer (10 μM); 2 μl of water; and 2 μl of gDNA. The optimal volume and concentration of input gDNA for the PCR assay were determined using 6 different concentrations of 8 B. sudanica DNA extracts (DNA extract concentrations between 101 to 236 ng/μl; Suppl. File S1). Following the comparable PCR results of 2 μl volume of 1:10 and 1:100 dilution of input DNA, 1:10 was selected because it contains a relatively higher amount of S. mansoni DNA than a 1:100 dilution, while preserving B. sudanica ND4 band amplification that was lost in the full concentration input DNA (File S1). Thermocycling conditions followed the manufacturer’s instructions: 98 C for 30 sec; 25 cycles of 98 C for 5 sec; 60 C for 5 sec; 72 C for 15 sec; and final elongation of 72 C for 60 sec. The total time to complete the full PCR cycle on an Eppendorf® Mastercycler® Nexus Thermal Cycler (Eppendorf, Germany) is 43 min. Annealing temperatures were optimized by trialing 6 temperatures in a gradient thermocycler (52, 5, 54, 55, 56, 58, and 60 C; File S2). Consistent amplification of the B. sudanica ND4 fragment in both S. mansoni–positive and S. mansoni–negative samples was achieved using an annealing temperature of 60 C (File S2). The PCR amplicons (5 μl) were visualized on a 2% agarose, SYBR safe E-Gel™ (Thermo Fisher Scientific), run on a Mother E-Base (Thermo Fisher Scientific) for 20 min using the EG electrophoresis mode, according to manufacturer’s instructions. This optimized protocol was also used for B. glabrata snails using the alternative primer pairs.

One of the advantages of PCR diagnostics is detection of even small amounts of parasite DNA; however, this quality can be problematic if the PCR amplifies DNA remaining from parasites that are removed by the immune system (Théron et al., 1997; Lu et al., 2016). The presence of schistosome DNA in the absence of shedding cercariae can be interpreted broadly as the following: the remaining schistosome DNA from failed infections (Lu et al., 2016); developing larval schistosomes that have a longer prepatent period than the study assessment period (Tavalire et al., 2016); or latent infections, defined here as nondeveloping intramolluscan schistosome miracidia or sporocysts that are not completely killed or cleared (Théron et al., 1997), but could be resolved or reactivated later (see Fig. 1A). Dormancy of S. mansoni is known to occur during periods of snail estivation (Barbosa and Barbosa, 1958), and although snail immunity-driven long-term developmental stasis and eventual reactivation of miracidia or sporocysts is not documented in schistosomes, it has been documented for other trematodes (Southgate et al., 1989; Laidemitt et al., 2019). The term latent is used here in a similar manner that it is used for latent Mycobacterium tuberculosis infections in humans, in which latent pathogens are dormant but may become reactivated later.

Next, the prevalence of schistosome infections in exposed snail populations determined by either PCR within the first week of exposure or microscopy for patent infections after 14 wk postexposure was compared. For the snails assessed using microscopy (referred to as the holdout population), snails were assessed at 8 and 14 wk postexposure, and infection prevalence was calculated as in previous studies (Spaan et al., 2023) by dividing the total number of infected snails (8 and 14 wk postexposure) by the number of snails surviving to week 8, expressed as a percentage. We acknowledge that this methodology is an imperfect estimation of prevalence, because it does not account for the survivorship of snails up to 8 wk and between the 8- and 14-wk assessments. Thus, it could lead to an underestimation of prevalence in exposed snails if infected snails have a lower survivorship than uninfected snails, although previous studies suggest that this is likely not the case (Anderson and Crombie, 1984; Blair and Webster, 2007). For the experimental infections, snails were exposed to 8 miracidia of S. mansoni of the Naval Medical Research Institute (NMRI) line obtained from the Schistosomiasis Resource Center (Lewis et al., 2008). Snails included a compatible and incompatible line of B. sudanica, Bs110s1 (selected for increased susceptibility from original line 110) and Bs163 (Table I), respectively, that were originally collected from Lake Victoria, Kenya, and purposely inbred in the lab with 3 generations of selfing (Spaan et al., 2023). Snails also included a highly susceptible B. glabrata line (Bg151) that originated from Guadeloupe (Spaan et al., 2022). The PCR assay was used to assess S. mansoni presence in snails selected from the population at 4 and 7 days postexposure (DPE; File S3) and compared these prevalences to the holdout population assessed by microscopy. Contingency table analysis with 1-tailed Fisher exact probability tests comparing PCR vs. microscopy positive and negative snails were performed to assess statistical significance. The hypothesis was that the rapid diagnostic PCR assay would detect S. mansoni DNA in compatible snails (Bs110s1 and Bg151) at 4 and 7 DPE at a prevalence equivalent to that observed in the holdout populations and that no S. mansoni DNA at 4 and 7 DPE or patent snails in holdout populations would be identified in the incompatible snails (Bs163). As expected, incompatible Bs163 snails at 4 and 7 DPE showed no PCR S. mansoni–positive individuals and no microscopy-positive (holdout) individuals by 14 wk (Table I). The S. mansoni–susceptible B. sudanica and B. glabrata lines both showed higher prevalence via PCR than the respective holdout samples, although only the 7 DPE PCR prevalence for both snail lines was significantly higher (Table I). This statistical significance however is marginal, and lost in either, if just 1 of the 7 DPE PCR-positive B. sudanica or B. glabrata was instead negative. In addition, no significant difference in PCR-positive rates between 4 DPE and 7 DPE for B. sudanica or B. glabrata was observed (P = 0.070 and 0.500, respectively). The survivorship of exposed snails was 100% for snails assessed at 4 and 7 DPE, whereas by week 8 in holdout populations, survivorship was 65.5, 62.8, and 81.4% for Bs110s1, Bs163, and Bg151, respectively (File S4, File S1), resulting in a significant loss of samples.

Because PCR can detect small amounts of DNA, our rapid diagnostic PCR assay will detect S. mansoni DNA from parasites at early time points postexposure that do not result in established infections (i.e., see the routes to failed infection, Fig. 1A). To determine if the rapid diagnostic assay could detect such small amounts of S. mansoni DNA at these early time points, when snails are still clearing infection, 4–4.9 mm B. sudanica snails of the B. sudanica Kenya Medical Research Institute (BsKEMRI; the line originating from Lake Victoria, Kenya, in 2010 and maintained in captivity since; see Spaan et al., 2023) were exposed to 1 of 2 lines of S. mansoni. These S. mansoni lines included the NMRI line, which is incompatible with the BsKEMRI snail line and the University New Mexico–Kenya (UNMKenya) line, which is ∼50% compatible with the BsKEMRI snail line (Spaan et al., 2023). Snails were exposed to miracidia for either 0.5- or 3-hr, and a subset of each group was used for the rapid PCR diagnostic, while the remainder were diagnosed by examining them for shedding via microscopy after 14 wk postexposure (Table II). An additional postexposure group (24 hr) was included for the compatible combination (Table II). The rapid PCR diagnostic detected a significantly higher proportion of S. mansoni DNA in all compatible and incompatible snail–schistosome combinations ≤24 hr postexposure than holdout snails observed to have patent infections (Table II).

Overall, these data indicate that (1) the rapid diagnostic PCR assay is a quick and effective way to detect true S. mansoni infections in Biomphalaria sp. by 4 DPE and (2) that S. mansoni DNA-based assays will overestimate the number S. mansoni infections that will become established and underestimate the number of failed infections in snails during the first 24 hr of infection. This trait of parasite clearance by 4 DPE could be variable among other combinations of snails and schistosomes; therefore, optimization is recommended to minimize both mortality (as is the issue for detecting patent infections using microscopy) and the probability of detecting DNA from parasites that are undergoing immune clearance (Fig. 1A).

For applying this assay for xenomonitoring purposes for Biomphalaria collected from Lake Victoria as in previous studies (Archer et al., 2024), the primers designed for B. sudanica likely will work for its sister taxon and morphospecies, Biomphalaria choanomphala, a snail endemic to the deeper waters of Lake Victoria that also plays a role in the transmission of S. mansoni (Gouvras et al., 2017; Mutuku et al., 2021). The mitogenomes of B. sudanica and B. choanomphala share over 98% nucleotide identity, including in the ND4 gene region, where the nucleotide sequence in the primer binding regions are identical, although 10 SNPs are present in the amplified region, differentiating the reference sequences used for each species (Zhang et al., 2018). As for Biomphalaria pfeifferi, another African Biomphalaria species, 2 and 3 SNPs are present in the forward and reverse primer binding regions, respectively, and as such, modified primers may be needed to optimize this PCR diagnostic for this species. Second, the S. mansoni ND5 primers may also amplify certain geographic isolates of Schistosoma rodhaini, a rodent parasite that also develops within species of Biomphalaria; however, previous studies demonstrate that S. rodhaini produces an ∼800-bp amplicon distinguishable on the agarose gel from the 302-bp S. mansoni (Lu et al., 2016) or may not produce any amplicon (Archer et al., 2024).

In conclusion, this rapid diagnostic PCR assay to determine S. mansoni infections in B. sudanica and B. glabrata snails provides a quick and effective tool for determining infection status of Biomphalaria at least 4 days following exposure. The assay provides reassurance over singleplex reactions because the internal control (snail-targeted DNA amplicon) accounts for extraction and PCR success, such as comparable PCR assays (Schols et al., 2019; Pennance et al., 2020; Archer et al., 2024). Furthermore, this assay can provide results within ∼1.5 hr, improving the ∼2.5 hr taken for the original singleplex S. mansoni ND5 (Lu et al., 2016) and other methods (Schols et al., 2019; Archer et al., 2024). This will be beneficial both in the laboratory, where snail infection status can be incorporated easily into other methods, and in the field, where thermocyclers are available and E-Gel™ systems can be easily transported. The choice of diagnostic methods used will be dependent on the study design (including room for inherent error), intended goals, and budget. During xenomonitoring and surveillance for elimination, any evidence of schistosomes in the environment is valuable information for control program managers; therefore, this PCR assay could be highly beneficial. Within laboratory experiments, if one wants to compare infection prevalence among treatments, PCR diagnosis could be a more powerful study design than microscopy, especially if survivorship is likely to be low, and development times are lengthy. However, if one wants to better distinguish the specific infection status of individual snails, a combination of microscopy and PCR may be best to distinguish patent and prepatent or latent infections (Fig. 1B). The additional costs of molecular PCR assays in comparison to those needed for classical microscopy methods need to be considered in study design but are certainly imperative in making accurate epidemiologic and genetic inferences.

Biomphalaria sudanica snails used in this study to optimize primers and the University New Mexico–Kenya schistosome miracidia used to challenge snails, all originated from Kenya and were originally collected following approval from Kenya Medical Research Institute (KEMRI) Scientific Review Unit (permit KEMRI/RES/7/3/1), Kenya’s National Commission for Science, Technology, and Innovation (permit NACOSTI/P/22/148/39 and NACOSTI/P/15/9609/4270), Kenya Wildlife Services (permit WRTI-0136-02-22 and 0004754), and the National Environment, Management Authority (permit NEMA/AGR/159/2022 and NEMA/AGR/46/2014; registration 0178). Informed consent was obtained from the parents of 5 children who were positive for Schistosoma mansoni from fecal samples using egg microscopy, of which the remaining samples were used for S. mansoni miracidial hatching. All 5 children were treated with praziquantel following diagnosis.

Funding for this project was provided by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Disease (NIAID; R01AI141862). The Naval Medical Research Institute schistosomes were provided by the NIAID Schistosomiasis Resource Center of the Biotechnological Research Institute (Rockville, Maryland) through NIH-NIAID (contract HHSN272201700014I).