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. 2022 Jul 26;16(7):e0010632. doi: 10.1371/journal.pntd.0010632

Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS

Alexander James Webb 1,‡, Fiona Allan 2,‡, Richard J R Kelwick 1,‡, Feleke Zewge Beshah 3, Safari Methusela Kinung’hi 4, Michael R Templeton 5, Aidan Mark Emery 2,*, Paul S Freemont 1,6,7,*
Editor: Neil D Young8
PMCID: PMC9355235  PMID: 35881651

Abstract

Schistosomiasis, also known as bilharzia or snail fever, is a debilitating neglected tropical disease (NTD), caused by parasitic trematode flatworms of the genus Schistosoma, that has an annual mortality rate of 280,000 people in sub-Saharan Africa alone. Schistosomiasis is transmitted via contact with water bodies that are home to the intermediate host snail which shed the infective cercariae into the water. Schistosome lifecycles are complex, and while not all schistosome species cause human disease, endemic regions also typically feature animal-infecting schistosomes that can have broader economic and/or food security implications. Therefore, the development of species-specific Schistosoma detection technologies may help to inform evidence-based local environmental, food security and health systems policy making. Crucially, schistosomiasis disproportionally affects low- and middle-income (LMIC) countries and for that reason, environmental screening of water bodies for schistosomes may aid with the targeting of water, sanitation, and hygiene (WASH) interventions and preventive chemotherapy to regions at highest risk of schistosomiasis transmission, and to monitor the effectiveness of such interventions at reducing the risk over time. To this end, we developed a DNA-based biosensor termed Specific Nucleic AcId Ligation for the detection of Schistosomes or ‘SNAILS’. Here we show that ‘SNAILS’ enables species-specific detection from genomic DNA (gDNA) samples that were collected from the field in endemic areas.

Author summary

Schistosomiasis is a neglected tropical disease, caused by the parasitic trematodes of the genus Schistosoma. Schistosomiasis is endemic to regions within Africa, Asia and South America with at least 250 million people infected and a further 779 million at risk of infection. The lifecycle of schistosomes are complex and involve specific freshwater intermediate snail hosts which shed infective cercariae into the waterbodies they inhabit. Schistosomiasis is subsequently transmitted to humans or animals that contact cercariae contaminated water. In Africa, human disease is largely caused by Schistosoma mansoni and Schistosoma haematobium. However, endemic regions also typically feature animal-infecting schistosomes that can have broader economic and/or food security implications. Therefore, the development of species-specific Schistosoma detection technologies may help to inform local environmental, food security and health programmes. To this end, we re-purposed a nucleic acid detection technology to enable the detection of different schistosome species. Our DNA-biosensor, abbreviated as ‘SNAILS’, employs carefully designed probes that recognise species-specific DNA sequences, coupled with enzymatic amplification steps, and a fluorescent signal-dye to indicate a positive detection. ‘SNAILS’ successfully differentiates between S. mansoni and S. haematobium samples and could conceivably be employed within future global health programmes.

Introduction

Schistosomiasis is a neglected tropical disease (NTD), caused by parasitic flatworm trematodes of the genus Schistosoma [1]. Worldwide, over 250 million people are infected with a further 779 million people at risk of infection, and an annual mortality thought to be 280,000 people in sub-Saharan Africa alone [2–4]. As such, it continues to be a considerable cause of concern to global health, particularly in low- and middle-income (LMIC) countries. Indeed, it has been described as second only to malaria worldwide in terms of human suffering caused by tropical disease [1]. As well as Africa, schistosomes are found in the Arabian Peninsula, South America, China, the Philippines and Indonesia, and more recently southern Europe (Corsica) [5,6]. ‘Schistosomiasis’ is something of a blanket term covering a series of related but distinct diseases, caused by several different species of schistosome, and not all species of schistosome cause human disease. Whilst causing considerable damage to human health, schistosomiasis also causes considerable harm to cattle and other livestock, thus negatively impacting on local economies and food security. However, different combinations of human, veterinary and wildlife infective schistosomes can co-exist, giving rise to a highly complex and dynamic picture, thereby complicating their detection [7]. Therefore, the ability to rapidly identify species of schistosomes relevant to human health, as well as differentiate them from related sympatric schistosome species is highly desirable. In Africa, the human disease can be broadly differentiated into intestinal schistosomiasis (predominantly caused by Schistosoma mansoni) and urogenital schistosomiasis (caused by Schistosoma haematobium) [3]. It is important to also be able to differentiate between these two species, particularly as there is data suggesting that S. mansoni and S. haematobium require different treatment regimens [8].

The main control intervention for schistosomiasis for the past 20 years has been preventive chemotherapy via the mass drug administration (MDA) of praziquantel. Other interventions within the control toolbox have recently become more popular for example, water, sanitation and hygiene (WASH) interventions (including behavioural change) and snail control to reduce schistosomiasis transmission [9]. However, this disease disproportionally affects LMIC countries, which may have inadequate infrastructure for WASH provisions and other preventive measures [4,9]. The presence, and persistence, of this disease has a direct impact on the realisation and application of some of the UN Sustainable Development Goals; goal 3 “Good health and well-being: ensure healthy lives and promote wellbeing for all at all ages”, and goal 6 “Clean water and sanitation: clean accessible water for all” [10].

As the main control intervention for schistosomiasis has been preventive chemotherapy via MDA, most diagnostics available have focused on the detection of the parasites in human and to a lesser degree, veterinary relevant animal populations. However, human or veterinary parasitological surveys can take time and to progress towards elimination it may desirable to also detect transmission in less traditional ways, such as using environmental DNA (eDNA) in water or soil, in order to gain a more complete perspective on schistosomiasis transmission. This study, as part of the Water Infrastructure for Schistosomiasis-Endemic Regions (WISER) project, has therefore, centred on a water and environmental monitoring approach for the detection of schistosomes. Such environmental surveillance data may ultimately inform local environmental, food security and health-based policy making [11] and aid in the realisation of the UN Sustainable Development Goals. Standard snail surveys (collecting snails and looking for infection) [12] and water sampling (looking for free swimming cercariae) [13], which can also include the use of natural oil laced traps [14], have been undertaken. These techniques, though, are labour intensive and lack specificity, as many species of cercariae are morphologically similar. The use of quantifiable mouse bioassays, where the mice are in a cage and dipped into water, so that their tails, paws and portions of the lower abdomen are wetted in either in situ water samples or pre-collected water samples, have also been employed [15]. The draw-back of mouse bioassays is the considerable time lag between infection and observation of possible infection, which requires the killing and dissection of the mice and then the counting of any adult schistosomes present. In regards to snail surveys, if snails collected in situ show no visible signs of infection i.e. shedding cercariae, they can be further analysed for prepatency (the presence of Schistosoma sporocysts), whereby the snails are crushed and analysed microscopically [16] and/or via molecular techniques such as using PCR-based methods [17–19] or loop-mediated isothermal amplification (LAMP) assays [20,21]. Furthermore, the detection of S. mansoni via the proteolytic activity of cercarial elastase, an enzyme it uses to invade thedefinitive host [22,23], on whole-cell bioreporters (WCBs) [24] or functionalised polyhydroxyalkanoates (PHAs)-based bioplastic beads [25] has also been tested. However, the use of WCBs within global health settings is challenging with many complex practical, societal, cultural, regulatory and data protection concerns needing to be addressed [26].

Recently, molecular approaches have emerged that involve the analysis of parasite-specific eDNA using water or soil samples [27,28]. These collection methods are relatively straightforward, and these samples can be analysed for the presence of schistosomes using a range of molecular techniques including polymerase chain reaction (PCR) [29,30], real-time PCR (RT-PCR) [31–33], multiplex RT-PCR [34], DNA sequencing [35], oligochromatographic dipsticks [33], LAMP assays [20,21] and a recombinase polymerase amplification fluorescence assay (RPA)[36]. These techniques such as RT-PCR, would, typically, also be followed by a DNA sequencing step for validation and to enable species level identification. Although DNA sequencing is a useful tool, it is however, expensive, and furthermore, the probes used for techniques such as RT-PCR may not be able to differentiate very small changes in target DNA sequences. More recent nucleic acid detection technologies, including SHERLOCK [37] and INSPECTR [38], have undergone accelerated development cycles towards viral diagnostic applications. However, to the best of our knowledge these technologies have not yet been tested for the detection of Schistosoma [39]. Therefore, we developed and validated a species-specific DNA-based biosensor for detecting schistosomes via small changes in DNA sequences over a short target length (22 bases). We termed our novel DNA target-specific biosensor: Specific Nucleic AcId Ligation for the detection of Schistosomes or ‘SNAILS’. Here, we report that the probes designed for S. mansoni and S. haematobium are specific to their respective species, using synthetic single stranded DNA (ssDNA) targets as well as adult worm genomic DNA (gDNA) from samples isolated in schistosomiasis-endemic areas.

Results

‘SNAILS’ DNA-based biosensor development and target identification

The ‘SNAILS’ DNA ligation-based biosensor system described here is adapted from a light-up ‘Spinach’ aptamer-based biosensor for the detection of micro RNAs (miRNA) [40], which has recently been employed to detect SARS-CoV-2 in clinical samples [41]. Although multiple miRNAs have been identified in and are released by schistosomes at different stages of their life cycle [42–44], we decided that due to potential abundancy, lifespan and stability issues with miRNA samples taken from study sites, we would target single-stranded genomic DNA (ssDNA). As such the biosensor design was revised and optimised for ssDNA detection. The biosensor comprises two probes, A and B, that each recognise a different half (11 bases) of a 22-base target region (Fig 1A). Probe A is phosphorylated at the 5’ end which also encompasses the 11-base target-complementary region, whilst the target-complementary region of probe B encompasses the 3’ hydroxylated (-OH) end (S1A Table). If the two probes recognise a ssDNA target, they will anneal to their specific target sequence. Addition of T4 DNA ligase will only catalyse the formation of a covalent bond between the 5’ and 3’ends of the two probes, if they specifically recognise and anneal to the target sequence. Probe A also encodes a 3’ T7 promoter, which after successful ligation with Probe B and addition of T7 RNA polymerase will result in transcription of the ‘Spinach’ aptamer and fluorogen binding enabling a real-time fluorescence measurement (Fig 1A).

Fig 1. “SNAILS” biosensor workflow and validation.

Fig 1

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a, Workflow of the ‘SNAILS’ biosensors assay. b, Validation workflow followed for the ‘SNAILS’ assay. c, ‘SNAILS’ biosensor sensitivity. Sensitivity of S. mansoni probe set 1 (SM_A1/SM_B1) against a range of concentrations of the 22-base S. mansoni target (SM_WT, AJW791). Probe concentrations were set to 200 nM of each half probe. n = 12 (4 replicates per reaction, each reaction split into triplicate runs). d, Optimisation of probe concentration. Concentration of probes required to detect 50 nM of S. mansoni 22-base target (SM_WT, AJW791). n = 9 (3 replicates per reaction, each reaction split into triplicate runs). Error bars denote standard error of the mean. The sequences of the probes and target are available in S1A Table.

For this proof-of-concept study, we targeted the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene of S. mansoni [45] and S. haematobium [46]. Representative cox1 sequences from S. mansoni and S. haematobium, along with those of the related species Schistosoma bovis, Schistosoma guineensis, Schistosoma curassoni, Schistosoma rodhaini, Schistosoma japonicum, Schistosoma malayensis and Schistosoma mekongi (S1B Table) were aligned using MUSCLE [47] to identify 22-base target regions that would differentiate between species (S1A). In order to decrease the likelihood of false positive or off-target detection, the probes were designed to maximise sequence divergence at the target region and in particular around the ligation junction (Figs 1A & 2A).

Fig 2. Biosensor ligation junction and S. mansoni probe set specificity.

Fig 2

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a, DNA targets tested. SM_WT is the target region probes SM_A1 and SM_B1 are designed to recognise. SM_B to SM_S have single, double or multiple base changes from that of SM_WT as indicated in red. The ligation junction for the probes is underlined. The corresponding target region for the other related Schistosoma species are indicated as follows: SR_WT (Schistosoma rodhaini), SH_WT (Schistosoma haematobium), SB_WT (Schistosoma bovis), SG_WT (Schistosoma guineensis), SC_WT (Schistosoma curassoni), SJ_WT (Schistosoma japonicum) and Smek_WT (Schistosoma mekongi). b, S. mansoni probes are specific for the S. mansoni wild-type target region. Both half probes and the target concentrations were tested at 50 nM. n = 9 (3 replicates per reaction, each reaction split into triplicate runs). Error bars denote standard error of the mean. The sequences of the probes are available in S1A Table.

Validation of the ‘SNAILS’ biosensor workflow, biosensor optimisation and specificity of the Schistosoma probes

To optimise the ‘SNAILS’ biosensor assay, a validation workflow was designed (Fig 1B). This workflow enabled the optimum target (Fig 1C; S1B; S1 Data) and probe concentrations (Fig 1D; S1C; S1 Data) to be identified as 50 nM of each. To test the specificity of the S. mansoni-specific probes, a range of single, double or multiple base changes were made to the S. mansoni wild-type target region (SM_WT), as indicated in red capitals (SM_B to SM_S; Fig 2A; S1A Table). The probes were specific for detecting the S. mansoni wild-type target (SM_WT), although there was some, albeit very low, signal detection for targets SM_C to SM_G, which only have 1-base difference from SM_WT (Fig 2A and 2B; S1D; S1 Data). However, when these S. mansoni-specific probes were tested against the corresponding target region of S. haematobium (SH_WT), S. rodhaini (SR_WT), S. bovis (SB_WT), S. guineensis (SG_WT), S. curassoni (SC_WT), S. japonicum (SJ_WT) and S. mekongi (Smek_WT), no signal was detected indicating that the probes were unable to recognise the corresponding target region of these related species (Fig 2A and 2B; S1D; S1 Data).

Probes were also designed to recognise and detect the corresponding region of cox1 in S. haematobium (SH_A1 and SH_B1; S1A Table). The probes were specific for S. haematobium (Fig 3A and 3B; S1E; S1 Data), though there was an off-target response to the S. curassoni target sequence (Fig 3B). However, this output is markedly less and equated to only ~8.9% of the output observed for the S. haematobium target sequence. These data, therefore, validated the general ‘SNAILS’ biosensor assay.

Fig 3. S. haematobium probe set specificity.

Fig 3

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a, DNA targets tested against S. haematobium probes. SH_WT is the target region that probes SH_A1 and SH_B1 are designed to recognise. The corresponding target region for the other related Schistosoma species are indicated as follows: SM_WT (S. mansoni), SR_WT (S. rodhaini), SB_WT (S. bovis), SG_WT (S. guineensis), SC_WT (S. curassoni), SJ_WT (S. japonicum) and Smek_WT (S. mekongi) with sequence variations indicated in red. The ligation junction for the probes is underlined. b, S. haematobium probes are specific for the S. haematobium wild-type target region. Both half probes and the target concentrations were tested at 50 nM. n = 9 (3 replicates per reaction, each reaction split into triplicate runs). Error bars denote standard error of the mean. The sequences of the probes are available in S1A Table.

Validation of biosensor detection of purified target ssDNA

To test real-world parasitic gDNA and eDNA samples, the ‘SNAILS’ assay requires a PCR amplification step followed by an enzymatic step to produce a pool of ssDNA targets (Figs 1B & 4A). In this study, we chose to amplify a 180-base target region to validate the probes for S. mansoni and S. haematobium (S1A Fig). To assess whether the probes could recognise and bind to targets in 180-base long regions, the probes were tested against varying amounts (ng) of their respective synthetic 180-base long ssDNA targets (SM_180 and SH_180; S1A Table) and were able to bind resulting in detectable outputs (Fig 4B and 4C; S1F, S1G; S1 Data). We tested target amounts (ng) as real-world samples may have a range of nucleotide changes which could affect accurate molarity calculations. From the data (Fig 4B and 4C), 30 ng of target ssDNA appeared to be adequate for future ‘SNAILS’ assays.

Fig 4. SNAILS biosensor probe validation with oligonucleotide or PCR amplified ssDNA targets.

Fig 4

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a, Schematic of an extended ‘SNAILS’ biosensor workflow showing the extra steps required to amplify the target and obtain purified ssDNA. Purified DNA (plasmid or adult worm gDNA) is amplified via PCR. PCR products are validated via agarose gel electrophoresis and then treated with lambda exonuclease to remove the phosphorylated 3’ DNA strand. The resultant ssDNA is purified and then subsequently incubated with relevant probes in ‘SNAILS’ reactions, as described in Fig 1A. b, S. mansoni oligonucleotide target. S. mansoni probes recognise and bind to a synthetic 180-base S. mansoni target. 50 nM of each probe half (SM_A1/SM_B1) were incubated with a range of different amounts (ng) of the SM_180 target oligonucleotide (AJW874). c, S. haematobium oligonucleotide target. S. haematobium probes recognise and bind to a synthetic 180-base S. haematobium target. 50 nM of each probe half (SH_A1/SH_B1) were incubated with a range of different amounts (ng) of the SH_180 target oligonucleotide (AJW875). For both b & c, n = 9 (3 replicates per reaction, each reaction split into triplicate runs). d, S. mansoni PCR-derived target. e, S. haematobium PCR-derived target. d & e, S. mansoni and S. haematobium probes ability (respectively) to bind to ssDNA derived from plasmid DNA. The 180-base target regions were amplified from plasmids pAJW250 (S. mansoni insert DNA) and pAJW251 (S. haematobium insert DNA) and treated as described in Fig 4A to produce purified ssDNA. Six PCR reactions per plasmid sample were tested against both probe pairs. 50 nM of each probe half was incubated with 30 ng of target ssDNA. As controls, both probe pairs were also incubated against 30 ng of the synthetic targets SM_180 and SH_180. Reactions are identified as follows: pSM_SM1-6 (S. mansoni probes against S. mansoni plasmid derived ssDNA), pSM_SM+ (S. mansoni probes against S. mansoni synthetic target), pSM_SH1-6 (S. mansoni probes against S. haematobium plasmid derived ssDNA), pSM_SH+ (S. mansoni probes against S. haematobium synthetic target), pSH_SM1-6 (S. haematobium probes against S. mansoni plasmid derived ssDNA), pSH_SM+ (S. haematobium probes against S. mansoni synthetic target), pSH_SH1-6 (S. haematobium probes against S. haematobium plasmid derived ssDNA), pSH_SH+ (S. haematobium probes against S. haematobium synthetic target). n = 3 (each reaction split into triplicate runs). Error bars denote standard error of the mean. The sequences of the probes and targets are available in S1A Table.

To analyse the ‘SNAILS’ Schistosoma probes ability to recognise and anneal to PCR-derived 180-base long ssDNA samples (Figs 1B & 4A), we required S. mansoni- and S. haematobium-derived control DNA. We, therefore, amplified cox1 from the gDNA of S. mansoni adult worm 1 (SM_AW1) and S. haematobium adult worm 1 (SH_AW1) and cloned the products into pCR-Blunt plasmids (pAJW250 and pAJW251; S1C Table). After sequence verification (S1D Table), we then amplified the 180-base target regions. The 3’ primers are phosphorylated, enabling the removal of the 3’ DNA strand via lambda exonuclease, leaving 180-base long ssDNA target fragments (Fig 1B & 4A). The S. haematobium-specific probes only detected the S. haematobium ssDNA samples, with no detection observed for the S. mansoni ssDNA samples (Fig 4E; S1J; S1 Data). However, the S. mansoni-specific probes compared to the respective positive control only, showed low detectable signals (an average of ~15.6% of that observed for the positive control) suggesting they were unable to sufficiently anneal to the PCR product (Fig 4D; S1I; S1 Data).

Validation of alternative S. mansoni-specific targets and probes

Further analysis of the S. mansoni 180-base target region identified three new targets (S1K Fig). New probe pairs (S1A Table) were designed and validated against the 180-base ssDNA samples tested previously (Fig 4D & 4E), as well as the 180-base control oligonucleotides. S. mansoni probe sets 2, 3 and 5 successfully detected only S. mansoni-specific ssDNA samples, with no fluorescence output detected for the S. haematobium-specific samples (Fig 5A, 5B & 5C; S1L; S1 Data).

Fig 5.

Fig 5

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Validation of re-iterated S. mansoni-specific probes. a, b & c, Re-iterated S. mansoni-specific probe set designs 2, 3 and 5 respectively, only recognised and bound to S. mansoni-specific target ssDNA. 50 nM of each probe half was incubated with 30 ng of the purified ssDNA samples used in Fig 4D & 4E. Reactions are identified as follows: pSM_SM1-6 (S. mansoni probes against S. mansoni plasmid derived ssDNA), pSM_SM+ (S. mansoni probes against S. mansoni synthetic target), pSM_SH1-6 (S. mansoni probes against S. haematobium plasmid derived ssDNA), pSM_SH+ (S. mansoni probes against S. haematobium synthetic target). n = 3 (each reaction split into triplicate runs). d, e & f, S. mansoni probe sets 2, 3 and 5 respectively, were tested for their ability to recognise the corresponding 22-base target regions from other Schistosomes, which are indicated as follows: SM_WT2, SM_WT3 and SM_WT5 (S. mansoni), SR_WT2, SR_WT3 and SR_WT5 (S. rodhaini), SH_WT2, SH_WT3 and SH_WT5 (S. haematobium), SG_WT2a and SG_WT3a (S. guineensis AJ519523), SG_WT2b and SG_WT3b (S. guineensis AJ519522), SC_WT2 and SC_WT3 (S. curassoni), SB_WT2 (S. bovis), SB_WT3a (S. bovis MH647124), SB_WT3b (S. bovis FJ897160), SGCB_WT_5 (target sequence for S. guineensis, S. curassoni, and S. bovis), SJ_WT2, SJ_WT3 and SJ_WT5 (S. japonicum) and Smek_WT2, Smek_WT3 and Smek_WT5 (S. mekongi). Probe sets and the target concentrations were tested at 50 nM. n = 9 (3 replicates per reaction, each reaction split into triplicate runs). a, b, c, d, e &f, Error bars denote standard error of the mean. g, h & i, DNA targets tested against S. mansoni probes 2 (g), 3 (h) and 5 (i). Sequence variations with respect to the corresponding target sequence for the S. mansoni probes are indicated in red. The ligation junction for the probes is underlined. The sequences of the probes are available in S1A Table.

We then further analysed the probes for their specificity (Fig 5G, 5H & 5I; S1A Table). Probe sets 2 and 3 were able to recognise corresponding target regions of S. rodhaini (Fig 5D & 5E; S1M, S1N; S1 Data). However, probe set 5 was specific for S. mansoni (Fig 5F; S1O Fig; S1 Data). Therefore, S. mansoni-specific probe set 5 and S. haematobium-specific probe set 1 were further tested on species-specific ssDNA samples obtained from adult worm gDNA.

Probes can differentiate ssDNA targets from adult schistosome worms

gDNA samples from S. mansoni and S. haematobium adult worms 1 and 2 were PCR amplified to generate 180-base target regions (Fig 6A & 6B; S1E Table). The target amplified from S. haematobium adult worm 1 gDNA (SH_AW1) had 2 base changes in the target region of S. mansoni probe set 5, one of which made it more similar to the S. mansoni sequence (Fig 6A; S1E Table). The sequences of the target regions for the other samples were as expected (Fig 6A & 6B; S1E Table). After generating ssDNA target samples, S. mansoni-specific probe set 5 and S. haematobium-specific probe set 1 were able to detect and differentiate between the target samples (Fig 6C & 6D; S1P, S1Q; S1 Data). There was some limited off-target detection for both probe sets (Fig 6C & 6D). However, we observed that the signal obtained for off-target probe detection was less than obtained for a true positive detection.

Fig 6. Species specific detection of S. mansoni and S. haematobium from gDNA derived biological samples.

Fig 6

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a, S. mansoni target region and b, S. haematobium target region and the corresponding regions from the four adult worm samples tested: S. mansoni adult worms 1 and 2 (SM_AW1 and SM_AW2 respectively) and S. haematobium adult worms 1 and 2 (SH_AW1 and SH_AW2 respectively). The differences between the target region and the corresponding regions of the adult worms are indicated in red. The probe ligation junctions are underlined. c, S. mansoni probe set 5 can differentiate between ssDNA derived from adult worm gDNA. 50 nM of each probe half was incubated with 30 ng of purified ssDNA. Reactions are identified as follows: SM_A1-6 (S. mansoni adult worm 1 derived ssDNA samples), SM_B1-6 (S. mansoni adult worm 2 derived ssDNA samples), SH_A1-6 (S. haematobium adult worm 1 derived ssDNA samples), SH_B1-6 (S. haematobium adult worm 2 derived ssDNA samples). n = 3 (each reaction split into triplicate runs). d, S. haematobium probe set 1 can differentiate between ssDNA derived from adult worm gDNA. 50 nM of each probe half was incubated with 30 ng of purified ssDNA. Reactions are identified as follows: SM_A1-6 (S. mansoni adult worm 1 derived ssDNA samples), SM_B1-6 (S. mansoni adult worm 2 derived ssDNA samples), SH_A1-6 (S. haematobium adult worm 1 derived ssDNA samples), SH_B1-6 (S. haematobium adult worm 2 derived ssDNA samples). n = 3 (each reaction split into triplicate runs). e, intraspecies sequence variation of the S. mansoni target region. f, intraspecies sequence variation of the S. haematobium target region. e & f, nucleotide sequences used to identify sequence variation subsets are listed in S1F and S1G Table. g, S. mansoni probe set 5 detection of ssDNA derived from gDNA of S. mansoni isolated from disparate geographical locations. 50 nM of each probe half was incubated with 30 ng of purified ssDNA. Reactions are identified as follows: SM_C1-3 (S. mansoni 2740), SM_D1-3 (S. mansoni 3112), SM_E1-3 (S. mansoni 4209), SM_F1-3 (S. mansoni N1g274), SM_G1-3 (S. mansoni 4464–2), SM_H1-3 (S. mansoni 4464–4). h, S. haematobium probe set 1 detection of ssDNA derived from gDNA of S. mansoni isolated from disparate geographical locations. 50 nM of each probe half was incubated with 30 ng of purified ssDNA. Reactions are identified as follows: SM_C1-3 (S. mansoni 2740), SM_D1-3 (S. mansoni 3112), SM_E1-3 (S. mansoni 4209), SM_F1-3 (S. mansoni N1g274), SM_G1-3 (S. mansoni 4464–2), SM_H1-3 (S. mansoni 4464–4). n = 3 (each reaction split into triplicate runs). Error bars denote standard error of the mean. The sequences of the probes are available in S1A Table.

It has been established that there is natural intraspecies sequence variation in schistosomes [48]. We, therefore, analysed the target sequences of a range of available S. mansoni and S. haematobium isolates (Fig 6E & 6F; S1F and S1G Table). As well as the wild-type target sequence, that is the specific sequence the probes were designed to target, we identified 6 target sequence subsets for S. mansoni and 3 target sequence subsets for S. haematobium (Fig 6E & 6F; S1F and S1G Table). Next, we tested the ability of the S. mansoni-specific probe set 5 and the S. haematobium-specific probe set 1 to detect 6 more S. mansoni gDNA samples isolated from disparate geographical locations. S. mansoni-specific probe set 5 was able to successfully detect 3 of these samples (worm SM_C: S. mansoni 2740 isolated from Kenya, worm SM_D: S. mansoni 3112 isolated from Senegal, and worm SM_E: S. mansoni 4209 isolated from Oman; Fig 6 G; S1R; S1 Data). S. haematobium-specific probe set 1 had an off-target response to S. mansoni worm SM_D isolated from Senegal (Fig 6 H; S1S; S1 Data), however this output signal equated to only ~9.1% of the output observed for S. mansoni-specific probe set 5 for the same target sample. The variations in the target sequences for these further S. mansoni worm gDNA samples are highlighted in S1E Table.

For the probes tested in this study (Fig 6C, 6D, 6G & 6H) we observed that a fluorescence cut-off value (Δ fluorescence of ~4500 a.u. per hour) as well as using the different species-specific probe sets in tandem, allowed differentiation of true positives and off-target positives, thereby enabling the SNAILS protocol to discriminate ssDNA targets from different Schistosoma species (S1P, S1Q, S1R & S1S; S1 Data).

Discussion

Schistosomiasis is a neglected tropical disease that is endemic to regions within Africa, Asia and South America, and is of an increasing burden to global health in that at least 250 million people are infected and a further 779 million at risk of infection [2]. In Africa, human disease is largely caused by S. mansoni and S. haematobium. However, endemic regions also typically feature animal-infecting schistosomes that can have broader economic and/or food security implications [7]. Therefore, the development of species-specific Schistosoma detection technologies may help to inform local environmental, food security and health programmes. However, as with other NTDs, historically there has been a significant underinvestment in commercial research and development programmes towards schistosomiasis diagnostics [49]. There are also limitations with currently used methodologies, several of which involve microscopic examinations of clinical or environmental Schistosoma samples. Whilst useful, microscopic examinations are labour intensive, highly subjective and do not enable the deeper insights that molecular approaches can conceivably provide. For example, the emergence of genomic DNA sequence variations within drug resistant (e.g. OXA) [50,51] or hybrid Schistosoma species [52] may not clearly manifest themselves at the morphological level but have important implications for global health programmes and affected patients.

To this end, we re-purposed a nucleic acid detection technology to enable the detection of different schistosome species. Our proof-of-concept data shows that our probe designs can detect and differentiate between two human health relevant schistosomes, S. mansoni (probe set 5) and S. haematobium (probe set 1), as well as discriminate these schistosomes from other closely related species including S. bovis, S. guineensis, S. curassoni, S. rodhaini, S. japonicum, and S. mekongi. Importantly, this study demonstrates that the ‘SNAILS’ probes are amenable to rapid design iterations that lead to improved biosensor sensitivity and specificity—that might also enable further improvements in base sequence resolution detection. Whilst the optimisation of biosensor probes has been an important aspect of this study, it is conceivable that other aspects of the ‘SNAILS’ assay could be refined in future studies. For example, the volumes or concentrations of assay reagents could be further optimised and open access enzymes (e.g., T7 polymerase) [53,54] could also be tested to reduce costs even further. Likewise, additional fluorescent molecule-binding aptamers could be utilised to enable compatibility with additional laboratory or in-field equipment [54,55] or enable assay multiplexing (detection of several DNA sequences within the same assay well). The global availability of PCR equipment has also rapidly expanded during the last several years and could be exploited to improve the accessibility of ‘SNAILS’ biosensor assays in different geographical locations. Beneficially, in contrast to PCR alone, ‘SNAILS’ biosensor assays can potentially provide single base resolution detection. Although crucial for the ‘SNAILS’ assay, the PCR amplification step is also useful in that it could also enable for positive detection at sites of low parasitic levels, that might be undetected by other methods without an amplification step.

We envision our ‘SNAILS’ biosensors as a complementary addition to the current schistosomiasis detection toolbox, in that it can provide species differentiation data which can highlight samples of interest for further analysis, such as full genomic sequencing. Likewise, bioinformatic insights from genomic sequencing studies can also be used to further refine or increase the number of validated ‘SNAILS’ biosensor probes—thus enhancing species-specific detection capabilities. Genomic sequencing and clinical validation of schistosome drug resistance markers, along with new workflows for integrating patient or veterinary samples (e.g. urine) into ‘SNAILS’ biosensor workflows, might also enable future clinical or agricultural point-of-care applications. In conclusion, we believe that the inclusion of ‘SNAILS’ biosensor assays may, in the future, enable epidemiological screening of test sites for basic research, environmental monitoring and WASH interventions, as well as monitoring the effectiveness of such interventions at these sites.

Methods

Identification of schistosome targets and probe design

S. mansoni AJ519524 [56] and MG562513 (GenBank), S. rodhaini AY157202 [57], S. haematobium GU257336 and GU257338 [35], S. bovis MH647124 [58] and FJ897160 [59], S. guineensis AJ519517, AJ519522 and AJ519223 [56], S. curassoni AJ519516 [56] and AY157210 [57], S. japonicum EU325878 (GenBank), S. malayensis EF635956 [60] and S. mekongi EF635955[60] cox1 nucleotide sequences were obtained from GenBank and aligned using MUSCLE [47] with default parameters (S1A Fig). Resultant schistosome targets identified and probe designs are listed in S1A Table. Oligonucleotide primers used as synthetic biosensor targets, for cloning and for sequencing, as well as those comprising the biosensor probes are listed in S1A Table and were ordered from Integrated DNA Technologies (IDT, USA).

Adult Schistosoma worm samples

S. mansoni adult worms 1 and 2 (SM_AW1 and SM_AW2), S. mansoni adult worm isolates 2740 (SM_C; isolated from Kenya), 3112 (SM_D; isolated from Senegal), 4209 (SM_E; isolated from Oman), N1g274 (SM_F; isolated from Nigeria), 4664–2 and 4464–4 (SM_G and SM_H; both isolated from Uganda) and S. haematobium adult worms 1 and 2 (SH_AW1 and SH_AW2) were obtained from the Schistosomiasis collection at the Natural History Museum (SCAN) [61].

Purification and elution of Schistosoma worm gDNA

Schistosoma adult worm gDNA was obtained using the DNeasy blood and tissue kit (catalogue #69504; Qiagen, UK) following the manufacturer’s instructions, with the purified gDNA eluted into 100 μl nuclease-free water (nH2O).

Amplification of parasite gDNA

Polymerase chain reactions (PCR) were used to amplify both the longer cox1 gene fragment and the 180-base target regions. For the longer gene fragment, gDNA from adult worms were amplified using Q5 high-fidelity DNA polymerase (catalogue #M0491S; New England Biolabs, USA) following the manufacturer’s instructions for 50 μl reactions. Essentially, 1 μl of gDNA sample was amplified using 2.5 μl (10 μM working stock) each ofprimers Cox1_schist_5’ (AJW485) and Cox1_schist_3’ (AJW486, 57] in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, USA) with the following protocol: 1 cycle of 98°C for 30 seconds, 30 cycles of 98°C for 10 seconds, 57°C for 30 seconds and 72°C for 90 seconds, followed by a final cycle of 72°C for 2 minutes. For the 180-base target region, 1 μl of gDNA (for all adult worm samples) or 1 μl of plasmid DNA (10 ng DNA total) were amplified using primer pairs 5-SM-PCR/3-SM-PCR (AJW870/AJW871; S. mansoni-specific) or 5-SH-PCR/3-SH-PCR (AJW872/AJW873; S. haematobium-specific) in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, MA, USA) with the following protocol: 1 cycle of 98°C for 30 seconds, 40 cycles of 98°C for 10 seconds, 57°C for 30 seconds and 72°C for 30 seconds, followed by a final cycle of 72°C for 2 minutes.

DNA samples to be cloned into the pCR-Blunt or pCR-Blunt II-TOPO plasmids were purified using the DNA clean & concentrator kit (catalogue #D4033; Zymo Research, USA). Visualisation for the validation of the 180-base target PCR products was undertaken using agarose gel electrophoresis: 5 μl of PCR product was separated using a 1.5% agarose gel with SYBR safe DNA stain (catalogue #S33102; Invitrogen, Thermo Fisher Scientific, MA, USA) in 1X Tris-acetate-EDTA buffer for 30 minutes at 100 volts. Quick-load 1 kb plus DNA ladder (100 bp-10 kb; catalogue #N0469S; New England Biolabs, USA) was also loaded onto the agarose gel to enable sizing of the PCR product. Bands on the agarose gel were visualised using the Gel Doc XR+ system with Image Lab software (model # Universal Hood II; Bio-Rad Laboratories Inc., USA).

Cloning of PCR products into pCR-Blunt or pCR-Blunt II-TOPO

PCR products amplified were cloned into either pCR-Blunt or pCR-Blunt II-TOPO plasmids following the manufacturer’s instructions (catalogue #K270020 & #450245; Invitrogen, Thermo Fisher Scientific, MA, USA). Reactions were transformed into Escherichia coli NEB10-beta or NEB5-alpha. For plasmid recovery, E. coli strains were grown in Luria-Bertani (LB) medium supplemented with 35 μg/ml Kanamycin and cultured at 37°C with shaking (220 rpm). The plasmid DNA from these cultures were purified using the QIAprep spin miniprep kit as per the manufacturer’s instructions (catalogue #27106; Qiagen, UK), with samples eluted in 50 μl of nH2O. The DNA sequences of the inserts were verified by the sequencing service provided by Eurofins Genomics GmbH (Ebersberg, Germany). The resulting plasmids are detailed in S1C Table.

Generation of ssDNA

After validation by agarose gel electrophoresis, PCR reactions for the 180-base target regions were enzymatically treated with Lambda Exonuclease (catalogue# EN0562; Thermo Fisher Scientific, MA, USA) to generate ssDNA. To the 45 μl PCR reactions 1 μl Lambda exonuclease (10 U/μl), 6 μl 10X reaction buffer (670 mM glycine-KOH (pH 9.4), 25 mM MgCl2, 0.1% (v/v) Triton X-100) and 8 μl nH2O were added to give a total reaction volume of 60 μl. The reactions were mixed gently via pipetting and incubated at 37°C for 30 minutes, 80°C for 10 minutes to inactivate the lambda exonuclease and then 10°C for 5 minutes in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, MA, USA).

The ssDNA produced was subsequently purified using the ssDNA/RNA clean & concentrator kit (catalogue #D7011; Zymo Research, USA) as per the manufacturer’s instructions, with ssDNA samples being eluted in 12 μl nH2O. The concentration of the purified ssDNA samples was determined, according to the manufacturer’s instructions, using a Qubit 3 Fluorometer (Thermo Fisher Scientific, MA, USA) and a Qubit ssDNA assay kit (catalogue #Q10212; Thermo Fisher Scientific, MA, USA).

SNAILS assay

The SNAILS assay developed in this study is adapted from a spinach-based fluorescent light-up assay for microRNAs [40]. However, in this study we use ssDNA as a target. The assay has four steps: preparation of the probes and ssDNA targets, annealing of probes and targets, ligation of the two half probes, aptamer transcription and fluorescence detection of the aptamer.

Preparation of the probes, ssDNA targets and DFHBI-1T fluorogen

Probe DNA stock solutions (100 μM in nH2O) were diluted in nuclease-free duplex buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate (CH3CO2K); catalogue #11-01-03-01, IDT, USA) to the required concentration for the reaction tested. The ssDNA target stock solutions (100 μM in nH2O) were diluted in nH2O to the required concentration for the reaction tested. The ‘2nd T7 promoter sequence’ oligonucleotide (AJW694) stock solution (100 μM in nH2O) was diluted 1 in 100 in nuclease-free duplex buffer (30 mM HEPES, pH 7.5; 100 mM CH3CO2K; catalogue #11-01-03-01, IDT, USA) to give a 1 μM working solution. DFHBI-1T fluorogen (catalogue #5610/10; R & D systems, Bio-Techne, MN, USA) stock solution (100 mM in dimethyl sulphoxide (DMSO)) was diluted 1 in 200 in nH2O to give a 500 μM working solution.

Annealing of probes and target ssDNA

Annealing reactions (7 μl) comprised 1 μl probe half A, 1 μl probe half B, 1 μl target DNA (or 1 μl nH2O for negative controls) and 4 μl nuclease-free duplex buffer (30 mM HEPES, pH 7.5; 100 mM CH3CO2K; catalogue #11-01-03-01, IDT, USA) and mixed gently via pipetting. Concentrations of probes and target ssDNA added are based on the 10 μl volume of the ligation reaction. For example, for reactions requiring 200 nM of each half probe in the final 10 μl ligation reaction, 1 μl of 2 μM probe DNA in nuclease-free duplex buffer (30 mM HEPES, pH 7.5; 100 mM CH3CO2K; catalogue #11-01-03-01, IDT, MA, USA) is added to the annealing reaction. Likewise, for reactions requiring 10 μM of target ssDNA in the final 10 μl ligation reaction, 1 μl of 100 μM target ssDNA in nH2O is added to the annealing reaction. For reactions using ssDNA samples generated from PCR reactions or the equivalent control oligonucleotide, 1 μl of the required sample amount (ng/μl) was added. Annealing reactions were incubated at 95°C for 3 minutes in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, USA) and then incubated on ice for 5 minutes.

Ligation of probes

To the 7 μl annealing reactions, 1 μl nH2O, 1 μl T4 DNA ligase (400 U; catalogue #M0202L; New England Biolabs) and 1 μl 10X T4 DNA ligase reaction buffer (final 1X concentration in reaction: 50 mM Tris-HCL, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5) were added and reactions mixed gently via pipetting. Ligation reactions were incubated at 25°C for 20 minutes, 65°C for 10 minutes to inactivate the T4 DNA ligase and then 25°C for 5 minutes in a ProFlex PCR System (Applied Biosystems, Thermo Fisher Scientific, USA).

T7 transcription and real-time fluorescence detection

T7 transcription reactions (60 μl final volume) using the TranscriptAid T7 high yield transcription kit (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), were set up as follows: 1.5 μl RNase Inhibitor (60 U; catalogue #M0314L; New England Biolabs), 24 μl NTP mix (final concentration of 10 mM each of ATP, CTP, GTP and UTP), 12 μl 5X TranscriptAid reaction buffer (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), 6 μl dithiothreitol (DTT; 0.1 M solution; catalogue #707265ML; Thermo Scientific, Thermo Fisher Scientific, USA), 6 μl TranscriptAid enzyme mix (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), 3 μl 2nd T7 promoter sequence oligonucleotide (1 μM stock solution), 6.3 μl of the ligation product, and 1.2 μl DFHBI-1T (10 μM final concentration). Reactions were mixed gently via pipetting. Three 15 μl aliquots of each reaction were loaded into 384-well plates (catalogue #781096, Greiner Bio-One, Kremsmünster, Austria) and measured using a CLARIOstar plate reader (BMG, Ortenberg, Germany) with the following settings: Excitation 440–15 nm/Emission 510–20 nm, orbital shaking for 5 seconds at 500 rpm before measurement every 5 minutes. Delta (Δ) fluorescence measurements (a.u.) per hour were calculated between 20 and 80 minutes of the reactions.

Statistics

Statistical analysis (standard error of the mean (s.e.m.)) was carried out using GraphPad Prism 9.1.1 (GraphPad Software Inc., La Jolla, California).

Supporting information

S1 Text. Supplementary figures, data and tables.

(DOCX)

Click here for additional data file. (7.1MB, docx)
S1 Data. Excel spreadsheet.

Excel spreadsheet showing biosensor assay data. The label on the spreadsheet tabs indicate the corresponding figure in the main text.

(XLSX)

Click here for additional data file. (38.4KB, xlsx)

Acknowledgments

We would like to thank our WISER project colleagues for their expertise and supportive comments. We also thank colleagues in the Section of Structural and Synthetic Biology within the Department for Infectious Disease at Imperial College London for their advice and helpful comments.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

AJW, FA, FZB, SMK, MRT, AME and PSF were funded by the UK Government’s Global Challenges Research Fund (GCRF) through the Engineering and Physical Sciences Research Council (EPSRC) grant [EP/P028519/1] as part of the WISER project. https://gow.epsrc.ukri.org/NGBOViewGrant.aspx?GrantRef=EP/P028519/1 AJW and PSF received funding from the Imperial College London EPSRC Impact Acceleration Account [EP/R511547/1] https://www.imperial.ac.uk/research-and-innovation/research-office/funder-information/funding-opportunities/internal-funding-opportunities/iaa/epsrc/. RJRK and PSF received funding from Biotechnology and Biological Sciences Research Council (BBSRC) grant [BB/T017147] and [BB/W012987/1]. https://bbsrc.ukri.org/research/grants/grants/PersonDetails.aspx?Personid=-27428 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0010632.r001

Decision Letter 0

Walderez O Dutra, Neil D Young

9 Mar 2022

Dear Prof. Freemont,

Thank you very much for submitting your manuscript "Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Neil D. Young

Associate Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objective of the study is to demonstrate proof-of-concept for a DNA-based biosensor for species-specific detection of i) synthetic DNA targets; and ii) field-collected samples, from two species of schistosome that cause human health problems. This is testable and the study design is appropriate to address the stated objectives for the synthetic targets derived from reference sequences as the authors use a total of nine closely related species (some with multiple sequenced genomes) to ensure specificity and they go on to extensively optimise a number of relevant parameters.

However, the use of only two adult worms of each species collected in the field is a concern as one of the two S. haematobium already had two base changes in a probe target region. There is no discussion of the potential or measured rate of variation in nature at those key base positions. They artificially mutated a range of single, double or multiple base changes to the S. mansoni wild-type target region to demonstrate that the probes retained specificity but this strand could be tied more clearly to robustness against likely natural variation.

It is also unclear why DNA from only two organisms for each species was available for the study as this is not a large sample size. Without further understanding of natural sequence variation it is difficult to conclude that the probes would be specific in a large scale study of samples from nature so I would expect to see this addressed in the discussion.

The statistical methods deployed are appropriate to support the conclusions. There are no concerns around ethical or regulatory requirements.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis presented matched the analysis plan and results are clearly and completely presented but may be too complete in that some of the information in the captions is easily discoverable in the methods so need not be repeated in each figure.

For example, most figures contain “Fluorescence outputs of reactions were analysed using a CLARIOstar plate reader (440-15 nm/510-20 nm; Gain 1500).” This information is already presented in the methods and could be removed from the captions. Likewise, “Mean and Standard error of the mean calculated using GraphPad Prism 9.1.1.” In the supplementary table captions is redundant to the methods section.

I request that the authors include a reference to the supplementary information in Fig 2,3,5 and 6 captions where their probe sequence data is included as a non-copyable image, so the reader knows that they can find a text version elsewhere.

Supplementary Figure 9, 10, 16, 17 are not high resolution in the PDF provided.

The PDF contains a very large number of tables with hourly plate reader date and calculations. This makes it difficult to find the other information that the reader is more likely to need, like the tables of nucleic acid sequences. Given this and that PLOS recommends that spreadsheets are preferable to PDFs or images when providing tabulated data, I would suggest providing a spreadsheet containing all of this experimental data (e.g. one experiment per sheet) rather than a PDF table, leaving the PDF shorter and containing only figures and resource tables.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusion is that the biosensor can detect and differentiate between two human health relevant schistosomes, S. mansoni and S. haematobium, as well as discriminate these from other closely related species such as S. bovis and S. rodhaini and that ‘SNAILS’ probes are amenable to rapid design iterations. This is supported by the data presented but I would further discuss the very limited sample size for field-collected samples and describe the level of intraspecies sequence variation that exists (if known) or may potentially exist, given one of the four samples tested had two base pairs variance. The rapid design iterations will mitigate against this causing issues during the design phase but not in field use if the natural population has high variance – for this I would expect to see a much larger sample size. The public health relevance of the study is described in terms of being an environmental surveillance tool to monitor presence of the schistosomes and the effectiveness of control measures.

I would also encourage some discussion of future developments which would be needed to advance the biosensor design as the introduction states that the authors concentrated "on the need for a species-specific point-of-care (PoC)/point-of-test (PoT) diagnostic" but there is then no further mention of the PoT potential for this assay, which currently uses PCR, one of the harder amplification methods to convert to PoT, followed by a further enzymatic step and then addition of the probe and ligase. It is also unclear if the fluorescent output is easy to read without a real-time system. These could be limitations for a PoT approach.

SNAILS is a redesign of the spinach aptamer assay for ssDNA detection and applications of this to other targets could be mentioned in the discussion alongside any planned further research.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Lines 207-250 belong in the introduction as they provide further detail about existing control measures and the landscape of existing molecular diagnostics into which this project fits, adding context and situating it within the literature. I would include content from line 250 onwards as discussion and add some further detail on natural sequence variation in populations and its potential effect on the assay specificity.

L.244 the comment that sequencing is expensive should be put in context and prefaced by a comment that the previous techniques would typically need to be followed by sequencing to enable species level identification.

There are a number of typos in the text but they all look like they should be detected by spell check so I haven’t listed them.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Overall this is a good lab-level proof of concept for a diagnostic biosensor design with good potential for impacting surveillance efforts in schistosomiasis. The design is a novel and original adaptation of an existing assay to detect ssDNA instead of RNA which may be of interest to other biosensor designers. It is intended to eventually be PoT but the proof of concept was performed with standard lab instrumentation and multiple steps in the lab with no further information provided on PoT potential, which makes it less relevant in its current form to a wide audience of practitioners.

Being species-specific without the need for sequencing is a compelling alternative to existing assays that require sequencing to provide that level of specificity. The major weakness of the study is the aforementioned small sample size for organismal DNA and lack of clarity on intraspecies sequence variation in the field which means it falls short of demonstrating broader application. However, this is consistent with the claims of the authors, who clearly describe it as a proof of concept. The experiments appear to have been well executed and data is provided in full. I have no further comments on the data presented and its analysis and interpretation.

Demonstrating successful detection across a much wider set of field samples would improve the paper's importance and relevance to researchers, practitioners, or policy makers in the field of NTDs. This could involve further collection of field samples if these are not already available to the authors, which could take significant amounts of time. The paper stands alone in terms of the biosensor design so could be published in another journal with different criteria with quite minor revisions, particularly recognising limitations in the discussion section, but I feel this major revision would substantially improve its fit with the PLOS NTD criteria.

An exception would be if the authors can convincingly demonstrate without additional experimental work (e.g. through presentation of secondary data from schistosome genetic/genomic studies or other means) that the likelihood of sequence mutations across a broad population within each species is low enough that the lab results obtained with synthetic DNA from reference genomes is representative of likely performance in the field. It is possible that this data exists but was not presented clearly in the paper. They did artificially mutate a range of single, double or multiple base changes to the S. mansoni wild-type target region to demonstrate specificity and may consider this is sufficient evidence that natural variation will not perturb the function of the assay. Discussing this in more detail and why it is likely a good proxy for natural variation could also be sufficient.

--------------------

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Reviewer #1: No

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0010632.r003

Decision Letter 1

Walderez O Dutra, Neil D Young

4 Jul 2022

Dear Prof. Freemont,

We are pleased to inform you that your manuscript 'Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Neil D. Young

Associate Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: I have reviewed the revisions made by the authors in response to Reviewer #1’s comments. I am in agreement with the issues raised by Reviewer #1 and I would have raised the same issues regarding the small sample size. The authors have carried out additional bioinformatic analyses on a range of publicly available datasets. I am satisfied that these additional analyses address the concerns of Reviewer #1.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: Yes, The updated images have been improved as suggested by Reviewer #1

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: Yes, these factors has been addressed

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0010632.r004

Acceptance letter

Walderez O Dutra, Neil D Young

19 Jul 2022

Dear Prof. Freemont,

We are delighted to inform you that your manuscript, "Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Text. Supplementary figures, data and tables.

    (DOCX)

    Click here for additional data file. (7.1MB, docx)
    S1 Data. Excel spreadsheet.

    Excel spreadsheet showing biosensor assay data. The label on the spreadsheet tabs indicate the corresponding figure in the main text.

    (XLSX)

    Click here for additional data file. (38.4KB, xlsx)
    Attachment

    Submitted filename: Webb_etal_SNAILS-response to reviewer.docx

    Click here for additional data file. (40.2KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS Neglected Tropical Diseases are provided here courtesy of PLOS

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