Introduction and background

Schistosomiasis is one of the world’s most prevalent neglected tropical diseases. Although estimates vary considerably, it is generally considered that 800 million people are at risk of infection, with a global prevalence of 229 million cases [1], of which 200 million live in sub-Saharan Africa [2–5]. It has an impact more significant than generally perceived on human health, ranking third among the Neglected Topical Diseases (NTDs) in disability-adjusted life years [6,7]. The Schistosoma parasites responsible for causing the disease are found as adult worms in the vascular system of humans and other mammals. The adult worms produce eggs that are passed in the host’s feces in the case of S. mansoni or S. japonicum, or the urine in the case of S. haematobium. The eggs hatch in water and release swimming miracidia that may locate and penetrate an appropriate freshwater or amphibious snail species. Specificity is evident as S. mansoni successfully infects only Biomphalaria snails, S. haematobium develops in Bulinus snails, and S. japonicum in amphibious snails in the genus Oncomelania. Asexual development occurs in sporocysts in the snail host, culminating in the production of fork-tailed cercariae that leave the snail host and swim towards and penetrate unbroken human skin to initiate new infections. Infected snails can produce tens of thousands of cercariae over a period of several months [8].

Schistosomiasis is enabled by poor sanitation, allowing schistosome eggs in feces or urine to pass into snail-containing habitats, and by the widespread use of such habitats for fishing or other occupations, bathing, recreation, washing of clothes, and as a source of drinking water. The long-term persistence of cercariae-producing snail infections in the water renders control more difficult. Even if infected people are successfully treated (usually with praziquantel) to eliminate their adult worms, they may quickly reacquire infections.

The World Health Organization has championed the view that the elimination of schistosomiasis is achievable [9]. In their new roadmap for sustainable development (WHO, 2020), they set a target to validate the elimination of schistosomiasis as a public health problem (defined as <1% proportion of heavy intensity schistosomiasis infections) in all 78 affected countries by 2030. A further goal concerning elimination is to eventually verify and declare the interruption of transmission in a country-by-country manner [10].

Given the importance of development and application of improved diagnostic and surveillance methods for the elimination effort, we have been asked by the WHO to review the suitability of available techniques to answer the following PICO (Problem Intervention Comparison Outcome) question: What techniques should be used to diagnose Schistosoma infections in snails and in the water in potential transmission sites? In other words, what are the best approaches for determining the presence and identity of schistosomes in populations of vector snails (frequently known as xenomonitoring), or of snails, schistosome life stages (like miracidia or cercariae) or their DNA in the water of suspected transmission sites? We did not consider literature pertaining to using human fecal bacteria as surrogates for assessing the likelihood of schistosome contamination of surface waters [11].

Such methods provide a needed alternative view to diagnosis or surveillance of infections in the human host. Snail or water-oriented methods might uniquely detect transmission if, for instance, eggs from non-human reservoir hosts were responsible for infecting snails. Monitoring events in snails or in the water also helps determine if particular schistosome species have been introduced into new areas or re-introduced into former endemic areas, define transmission hot spots spatially and temporally, and gauge the success of intervention methods targeted at the human population [12,13]. The successful effort to control Schistosoma japonicum in China has been aided by monitoring infections in snails and in the water of suspected transmission sites [14]. Considerations important to our evaluation include the points listed in Table 1.

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Table 1. The ideal diagnostic test to answer the stated PICO question should include the following characteristics.

https://doi.org/10.1371/journal.pntd.0009175.t001

A detailed list of the general diagnostic procedures we considered is provided in the results section. For those who may not be familiar with the general approaches taken over the years to detect and identify schistosome infections in snails or in the water column, we provide an overview in Fig 1 of the available diagnostic approaches that have been undertaken relative to our PICO question.

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Fig 1. Shown are methods for detecting schistosome infections in snails or for detecting schistosome or snail signals in the water.

Identification protocols rely on traditional morphological means or, increasingly, sequence-based identifications. With respect to finding infections in snails, the traditional technique of isolating and shedding snails for cercariae is widely practiced, but alternative methods, mostly relying on amplification of schistosome DNA sequences by a variety of means, is also widely practiced. Additional follow up techniques may be required to provide sufficient sequence information for identification. A variety of techniques has been devised to detect schistosomes in the water column ranging from using sentinel mice to collect cercariae from the water to collection of environmental DNA samples containing DNA from intact or disintegrated bodies of schistosomes or their snail hosts. Again, follow-up sequencing may be required to confirm species identifications.

https://doi.org/10.1371/journal.pntd.0009175.g001

Methods

Results

PRISMA flow diagram

The Prisma diagram (Fig 2) provides an overview of how we arrived at the final 158 articles included in our systematic review process.

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Fig 2. Overview of the PRISMA process.

https://doi.org/10.1371/journal.pntd.0009175.g002

Determination of certainty of evidence following GRADE criteria

We grouped the techniques covered in the 158 papers into 25 categories (Table 2). Some papers discussed more than one technique. As noted in Fig 1, the techniques cover a wide range of approaches. Some are directed towards detecting schistosome infections in snails, and some involve the detection of schistosomes (or snails) in water samples. In some cases, like shedding of snails, identifications of schistosomes can be undertaken using morphological criteria or by submitting them to molecular techniques. For some methods like eDNA, the process typically involves collecting a sample followed by extraction and the submission of the sample to some type of amplification protocol. The data supporting the scores provided in Table 2 are provided in S1 Table. The five criteria contributing to the overall certainty in the grade score provided were limitations, inconsistencies, imprecision, indirectness, and publication bias. As we did not detect the latter, it does not appear in the table. The remaining five columns (coverage, cost, support needs, species differentiation, and relevant detection limits) reflect our view of their importance, but they were not included in the GRADE scores. Note that by the GRADE criteria provided, many techniques were assessed as having very low or low overall certainty: in several cases, they reflect techniques reliant on older technology. Three techniques were scored as providing moderate certainty: LAMP, eDNA based techniques, and qPCR. One of the significant considerations diminishing confidence for several of the techniques was the lack of widescale testing and standardization considerations.

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Table 2. Grade Summary Table.

We used the numbers 0, -1, -2 to score every method, with 0 being adequate, -1, serious concern, -2 very serious concern. It is worth noting that publication bias is not part of this table, as we did not have any reason to suspect it has occurred with any of the methods. We defined species differentiation ability of each method as N.S = Nonspecific, H.S = Heterospecific, N.H.S = Narrowly Heterospecific, M.S = Monospecific. And relevant detection limits as Hig.S, High sensitivity, Mar.S = Marginal sensitivity, Ina.S = Inadequate sensitivity.

https://doi.org/10.1371/journal.pntd.0009175.t002

In S2 Table, following GRADE criteria, we present determinations of sensitivity and specificity using the definitions often applied to diagnostic techniques in the medical literature (see table legend for definitions). Insofar as most of the papers we reviewed simply did not provide this information, its relevance to our discussion is lessened. Lastly, in Table 3, with an eye on practical guidance, we provide an overview of the pros and cons of each of the techniques we classified.

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Table 3. A summary of perceived advantages and disadvantages of the techniques evaluated, with comments on possible improvements and potential applications.

https://doi.org/10.1371/journal.pntd.0009175.t003

Discussion

Successful application of any diagnostic technique for the determination of schistosome infection in snails or in the water of natural snail habitats requires accurate knowledge of the species of schistosomes and their snail vectors historically or currently present. A single schistosome and snail species might be involved in some locations, but often multiple Schistosoma or potential snail host species are present. For instance, S. rodhaini might complicate the diagnosis of S. mansoni in some parts of Africa, but not in South America. S. haematobium may be especially hard to differentiate from closely related animal schistosomes or their hybrids in some sub-Saharan Africa areas. Multiple species of bulinid snails might be implicated to one extent or another as vectors. Some snail species not susceptible to human schistosome infections like Planorbella duryi can easily be mistaken as vector species upon superficial examination [18], potentially resulting in misdirected sampling effort. Familiarity with the literature for a specific area, consultation with parasitologists or malacologists, and examining available keys are all helpful. Submission of voucher specimens of schistosomes and snails to an appropriate museum both for subsequent identification and to provide historical documentation should be encouraged.

Another fundamental reality is that the scale of the task required of the diagnostic technique will vary dramatically. The problems posed for ascertaining if and where schistosomiasis transmission might be occurring on a Caribbean island or in a desert country with few transmission sites are quite different compared to some countries where schistosomiasis transmission potentially occurs across vast geographical areas in almost innumerable freshwater habitats. Clearly, such situations may require different sampling and diagnostic approaches.

Especially in the context of verifying elimination, an all-important consideration is the habitat sampling protocol to be used, a topic requiring much further collective discussion. Given that schistosome transmission is often focal, sampling effort should be guided by available information about past transmission foci and any new evidence (such as positive serology results from recent human surveys) providing relevant locality data. One general goal though, should be to generate many samples from different locations and time points to avoid the possibility of overlooking new or unknown foci of transmission, possibly including ones involving animal reservoirs. Consequently, whatever methods will be chosen to detect positive snails or water samples should be compatible with efficient and low-cost sampling methods that provide realistic coverage to support an accurate assessment of achievement of elimination.

Spear et al. [19] noted, “the development of environmental diagnostics for the infective stage of parasites such as schistosomes is stunningly behind those of other parasites present in water.” In Table 1, we identified basic features required of the ideal diagnostic test to answer the stated PICO question. Although the ideal technique does not exist, considerable strides have been made in recent years in devising sensitive and specific detection methods and providing more straightforward methods for extractions, amplifications, and documentation. We are making progress.

As we move forward, we may learn that the application of a combination of diagnostic approaches is inescapable. For example, although frequently maligned, the snail shedding technique has some benefits that may be impossible to replace with even the most sophisticated molecular technique. For one thing, it is specimen-based, providing indisputable evidence about the presence of a particular snail vector species and possibly of the schistosome species it is transmitting. The specimens, or portions derived from them, can be used in follow-up molecular analyses and can be vouchered to become part of a more permanent record and facilitate their use in applications we cannot yet anticipate. The shedding method can also provide spatially and temporally explicit information from multiple locations about how common schistosomes might be. It could be argued that familiarity with this method should be part of the training of any person working in schistosome environmental diagnostics, simply to reinforce an understanding of the underlying biological realities of the system.

One of the most promising recent developments in schistosome environmental diagnostics is to use as starting material schistosome or snail eDNA as derived from water samples. Suppose eDNA samples are shown not to rapidly decay [20], not to be prone to inhibition and enable sensitive, specific, repeatable analyses across multiple aquatic habitat types.. In that case, it is easy to imagine eDNA as the preferred method of sample collection, perhaps supplemented with occasional snail specimen collection for validation purposes. One great appeal of eDNA samples is the potential for rapidly collecting many samples, each of which effectively integrates a signal over space and time, providing further coverage. Additional improvements to facilitate the collection of eDNA samples are sure to follow, further underscoring this approach’s value.

Once a DNA sample is in hand, then most modern diagnostic approaches proceed to amplify DNA via an ever-growing variety of techniques. This field is moving fast, and what seems best in 2021 may soon be dated. As noted in Table 3, there are pros and cons to each. One notable development to go along with the increasing use of eDNA is qPCR-based technology to analyze extracted specimens [20–24]. A quenchable fluorescent probe designed to detect a particular sequence of the target organism of interest is typically included (FRET-qPCR). Significant general advantages for qPCR are that amplification and detection are combined in a single step, it is a mature technique with well-defined protocols and data analysis, it is relatively quantitative in allowing the amount of amplified product to be determined (but requires reference samples and development of a standard curve), costs are relatively low per sample, and it can be set up for high-throughput and multiplexing (perhaps simultaneously detecting schistosome and snail signals in the same sample). It is supported commercially by the active development of new products. Smaller, less expensive qPCR machines are commercially available but are less amenable to high-throughput sampling. Droplet digital PCR has some key advantages over qPCR should eDNA samples prove difficult to analyze: ddPCR is much less subject to inhibition, sensitive to detection of rare targets, and provides absolute quantification.

qPCR and ddPCR offer many advantages, but acquiring and maintaining the instrumentation, procuring reagents in a cost-effective manner, and standardizing protocols among separate labs would remain a challenge. To avoid these problems, and to take advantage of purchasing reagents in bulk and using high-throughput protocols, submission of relatively stable eDNA/DNA samples to one or a few central facilities charged with the responsibility of extracting and amplifying the samples and interpreting the data seems to us to be the way forward. A strong case could also be made for LAMP or other isothermal techniques, including the possibility of having a more widely distributed system of labs employing the technique.

No matter the means of sampling or the technique chosen for detection, some key general points remain. It would be highly desirable from a global elimination program’s standpoint to ensure that the techniques employed have been widely tested on samples collected from many different habitat types on other continents. Standardization with respect to extraction methods, particular reagents or kits used, means to test for or prevent inhibition of detection reactions, amplification protocols, and data interpretation and presentation are all highly desirable.

In the case of amplification-based techniques such as PCR/qPCR strict precautions must be taken to prevent contamination of the lab environment with amplicons of the target species to avoid the recording of false positives. The inclusion of both positive and negative controls and standards in all protocols is essential. Aliquoting of key reagents is vital to minimize contamination risk. Physical separation of separate steps in preparation of amplification reactions, if possible, is also essential. If qPCR, ddPCR, or other complex equipment prone to noise-to-signal inaccuracies are to be involved, regular and careful calibration of the machines is critical.

With respect to testing the primers used for various techniques, to avoid misleading results, there is a need to be sure 1) the closest and most common relatives of the target species are tested, and 2) that spurious cross-reactions even with parasite species that are not close relatives of the target species are excluded as a possibility. Testing against local co-occurring snail and trematode species is preferred as these are the species likely to cause cross-reactions and false positives to occur [22]. Having additional genome sequences for the trematode species most likely to co-occur with targeted schistosome species, either in the same snail species or the same environment, would be helpful. It would help eliminate the possibility of designing primers based on schistosome genes or genomic repeats that quite unexpectedly share sequence similarity with other trematodes. Extraneous trematodes may be more of a diagnostic complication for detecting schistosomes in snails than presently envisioned because of the tendency of groups like echinostomes, xiphidiocercariae, and strigeids to encyst as metacercariae within field snails. Similar considerations apply to eDNA samples taken from environments supporting diverse trematode faunas, which is often the case.

Primer design often relies on in silico predictions to limit cross-reactivity, which may have pitfalls [25], underscoring the need for follow-up confirmation. Sanger or high-throughput amplicon sequencing with comparison to sequences in GenBank [26,27] will be particularly crucial during elimination and surveillance operations to ensure positive results that are recorded by a band, Ct value, or a color change in a tube definitively belong to the schistosome or snail species responsible for the positive sample. Cross-reactions could lead to erroneous conclusions about the persistence of transmission or lead to misidentification of hot spots, in turn leading to erroneous targeting of special control efforts and wasting limited resources.

It is also important to rule out the effect of inhibitors in diminishing or precluding amplification. Inhibitors can originate from the sample or can be introduced during extraction. The standard method to deal with inhibitors is through control reactions spiked with known amounts of a reliably amplifiable non-target sequence. By comparing the quantity of non-target detected to the originally spiked amount, the level of inhibition can be determined in any given sample. This helps to guard against obtaining false negatives with unknown samples. If the presence of inhibitors is deemed a persistent problem, then sample treatments may be required, or the use of ddPCR may then be preferred.

Regarding the molecular targets that have been used for schistosome detection (see [28] for one list), they are quite understandably typically based on sequences expected to be represented many times in a single schistosome cell, thereby increasing the ability to sensitively detect a signal. They include mitochondrial markers (such as 16S rRNA, Cox1, Cox3, Nad5 (ND5) [29]), or nuclear sequences including portions of the rRNA complex (18SrRNA, intergenic 28S-18S spacers) and repetitive sequences such as Sm1-7 and DraI [12]. Many of these sequences have also been used to detect schistosomes in human samples such as plasma [30], however snails and environmental samples pose added challenges due to presence of a multitude of potential competing sequences, that as noted above might have surprising homologies with schistosome target sequences. Certainly, there is additional scope for development of better target sequences that might avoid cross-reaction or resist degradation, but it is also important to more thoroughly test the primers we have. One recent comparative study found primer sets targeting ND5 or 28S to work better than primers targeting a repeat element [31], the latter potentially also favoring amplification of unknown products from other complex genomes also present in extracted samples.

Particularly needed for eDNA-water based approaches are studies comparing different primer sets under a diversity of realistic field conditions in which known quantities of schistosome specimens or DNA are present, preferably undertaken by the same research team. Such controlled comparative studies should include considerations of how eDNA moves and degrades over time. Included in the comparisons would be considerations of different extraction techniques and downstream amplification protocols such as LAMP and qPCR. An important consideration is tailoring primers to the amplification method: gel-based PCR detection can accommodate long amplicons which can be made highly specific, however qPCR and ddPCR have better performance using shorter amplicons in the 70–200 bp range, limiting the potential regions to amplify. Considering the plethora of genomic resources [32] and molecular targets already found for schistosomes, it is likely that the limiting factor to the identification of the best targets to use is the lack of comprehensive comparative studies rather than the lack of useful molecular targets.

It will be tempting to pool samples, especially when in a surveillance program, it becomes clear that most samples collected and analyzed prove to be negative. Pooling could allow expanded coverage and sharply lower expenses. For some snails like the tiny Oncomelania involved in S. japonicum transmission, this may prove more feasible, as some of the papers examined suggest (one cercaria can be detected per 1000 uninfected snails). For larger snails like Bulinus or Biomphalaria, pooling may prove more complicated, especially if rare and/or prepatent infections are represented among the pooled snails. Recognizing that a trade-off is involved in the pooling of samples, one way to check the ability of pooling to detect small schistosome signals is to spike some of these samples with various doses of parasites to see if they reliably detect positives. Similar considerations apply to pooled water samples.

For studies based on detection of eDNA from habitats, it is important to recognize that positive signals from filters could be derived from cercariae that were intact and alive at the time of sampling or from environmental DNA no longer associated with living cercariae or any other life cycle stage. Positive signals from both sources are valuable but tell different stories. If positive signals from living cercariae are being detected, then the techniques convey valuable information that infections are ongoing and where they are being acquired. If the signals originate from eDNA, they are more indicative of a signal integrated over time. The length of the persistence of eDNA signals is bound to be variable, from days to weeks [33], which will influence the extent to which signals may be distributed within a habitat. Developing techniques to detect eRNA, indicative of the presence of living specimens, may prove to be useful in some contexts.

Conclusions

Some important knowledge gaps going forward are:

1. We are not necessarily there yet with respect to identifying the best schistosome sequences to target for amplification. The optimum length of the target to be sequenced may well depend on the method of amplification. Further comparative studies to assess existing primers [31] and more testing to improve specificity with respect to the realistic identification challenges faced in a particular location are needed.
2. To field-test the most promising sample collection, extraction methods, primers, and detection methods in multiple locations, possibly with standardized spiked samples. This would be undertaken preferably in a coordinated, comparative fashion, under centralized supervision such that quality control and standardization can be promoted. This would help identify ways to reduce costs and identify the most suitable combination of techniques for broader application.
3. Further emphasis on the development of eDNA based methodology is warranted to determine: temporal stability of amplifiable signals in the water column; how far eDNA might be dispersed and still be detected; if robust and consistent signals be retrieved from complex tropical aquatic ecosystems, and; if the samples be easily shipped elsewhere for analysis?
4. Ideally, our diagnostic tool kit would eventually include approaches that might allow differentiation among live or dead specimens and different life cycle stages of the same species, for instance, differentiating between miracidia (indicative of contamination) or cercariae (indicative of the potential for infection) and determining if infections in snails derive from humans or reservoir hosts.
5. We must remain open-minded for dramatic new approaches that rely on robotics for sample collection/processing or for self-contained microfluidics devices that, if proven effective and made inexpensive, could solve many of the coverage and standardization problems.
6. Inducements to encourage more researchers to participate and communicate with one another to develop standardized new diagnostic techniques should be provided.

Supporting information

Acknowledgments

This PRISMA and GRADE analysis was commissioned and supported by the World Health Organization (WHO). We thank the 11 experts and previous prominent contributors for their input and information regarding this topic. In addition, this work has benefited from the critical discussions with Dr. Amadou Garba Djirmay of the World Health Organization, Dr. Nathan Lo, and Dr. Murad, M. Hassan, particularly with regard to the application of the GRADE framework. This work also benefited from the important discussion and input from the Technical Working Group on protocols of surveys to verify the interruption of transmission of Schistosomiasis at the WHO. The content for this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the World Health Organization.