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Schistosomiasis diagnosis: Challenges and opportunities for elimination [1]

['Ombeni Ally', 'Department Of Molecular Biology', 'Biotechnology', 'Pan African University Institute For Basic Sciences', 'Technology', 'Innovation', 'Pausti', 'Nairobi', 'Department Of Biology', 'College Of Natural']

Date: 2024-08 

The global prevalence of schistosomiasis remains too high, with a great burden being carried by sub-Saharan Africa [1,2]. According to World Health Organization (WHO), about 240 million people from 78 countries worldwide required preventive chemotherapy for schistosomiasis in 2020 [2,3], over 90% of whom live in Africa [1,3]. There are 2 major forms of schistosomiasis; intestinal, which is mainly caused by Schistosoma mansoni, S. mekongi, S. japonicum, S. intercalatum, and S. guineensis, and urogenital, caused by S. haematobium [3]. Of the 6 Schistosoma species, S. mansoni and S. haematobium remain the most prevalent [1]. S. mansoni causes liver inflammation, and undernutrition and growth retardation in children, while S. haematobium causes haematuria, painful and frequent urination, urinary tract infections, and in severe cases, bladder and kidney damage, and high infertility rates in regions with a high prevalence of schistosomiasis [1,4].

Schistosomiasis is a preventable and treatable disease [2,5], and sensitive diagnostics would enable early intervention, saving many lives, particularly those of small-scale agricultural and fishing communities. In the fight against neglected tropical diseases, many efforts have been made to eliminate schistosomiasis as one of the major public health concerns [1,3,6–8], and in 2022, the WHO updated guidelines for controlling and eliminating human schistosomiasis [3]. The WHO recommends an integrated approach, combining the extension of preventive chemotherapy to all people at risk from 2 years of age in communities with a ≥10% prevalence, sanitation, treatment in health facilities, and snail control to eliminate schistosomiasis as a public health problem. Treatment campaigns have been scaled-up in several sub-Saharan African countries where most people at risk live [1,8]. The evaluation of mass drug treatment campaigns conducted in 2022 indicated a significant reduction of schistosomiasis in Burundi, Eritrea, Eswatini, Gambia, Lesotho, and Rwanda [8].

Diagnosis plays a critical role in measuring the impact of interventions as it determines levels of reduction of disease, infections, and parasite transmission. The sensitive, affordable, and accurate diagnostics are necessary to monitor, treat, and perform continuous surveillance of schistosomiasis cases [2,3,9]. Efforts have been made to develop new diagnostic tools for schistosomiasis, but sensitivity, specificity, and affordability remain the major challenges [2,3,9].

In October 2019, WHO established a diagnostic technical advisory group (DTAG) to develop target product profiles (TPPs) for better diagnostic tests for neglected tropical diseases, including schistosomiasis, as most existing tests are inadequate [9]. The diagnostic target set by WHO requires a highly sensitive point of care diagnostics specific to S. mansoni or S. haematobium, and S. mansoni and S. haematobium, as acceptable and preferred criteria, respectively [3,9]. The test should also enable point-of-care (POC) detection with minimum infrastructure for a single field visit decision. Ideally, the POC should cost as less as microscopy (maximum of $3 per sample) in contrast to most laboratory-based tests, whose initial capital could be as high as $10,000 [9].

Diagnosis of schistosomiasis targets different stages of the parasites complex lifecycle as illustrated in Fig 1. In general, the diagnosis of schistosomiasis is routinely carried out using microscopy, which though affordable and highly specific, is less sensitive [9–12]. Recently, antibody-based assays, parasite antigen detection, and nucleic acid amplification assays have been developed [13–15], but none is robust enough to replace microscopy as the gold standard [3]. Therefore, there is an urgent need to develop rapid and affordable POC diagnostics that are highly sensitive, particularly in sub-Saharan Africa, where the disease is still a major public health burden.

Loop-mediated isothermal amplification (LAMP) allows simple and rapid detection without the requirement for expensive equipment such as a thermocycler and automated documentation systems [ 32 , 36 , 37 ]. In 2018, LAMP was evaluated for its potential to diagnose schistosomiasis in low transmission settings, and results indicated that LAMP was more efficient in detecting S. mansoni compared to KK in human samples and nested-PCR in snails [ 33 ], and Brazil and China have already employed the use of LAMP for snail surveillance [ 33 ]. The method has also been used for field monitoring of snails infected with S. mansoni, S. haematobium, and S. japonicum, and studies have also demonstrated that LAMP can detect schistosome DNA from human stool, urine, blood serum, and snail samples [ 22 , 36 , 38 ]. The use of specific inner and outer sets of primers makes LAMP assay highly specific in detecting the specific target of schistosome parasites. However, the use of multiple primers complicates the initial optimization of LAMP assays, and also if good laboratory practices are not followed carefully, aerosols with high concentrations of LAMP amplified products may easily be formed while handling reaction tubes, leading to contamination of the surrounding area. Subsequently, carryover contamination may occur through contaminated reagents, pipettes, working surfaces, or personal protective gear [ 20 , 23 ].

Real-time PCR allows the detection of schistosomes in real-time, and a multiplex version is available [ 12 , 35 ]. Real-time PCR offers higher sensitivity than conventional PCR and can indicate the infection intensity of an infected individual, but the major limitation of the PCR-based methods is caused by cumbersome DNA extraction and purification procedures and the need for sophisticated tools and experts [ 7 ].

Different PCR-based assays have been developed to target specific locations in the Schistosoma genome, including the ribosomal subunits 18S rDNA, SjR2 retrotransposon, SM1-7 tandem repeat sequence, Dra1 repeat sequence, cytochrome c oxidase-COX I, 28s rDNA, and SSU-rRNA, mitochondrial genes, and the internal transcriber-spacer-2 sequence–ITS2 [ 6 , 7 , 25 ]. The Sm1-7 tandem repeats and Dra-1 repeat sequences have been targeted for an improved sensitivity in diagnosing S. mansoni and S. haematobium infection, respectively [ 34 ].

Nucleic acid amplification tests use specific nucleotides targeting specific location(s) of the schistosome, hence enabling the amplification of the targeted region(s). Various nucleic acid amplification tests (NAATs) have been developed and evaluated to diagnose schistosomiasis [ 7 , 12 , 25 , 32 , 33 ]. There are 3 commonly used NAATs for detecting schistosomiasis in field and clinical settings [ 7 ]: conventional PCR, real-time PCR, and loop-mediated isothermal amplification assay (LAMP). Other NAATs-based assays include digital droplet-PCR, nested-PCR, and recombinase polymerase amplification (RPA) [ 7 ]. NAATs usually demonstrate much better sensitivity than microscopy, making them suitable for low infection burden [ 7 , 12 , 25 ].

Schistosoma infections may also induce the production of antibodies, which can be used as diagnostic markers. The detection of immunoglobin G (IgG) and immunoglobin M (IgM), which are produced by the body in response to the presence of cercaria secretions, schistosome egg antigens, or soluble adult antigens, are the most commonly used antibody-based methods [ 2 , 6 , 14 , 15 ]. Antibody-based methods are also highly sensitive compared to the microscopy approach; hence, they have been recommended for individuals with low infection intensity. The major challenge for antibody-based methods is that they are associated with false positive due to the continuous circulation of antibodies after the infection has been cleared [ 3 , 25 ]. Secondly, the level of antibodies does not necessarily indicate the level of parasite intensity, and there might be an occurrence of cross-reactivity since schistosomes share antigen epitopes with some helminths such as Filaria spp., Echinococcus spp., and Strongyloides [ 15 ].

Adult schistosomes and their eggs contain specific proteins that can be detected in blood circulation, urine, stool, and saliva [ 11 , 26 ]. Antigen-based assays have been developed and evaluated, targeting the circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) [ 18 , 27 – 30 ]. The CCA and CAA are regurgitated from the gut of schistosomes, excreted in the patient’s urine, and positive results indicate an active infection. The POC targeting CCA is commercially available; however, there are concerns over lack of sensitivity and specificity, caused by reported discrepancies when the assay was evaluated against confirmed Schistosoma-positive and negative samples [ 3 , 18 , 28 , 31 ]. One study reported that POC-CCA have high false positives in pregnant women (32.7%) and pre-school-aged children (46.5%) [ 19 ]. In addition, significant variation in sensitivity and specificity among different batches and versions have been reported, and frequent fluctuations in assay replicates have also been reported [ 18 , 28 , 29 ], imposing some questions to the current manufacturing process of a POC-CCA test.

Major challenges in diagnosing schistosomiasis

The need for a genus-specific POC test. Regardless of the causative agents, schistosomiasis treatment is based on using a single drug (Praziquantel), at least to date [2,3,8]. Researchers have been able to come up with multiplex-based PCR assay [12,35] but the need for DNA extraction, cold chains, and sophisticated machines such as thermocyclers are the 3 major challenges to its use, especially in remote settings areas, where the disease affects the most. CCA and CAA have been investigated as genus-specific biomarkers to detect schistosomes, and POC diagnostics based on the 2 antigens are available [29–31]. The POC-CCA test, which is commercially available, can accurately demonstrate moderate to heavy S. mansoni infection, and some studies have indicated that it cannot be used in low infection settings [28,39]. The other potential POC diagnostic is up-converting phosphor-lateral flow (UCP-LF)-CAA test, which has indicated much more sensitivity than microscopy and qPCR, and the current version allows for dry reagent formats for easy transport and storage at ambient temperature [13,30,40]. Still, the potential for its use as a POC test in its current format is limited as it is laboratory-based and requires trained personnel and centrifugation equipment [40]. CCA and CAA are also inappropriate biomarkers for detecting prepatent infection, and hence, their uses in surveillance studies for mass drug administration programs are restricted, especially where there is low transmission. Therefore, for elimination purposes, it is clear that there is a need to validate or develop and make publicly available an affordable and sensitive POC diagnostic that can simultaneously detect multiple schistosomes.

Sensitivity and specificity issues for the common diagnostic tools. The debate about the low sensitivity of microscopy has existed for several decades, and the WHO recommends much more sensitive approaches to diagnose schistosomiasis in low-transmission settings [9]. Although NAATs, POC-CCA, and antibody tests are reported to offer much better sensitivity than microscopy, studies have indicated a potential risk for these assays to designate negative for some positive samples, which might significantly affect the actual prevalence estimates and elimination strategies, particularly where Schistosoma infection burden is low [2,10,12,17]. According to the WHO, the overall performance of Schistosoma infection diagnosing tools remains moderate, with most tools having low to moderate sensitivity or specificity [2]. The report also called for further evaluation of immunological and molecular tests, as there is no enough data to make an informed decision on the performance of most diagnostics [2]. In contrast, the recently published systematic review and meta-analysis by Feleke and colleagues [17] evaluated the performance of PCR and ELISA, among other tests. Still, the conclusion made by the authors might need further analysis. For example, the authors performed a meta-analysis by pooling different PCR that target different genes or target(s) (single or multiple repeat targets), with variation in source of samples (urine or stool) or reference tests (urine examination or KK), which is likely to invalidate the estimation of the accuracy of the performance of PCR tests [17]. Few studies, however, have reported that the sensitivity of NAATs for Schistosoma, including qPCR, depends on the intensity of infection [12,41–43]. A study to compare the KK to Schistosoma-specific ITS2 qPCR found that percentages of PCR positives varied from 79% to 87% in the group with light-intensity infections (1 to 99 eggs per gram of feces (epg)), 83% to 97% in the moderate egg count group (100 to 399 epg), and 100% for heavy infections (≥400 epg) [12]. In another comparison, Keller and colleagues [44] reported that real-time PCR targeting the Dra1 repeat unit in S. haematobium resulted in an overall sensitivity of 89.5% with 82.8% specificity, missing 11 out of 105 urine samples with S. haematobium eggs, but the sensitivity increased to 96.4% (27/28) for ≥50 epg samples. Recent findings on isothermal amplification assays, including LAMP and RPA have also indicated a potential to offer comparable sensitivity to much more than PCR, and this shows their potential to replace PCR, not only based on their cost effectiveness but also sensitivity [33,36,45]. Protein-based biomarkers (CCA, CAA, and antibodies) are also ubiquitous targets for Schistosoma infection, with POC-CCA being WHO recommended for surveillance of S. mansoni [2]. When qPCR was compared to CCA and CAA-based tests, Armoo and colleagues [31] and Hoekstra and colleagues [10] reported reduced sensitivity of qPCR compared to urine CCA assay and (UCP-LF) CAA test, respectively. Nevertheless, (UCP-LF) CAA is not commercially available, and the reported variation in sensitivities and specificities between different versions and batches of the commercially available POC-CCA tests limits their reliability in diagnosing Schistosoma infection [18,29,46]. Graeff-Teixeira and colleagues [18] concluded, quoted, “the indicated limitations of specificity and reproducibility with POC–CCA prevents an unrestricted recommendation for its application not only as a cut-off point for MDA scheme but also as a reliable diagnostic tool for selective chemotherapy in low endemic areas and at final stages of transmission interruption,” highlighting the need for manufacturers to standardize the production, assuring quality and reproducibility of POC-CCA tests. Few studies have also reported that antibody-based tests offer much better sensitivity than microscopy [13–15]. Still, their specificity is much more reduced in communities where Schistosoma infection is co-infected with other related helminths or the availability of the circulating antibodies in some successfully treated individuals [9,13]. For instance, Hoermann and colleagues [15] reported the Schistosoma ICT IgG-IgM rapid diagnostic test to have 100% sensitivity in detecting single or multiple schistosomes, and the test indicated 100% specificity in serum from healthy individuals, while samples with helminths related to schistosomes lowered specificity to 85%. Discrepancies in the performance of the existing tests and the insufficient data to support the feasibility of using any new diagnostics beyond the conventional tools for diagnosis, KK, POC-CCA, and urine microscopy [2], make an open call for developing new diagnostic approaches for schistosome infections and evaluating existing ones.

Mis-diagnosis of male and female genital schistosomiasis. In 2015, the WHO published a pocket atlas for clinical healthcare professionals, highlighting the potential health complications related to female genital schistosomiasis (FGS) [47]. According to the WHO, genital schistosomiasis affects females and males, but the clinical signs and complications are much more prevalent in women. It is estimated that 56 million women and girls in the African region may be affected by FGS [48,49]. Furthermore, WHO recognizes existing complications in diagnosing FGS, pointing out that not every woman with schistosome eggs in their urine will have FGS, and not everyone with FGS has detectable eggs in their urine [48,50]. The WHO recognizes S. haematobium as the major cause of FGS, and most patients and health workers confuse the disease with infertility, cancer, and sexually transmitted infections (STIs) [16,48]. A retrospective study conducted in Tanzania also indicated an increase in the trend of schistosomiasis-related cancers [4] that might be confused with male and female genital schistosomiasis, hence necessitating the need for the urgent need for appropriate diagnostics.

Nature of samples used for diagnosing schistosomiasis. For improved sensitivity, WHO recommends using urine and stool samples to diagnose intestinal and urogenital schistosomiasis, respectively [2,9]. These samples are noninvasive to patients, but the odor they produce during diagnosis can concern health practitioners and reduce the frequency of disease monitoring. The major challenge for changing samples for diagnosing schistosomiasis is associated with invasiveness and low sensitivity when other samples, such as blood serum, dried blood samples (DBS), finger prick blood, and saliva, are used, depending on the test of choice [9,13,21,41,51]. For instance, in a study conducted by Fuss and colleagues [41], serum-based qPCR missed 4 out of 86 positive (4.6%), while DBS-qPCR failed to detect 55% of positive samples, and the detection performance varied based on infection intensities. In the same study, the authors reported that POC-CCA performed much better with detection sensitivity (96.6%) compared to serum-qPCR (89.3%) and DBS-qPCR (65.5%) for the 100 to 399 eggs per gram of KK confirmed stool samples. The explanation for the reduced sensitivity of NAATs-based blood serum, DBS, and saliva tests is likely that the amount of circulating DNA, which mainly originate from Schistosoma eggs, is lower in cell-free samples than in stool and urine samples [21,52]. For antibody and antigen-based assays, detecting antibodies requires blood samples, and CCA and CAA-based tests shows much better sensitivity using serum and urine [15,27,30,53,54]. Still, serum samples fit nicely with clinical settings where the use of serum is preferred over urine, as opposed to field surveillance, where people prefer to provide non-invasive samples. It is, therefore, clear that targeting higher sensitivity and the non-invasiveness nature of stool, and patient-friendly urine samples make them the 2 samples significant respective of the sample characteristics.

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