Bilharzia (schistosomiasis) is the second most devastating parasitic disease after malaria, affecting over 240 million people worldwide (World Health Organization [WHO], 2022a), of which 90% reside in Africa (Aula et al., 2021). Schistosoma mansoni and S. haematobium are the significant causes of schistosomiasis, resulting to intestinal and urogenital schistosomiasis, respectively (Aula et al., 2021). Intestinal schistosomiasis causes diarrhea, abdominal pain, bloody stool, and hepatosplenomegaly in advanced cases, and urogenital schistosomiasis manifests through the genital and urinary system, resulting in haematuria and bladder cancer when the disease is left untreated for a prolonged period (Yohana et al., 2023).

Despite the absence of an approved vaccine against schistosomiasis to date, praziquantel still remains the most widely used control measure for schistosomes (World Health Organization [WHO], 2022a; Kokaliaris et al., 2022), imposing the need for alternative species-specific and genus-specific diagnostic approaches, primarily for Schistosoma species-specific surveillance and elimination purposes, respectively. To date, despite its low sensitivity, microscopy remains the gold standard approach for diagnosing schistosomiasis, and this approach offers high specificity compared to the existing diagnostic approaches. The WHO’s recommendation on the use of microscopy Kato-Katz (KK) and urine examination is restricted to individuals with moderate to high parasite loads for consistent results. In individual with low parasite load, the number of schistosome eggs in stool and urine decreases, making it difficult to detect by microscopic KK or urine examination. Alternative methods have been evaluated including circulating cathodic antigen (CCA), the most common point-of care assay for diagnosing schistosomiasis, and the commercial version is available for field diagnosis (Hoekstra et al., 2021; De Dood et al., 2018). However, research findings and WHO reports have documented the significant bias of the CCA test for S. mansoni over other species and further batch-to-batch variation makes it difficult to accurately estimate the actual prevalence using CCA test, especially in low infectious settings (Hoekstra et al., 2021; World Health Organization [WHO], 2021). In a study by Graeff-Teixeira et al. (2021), for instance, POC-CCA resulted in 62.1% specificity, but when the samples were tested using two different batches and after being stored at -20°C, the specificities for the two batches were 34.3% and 75.0%, respectively. Herein, the weak agreement was an indicator of the urgent need for the standardization of the manufacturing process of the POC-CCA tests. Straily et al. (2022), as well, found that the POC-CCA assay often gave false negative results for samples that KK had confirmed to be positive. In their study, the authors reported that 356 KK-confirmed samples were designated as negative by POC-CCA test, representing a 2.8% false negative rate for all tested samples, with 326 (91.6%) of these samples originating from low-intensity individuals.

Nucleic acid amplification tests (NAATs) are also proposed to offer good sensitivity than microscopy, and real‐time polymerase chain reaction (qPCR), loop mediated isothermal amplification (LAMP) and conventional PCR (cPCR) are most common approaches for surveillance of schistosomes (Fernández-Soto et al., 2020; Weerakoon et al., 2015; Chala, 2023). Still, the sensitivity of NAATs can be significantly affected by the copy number of amplification targets (Raju et al., 2019), resulting in an underestimation of true Schistosoma infections (Hoekstra et al., 2022; Keller et al., 2020), especially in low transmission settings. The WHO, through its guidelines for control and elimination of human schistosomiasis, published online in 2022 (World Health Organization [WHO], 2022b), pointed that there is the need for further evaluation of sensitivity of NAATs, as little is known on their performance in comparison to the gold standard, KK and urine examination. Few studies however, have indicated the possibility of some NAATs, including qPCR, to designate negative to some microscopy-confirmed positive samples, particularly in low infectious burden (Hoekstra et al., 2022; Meurs et al., 2015; Feleke et al., 2023).

One study found that qPCR targeting the Dra1 repeat unit in S. haematobium had overall sensitivity of 89.5%, and 82.8% specificity, and the author reported that the assay missed 11 out of 105 urine examined positive samples, while the sensitivity increased to 96.5%, when samples with ≥50 eggs per gram (epg) were re-evaluated (Keller et al., 2020). Another study reported the ITS2 qPCR to miss some positive samples, and the author found that the sensitivity ranged from 79% to 87% for low infection burden (1-99 epg), 83% to 97% for moderate infection (100-399 epg), and 100% for heavy infection (≥400 epg) (Meurs et al., 2015). Hoekstra et al. (2022) and Armoo et al. (2020) reported a reduced sensitivity for qPCR compared to Up-Converting reporter Particle technology based, Lateral Flow (UCP-LF) CAA test and the urine-CCA assay, respectively. Unlike the CCA test, which is WHO-recommended for S. mansoni detection, the CAA test is reported to offer much better sensitivity against S. haematobium (De Dood et al., 2018; Hoekstra et al., 2022; Fasogbon et al., 2023). When Hoekstra et al., 2022 looked at the performance of the CAA test, urine microscopy, and PCR test, the authors found that more people were testing positive in light infections individual when UCP-CAA test was used instead of PCR (Hoekstra et al., 2022). Still, further studies are needed to validate and contextualize the use of the CAA test at POC settings (Fasogbon et al., 2023).

Isothermal amplification assays have also indicated comparable to much more sensitivity than the cPCR and qPCR (MacGregor et al., 2023; García-Bernalt Diego et al., 2021). For example, when LAMP was compared to other tests, findings indicated its better performance in sensitivity compared to nested PCR and KK in snail and human samples, respectively (Gandasegui et al., 2018). WHO (2022), however, adapted the findings from a systematic review and meta-analysis articles, on the performance of two LAMP assays, one with 87% sensitivity and 50% specificity against KK and the other with 66% sensitivity and 79% specificity based on the urine examination results (World Health Organization [WHO], 2022b). In this instance, the relatively low to moderate specificity may have resulted from the fact that microscopy is less sensitive, but the issue of low to moderate sensitivity of LAMP against samples with Schistosoma eggs needs attention to lower the possibility of false negatives. The need for a single temperature and shorter amplification times, most takes approximately 30 minutes (García-Bernalt Diego et al., 2021), gives isothermal amplification assays more potential to replace NAATs that need expensive machines (MacGregor et al., 2023; Gandasegui et al., 2015). However, the need for multiple pairs of primers complicates the initial optimization, and the reported high chance of carry-over contamination raises the risk of false positives in LAMP reactions (Quyen et al., 2022; Zen et al., 2020), which necessitates the need for cPCR and qPCR, especially in well-established laboratories with trained staff.

On the other hand, lengthy qPCR run times pose challenges when dealing with large sample volumes and limited resources. In most cases, a standard qPCR run takes approximately 45 to 120 minutes to complete (Nayab et al., 2008; Bustin and Nolan, 2020), and this includes the time taken for initial denaturation to activate the modified hot-start DNA polymerase, usually followed by 30-50 cycles of denaturation, annealing, and extension (Nayab et al., 2008; Aryeetey et al., 2013). Despite the enormous advances in qPCR machines and improved polymerization speed and processivity of polymerases, most existing qPCR assays takes approximately one hour on average (Bustin and Nolan, 2020).

In developing genus-specific NAATs, the primary concern is the availability of a shared nucleotide sequence among the pathogens or parasites of interest. Schistosomes, however, have a relatively large genome size, with about 409.57 megabase (mb) (49.23%) repeat contents and 400.27 mb (54.57%) repeat contents in S. mansoni and S. haematobium, respectively, and most nucleotide sequences are shared among the two species (Stroehlein et al., 2022). With the advances in genome-mining approaches that allow the identification of the repeated sequences in genomes, it is possible to improve the sensitivity of the NAATs-based genus-specific assay for diagnosing schistosomiasis, targeting the repeated sequences in schistosomes. In addition, the availability of modified hot-start polymerases, requiring 1 to 2 minutes activation time, and the enhanced performance speed ensures the reduction of qPCR run time without affecting the assay’s sensitivity.

To improve sensitivity and specificity of molecular diagnostic assays, Raju et al. (2019) proposed novel genome mining approach using the IMRS algorithm to identify new, pathogen-specific biomarker target regions called IMRS and designed nucleic acid-amplification assays using IMRS as primers. The IMRS initiate amplification from multiple loci across the genome improving overall analytical sensitivity. The IMRS platform technology was used to design ultrasensitive qPCR (Raju et al., 2019), iso-IMRS isothermal amplification assay for Plasmodium falciparum malaria (Kolluri et al., 2021) and other commercial TaqMan IVD assays (unpublished). In this study, we have used the IMRS genome mining approach to identify all possible repeat regions in the genomes of S. mansoni and S. haematobium and designed TaqMan qPCR assay by using best ranked IMRS region. This assay was 10-fold more sensitive than conventional assay targeting 16S rRNA for simultaneous detection of S. mansoni and S. haematobium with a time-to-result of 36 minutes. Herein, we report our findings.

Herein, we describe a rapid and sensitive qPCR assay for detecting schistosomes, leveraging on IMRS, and how targeting repeated sequences is essential for parasites with large genomes. Theoretically, the copy number of amplification targets is inversely proportional to the genome size (Formula 1). Hence, large genome sizes can significantly reduce the sensitivity of NAATs, especially when an assay has a single target in a given genome. The use of primers that target identical multi-repeat sequences (IMRS) is a significant way to reduce the impact of low genome copies, and this approach has resulted in an improved sensitivity in the detection of P. falciparum using intercalating-dye based real-time PCR (Raju et al., 2019) and isothermal amplification (Kolluri et al., 2021).

The in silico validation using NCBI-BLAST and UCSC in silico PCR indicated that the designed IMRS primers and probe are specific to the Schistosoma genus, being able to detect all schistosomes of medical importance except S. japonicum (World Health Organization [WHO], 2022c), and have multiple repeat targets in both S. mansoni and S. haematobium. These repeat targets were based on amplifiable region limited to less than 1000 bp with no mismatch because for the time-limited amplification, too large amplicons lower the efficiency of NAATs (Van Holm et al., 2021). The IMRS primers chosen for conventional PCR were amplifying complete 151 bp repeat region, whereas the primers chosen for qPCR were amplifying a smaller region of 101 bp within the 151 bp to increase the efficiency of qPCR.

In IMRS conventional PCR, the amplification of approximately 151 bp enabled detection as low as 0.0001 ng/µL of standard genomic DNA from S. mansoni and S. haematobium and 100% sensitivity for detecting positive Schistosoma snail and baboon stool samples. This represents a detection of as little as a single genome copy per microliter and a tenfold improvement over conventional PCR, targeting 143 bp in S. haematobium. The findings on the improved sensitivity of IMRS-based NAATs over the conventional approach are supported by other studies (Raju et al., 2019; Kolluri et al., 2021).

For the IMRS qPCR, the aim was to lower the amplification time to at most 40 minutes, as opposed to most standard qPCR assays, which takes up to 1 hour on average (Nayab et al., 2008; Bustin and Nolan, 2020). To attain abovementioned objective, the fast-cycling conditions were applied as described by Milosevic et al. (2021). In their study, however, Milosevic et al. (2021) managed to develop an ultra-sensitive reverse transcriptase (RT) qPCR, which could detect as little as 25 copies of SARS-CoV-2 RNA genome in 30 minutes. For our study, the qPCR detected as little as one genome copy per microliter of Schistosoma genome within 36 minutes. The difference in polymerase enzyme used accounts for six minutes amplification time difference. The SpeExcelTAR HS DNA Polymerase used by Milosevic et al. is optimized for a 10s/kb extension rate, while the standard DNA polymerase has a 60s/kb extension rate (Milosevic et al., 2021). Reducing the amplification time in qPCR will enable processing of many samples in a reasonable time, especially for surveillance studies.

The 36 minutes amplification time was based on the regression analysis, whereby there were no significant differences in amplification efficiency for the qPCR which took 47, 41 and 36 minutes, all giving R2 > 0.99 ( Figure 3 ), an acceptable amplification efficiency (90-110%), and as low as 0.0001 ng/µL was detected. When tested with field samples, the IMRS qPCR was positive for all positive Schistosoma snail samples, and negative Schistosoma snail samples that were positive for IMRS PCR turned positive with IMRS qPCR ( Supplementary Figure 9 ). This indicated agreement between IMRS PCR and IMRS qPCR. In most cases, however, the no template control (NTC) gave late ct value caused by background fluorescence ( Figure 4 ), and therefore, for reliable results, samples must be run in triplicates, and the NTC must be included to rule out positive and negative samples (Keller et al., 2020; Forootan et al., 2017).

The reported false negatives when NAATs are used to detect schistosomes from human stool and urine (Hoekstra et al., 2022; Meurs et al., 2015) might have been caused by the large genome size, which significantly reduces the copy number of amplification targets, and hence targeting many identical repeats should enhance sensitivity much better than targeting single to very few targets. Therefore, the ability of IMRS PCR and IMRS qPCR to amplify as little as one genome copy per microliter of standard DNA for both S. mansoni and S. haematobium standard DNA, all Schistosoma positive baboon’s stool and snail samples indicates the potential of IMRS primers to diagnose schistosomiasis caused by the two major species, S. mansoni and S. haematobium as well as individual with low parasite levels. Additionally, the IMRS primers detected 3/49 Schistosoma samples, previously confirmed negative by microscopy and cPCR that target 500 bp of the internal transcribed spacer region (ITS1) in schistosomes and 600 bp for snail DNA. This might indicate that ITS1 PCR might have missed some Schistosoma-positive samples.

The LLOD for IMRS PCR and qPCR for S. mansoni and S. haematobium was comparable to much higher than genus-specific LAMP assay for schistosomes that used standard DNA from BEI resources, whereby the authors reported detection limit 0.01 ng/µL, 0.001 ng/µL, 0.001 ng/µL and 0.0001 ng/µL for the detection of S. bovis, S. mansoni, S. intercalatum and S. haematobium, respectively (Fernández-Soto et al., 2020). For most studies that used control DNA extracted from in-house schistosomes, authors reported varying analytical sensitivity, with the detection limit ranging from 0.1 ng/µL to 0.0000001 ng/µL (García-Bernalt Diego et al., 2021; Gandasegui et al., 2015; Crego-Vicente et al., 2021; Abbasi et al., 2010; Kumagai et al., 2010; Rostron et al., 2019). This suggests that more research is needed to compare how well NAATs work in a single controlled setting. For instance, Gandasegui et al. (2015) reported 10 times better sensitivity using Schistosoma DNA from urine samples compared to the Schistosoma positive control from BEI resources, which further show how the DNA control might influence the LLOD estimates, and necessitate the WHO’s call for more data on sensitivity and specificity of NAATs (World Health Organization [WHO], 2022b).

This study has some limitations. First, the study doesn’t have S. mansoni stool samples to compare the IMRS tests with KK and Sm 1–7 PCR, or any human S. haematobium urine samples to compare the tests with Dra1 PCR and urine examination. Additionally, the in silico PCR to confirm the analytical specificity was limited by the genomic data available at primer-BLAST and UCSC genome browser, and only one sample was used to experimentally test the specificity of IMRS primers. Nevertheless, to the best level of our knowledge, the study reports the first rapid, and probe-based IMRS qPCR for schistosomes, and the findings presented might positively impact the development of sensitive assays for detecting parasites, particularly platyhelminths, whose genomes are large, and have many repeated sequences (International Helminth Genomes Consortium, 2019).

Our findings indicate that IMRS primers offer an improved sensitivity than conventional primers and microscopy, therefore the IMRS primers could be used when an improved sensitivity is a primary requirement, especially for parasites with large genomes. Nevertheless, further studies are required to assess the specificity and sensitivity of IMRS-based diagnostics compared to other diagnostic approaches, including qPCR methods, for Schistosoma spp. and Schistosoma-related species. These studies should include statistically significant human samples from areas with both high and low prevalence of circulating single and multiple Schistosoma species. Additionally, the assessment should encompass multiple sample types, such as stool, urine, blood serum, dried blood samples, and saliva.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Mount Kenya University Ethical Review Committee under reference MKU/ISERC/3022, approval date August 2023 and review number 2066. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from gifted from another research group. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements. The animal study was approved by Mount Kenya University Ethical Review Committee under reference MKU/ISERC/3022, approval date August 2023 and review number 2066. The study was conducted in accordance with the local legislation and institutional requirements.

OA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. BK: Conceptualization, Methodology, Resources, Validation, Writing – review & editing. SK: Conceptualization, Software, Writing – review & editing. CS: Conceptualization, Methodology, Resources, Validation, Writing – review & editing. EN: Resources, Validation, Writing – review & editing. MO: Resources, Validation, Writing – review & editing. SN: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – review & editing. CKK: Resources, Writing – review & editing. GM: Conceptualization, Methodology, Writing – review & editing. LO: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – review & editing. SL: Conceptualization, Software, Writing – review & editing. JG: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – review & editing.