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Panzner, U. Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis. Encyclopedia. Available online: https://encyclopedia.pub/entry/21188 (accessed on 27 July 2024).
Panzner U. Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis. Encyclopedia. Available at: https://encyclopedia.pub/entry/21188. Accessed July 27, 2024.
Panzner, Ursula. "Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis" Encyclopedia, https://encyclopedia.pub/entry/21188 (accessed July 27, 2024).
Panzner, U. (2022, March 31). Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis. In Encyclopedia. https://encyclopedia.pub/entry/21188
Panzner, Ursula. "Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis." Encyclopedia. Web. 31 March, 2022.
Peer Reviewed
Clinical Applications of Isothermal Diagnosis for Human Schistosomiasis
This is a peer-reviewed entry published in Encyclopedia Journal, full entry can be found at 10.3390/encyclopedia2020048

About 250 million people affected, 779 million people at risk of infection, and 440 million people with residual morbidity are globally attributable to schistosomiasis. Highly sensitive and specific, simple and fast to perform diagnostics are required for detecting trace infections, and applications in resource-poor settings and large-scale assessments. Research assessing isothermal diagnoses of S. japonicum, S. haematobium, S. mansoni, mixed infections, and schistosomal hybrids among clinical human specimens was investigated. Loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and combined techniques were identified. Both, LAMP and RPA reached species-dependent 100% sensitivity, and detection levels within femtogram and nanogram amounts for pure and hybridale breeds. Cross-reactivity among Schistosoma species and co-endemic pathogens was rare though research on diagnostic markers and primer optimization should continue. Operating with ready-to-use lyophilized reagents, simplified and inexpensive nucleic acid extraction, tolerability to likely inhibitors, and enzyme stability at ambient temperature is advantageous. RPA performed optimal at 35–39ºC within 5–10 minutes while LAMP operated at 61–65ºC for up to 120 minutes; properties are preferable over assays requiring expensive laboratory equipment. DNA degradation could be prevented by stabilizing substances. A limitation throughout warranting future research is the small sample size reaching a few hundred participants at the maximum. Isothermal diagnostics are highly valuable in detecting trace infections seen subsequent to chemotherapeutic treatment, and among apparently healthy individuals, both constituting likely sources of ongoing pathogen transmission. Its expansion to the vaccine field for assessing parasitological trial endpoints could be considered.

schistosomiasis Schistosoma isothermal diagnosis LAMP RPA Submit
Schistosomiasis as a neglected tropical helminthic disease causes large morbidity and mortality. Among the genus Schistosoma, S. haematobium, S. mansoni, and S. japonicum are the most widespread and clinically relevant species afflicting humans. S. haematobium and S. mansoni are prevalent in Africa and the Middle East; S. mansoni occur also and S. japonicum soley in Latin America and the Caribbean, as well as Asia, respectively [1][2]. The species S. mekongi, S. guineensis, S. intercalatum, and S. malayensis also affect humans, but to a lower extent [1][2]. Of growing concern is cross-species hybridization abated by natural and anthropogenic changes; it leads to a large diversity of novel inter-species and/or inter-lineages through instant acquisition of genetic information [3].
The blood-feeding flukes cause globally more than 250 million people affected, over 779 million people at risk of infection, about 440 million people with residual morbidity, and nearly 500,000 annual deaths [2][4][5]. Infections of vertebrates occur during contact with freshwater infested with skin-penetrating schistosomal cercariae disseminated by species-specific snails; larval cercariae transform into schistosomulae and migrate matured to adult worms to their oviposition sites within the vasculature for mating and sexual reproduction; see Figure 1 for more details. Intact adult worms can persist in immunocompetent definitive hosts for decades. Purposefully, eggs are shed via fecal or urinary routes to continue the transmission from vertebrates to molluscs for asexual reproduction upon hatching of miracidia into freshwater; see Figure 1 for more details [2][4][5].
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Figure 1. Generic Schistosoma life cycle.
Acute schistosomiasis or Katayama fever seen among individuals without past parasitic exposure presents as debilitating febrile illness coupled with, e.g., headache, myalgia, fatigue, diarrheal, as well as respiratory symptoms, and hepato- and/or splenomegaly following an incubation period of up to 10–12 weeks. Chronic schistosomiasis manifests as host immunoresponses to unreleased retained eggs, that can lead to long-term complications. Host reactivity presents as bleeding and scarring, chronic inflammations and granulomatous-fibrotic formations around eggs trapped in capillaries damaging species-dependent organs inclusive of liver, intestine, spleen, and the urinary bladder [2][3]. Intestinal chronic schistosomiasis may induce diarrhea or constipation including blood admixture with progression to ulcerations, hyperplasia, polyposis, and fibrosis. S. haematobium causes urogenital pathologies such as dysuria and hematuria and so called female genital schistosomiasis (FGS) if eggs are deposited in the female genitalia [6]. FGS impairs fertility, e.g., ectopic pregnancy and miscarriage, and susceptibility to viruses, e.g., human immunodeficiency virus [7] and papillomavirus, and progressing to malignancies augmented by calcification, e.g., squamous cell carcinomas and sandy patches [8][9][10][11][12]. Ectopic excess egg deposition or erroneous migrating adult worms within the central nervous system can induce cognitive and physical impairments as seen among infested children in endemic settings. Praziquantel (PZQ) is commonly used [7] to treat schistosomiasis and eliminate adult schistosomes by changing irreversibly the permeability and stability of their tegument, but it requires prevailing host immune defense mechanisms for complete efficacy [13]. Control and prevention measures should be complemented by vaccination given the advances in this field to achieve long-term protection against transmission, infection, and disease recurrence [8].
Conventional schistosomal diagnostics include microscopy as the gold standard to visualize fecal and urinary eggs, and the detection of antibodies, antigen(s) and genetic information. However, they vary in sensitivity and specificity depending on, e.g., disease status, endemicity/co-endemicity levels, and chemotherapeutic treatment [7], in particular post-treatment [2][14][15][16]. Oviposition is a common marker to indicate active infection. However, egg shedding starting approximately one month post-infection is impacted by day-to-day variations [7], and the acquisition of, e.g., single sexes, infertile females or senile worms, which make this a rather poor indicator [16][17][18]. Despite, reliable diagnostics are essential for disease monitoring to detect in particular low infection levels promoting pathogen transmission [7], and for evaluating the effectiveness of treatment and control measures [1][17]. Consensus exists that an optimal diagnostic tool must be highly sensitive and specific, simple, and fast to perform and interpret also on different specimen types; favored diagnostics should be capable of detecting acute phase and trace infections supporting early treatment, and cost-effective for use also in resource-limited endemic settings [19].
Nucleic acid amplification is a highly valuable tool for simultaneous detection and species identification at day one post-infection [7] since the introduction of the polymerase chain reaction (PCR) [2][15]. However, PCR-based assays require expensive laboratory and technical equipment besides highly skilled personnel, cold chain for reagent storage, and prolonged reaction times [19][20]. This impacts their large-scale implementation as a genetic high-throughput point-of-care diagnostic measure, especially in resource-constrained settings. Isothermal amplification techniques can overcome aforementioned limitations due to their advancement in speed, simplicity, sensitivity, and specificity [1][21][22]. They detect nucleic acid in an exponential manner without constraints of thermal cycling, and are adaptable to multiplex, quantitative real-time, and reverse transcriptase techniques [23][24]. This is because nucleic acid strands are not heat-denaturated to enable primer binding and initiate amplification reactions since a polymerase of, e.g., Bacillus stearothermophilus (Bst), Bacillus subtilis (Bsu), or Staphylococcus aureus (Sau) with strand-displacement enzymatic activity is used [2][21].
An isothermal amplification technique of great interest is the loop-mediated isothermal amplification (LAMP), first reported by Notomi et al. [25]. LAMP detects few copies of genetic material as seen in low-endemic or newly emerging settings and subsequent to mass drug administration (MDA) [1][17]. Assays exist for various pathogens, e.g., Plasmodium falciparum, Babesia spp., Leishmania spp., Trypanosoma brucei, Ascaris lumbricoides, Ancylostoma spp., Necator spp., Taenia spp., and Toxoplasma gondii [16][19][26][27][28][29]. LAMP produces in a one-step reaction large amounts of lengthy double-stranded genetic information with a mutually complementary sequence and an alternate repeated structure at a constant temperature of 60–65 °C during 60 min reaction time on average [30][31]. Dumbbell-shaped DNA with stem-loops at both ends is formed, which activates steps of polymerization and extension [32]. LAMP performs well with simple isothermal equipment, e.g., heat block or water bath [17]. A pair of highly specific internal primers for strand displacement and synthesis, and external primers are required to detect six distinct sequences among the cognate sites [33]. Amplification can be accelerated and targeted regions expanded through additional loop primers [2][25][34]. Amplified products are visualized by agarose gel electrophoresis, turbidity, and colorimetry based on metal ion indicators and dyes causing color changes that are visible to the naked eye or a fluorometer [1][35][36][37][38]. Reagents are storable at ambient temperature, and test reactions are robust against inhibitory compounds in specimens, and variations in pH and temperature [9][39][40].
Another isothermal amplification technique of growing interest is the recombinase polymerase amplification (RPA) [21][41]. It performs similar to LAMP, except it forms in the presence of a recombinase protein derived from, e.g., T4-like bacteriophages or Escherichia coli, and a high molecular crowding agent a recombinase-primer complex; both promote primer invasion into double-stranded DNA at cognate sites [42]. The invasion is stabilized by single-stranded binding proteins. Subsequent polymerization and extension of loop-like DNA are induced by a chain-replacement polymerase [42]. RPA operates at 22–45 °C, though best at 37–42 °C, that allows simple cycling equipment, e.g., incubator, heatblock, chemical heater, body heat, or ambient temperature, during a reaction time of less than 30 min [10][11]. RPA operates on many specimens, e.g., cultured organisms, body fluids, surgical biopsies, organ tissues, and animal or plant products, and with lyophilized reagent pellets [10][20][43]. Amplicons are visualized similar to LAMP or by oligo chromatographic lateral flow strips, and fluorescence-labeled probes, i.e., dT-fluorophores coupled with corresponding dT-quenchers [44].
Research performed on the isothermal detection of human schistosomiasis within clinical applications was investigated and findings are presented hereby. The progression of isothermal techniques as a high-throughput point-of-care diagnostic measure for single- and multiple-species identification, including schistosomal hybrids, is also addressed. Searches performed in PubMed, Embase and Web of Science, and details of assays identified for S. japonicum, S. haematobium, S. mansoni, and mixed infections are delineated in Figure 2, Figure 3, Figure 4 and Figure 5, respectively.
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Figure 2. Articles identified on the isothermal diagnosis of Schistosoma japonicum with details on participants, specimens, assay features, and test evaluation in terms of detection limits, sensitivity, specificity, PPV and NPV versus a comparator test by chronological order of publication date. Articles highlighted in light blue delineate schistosomal diagnosis based on loop-mediated isothermal amplification (LAMP) while articles highlighted in blue delineate schistosomal diagnosis based on recombinase polymerase amplification (RPA). We searched databases of PubMed, Embase and Web of Science for suitable publications on the isothermal diagnosis of schistosomiasis among clinical specimens collected from human subjects by applying the following terms: “schistosomiasis”, “Schistosoma”, “snail fever”, “schistosomiasis MeSH Terms”, ”isothermal”, “diagnosis/diagnostic”, “detection”, and “assay”. The last searches were performed on 31 December 2021. Publications included after removing duplicates, screening titles and abstracts, reading full-texts, and complementing through reference searches were not restricted by time period, but by the availability of full-texts available in English. Experimental animal studies, investigations on intermediate mollusk hosts, reviews and mathematical models were excluded unless considered highly relevant. Abbreviations: S. = Schistosoma, EPG = eggs per gram, KK = Kato-Katz, bp = basepair, min = minutes, EtBr = ethidium bromide, ♂ = male, ♀ = female, PCR = polymerase chain reaction, PPV = positive predictive value, NPV = negative predicitive value, ELISA = enzyme-linked immunosorbent assay, IHA = indirect haemagglutination assay, fg = femtogram, fM = femtomolar, pg = picogram, Bst = Bacillus stearothermophilus, NA = not available, EDTA = ethylenediaminetetraacetic acid, HCl = hydrogen chloride.
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Figure 3. Articles identified on the isothermal diagnosis of Schistosoma haematobium with details on participants, specimens, assay features, and test evaluation in terms of detection limits, sensitivity, specificity, PPV and NPV versus a comparator test by chronological order of publication date. Articles highlighted in light blue delineate schistosomal diagnosis based on loop-mediated isothermal amplification (LAMP) while articles highlighted in blue delineate schistosomal diagnosis based on recombinase polymerase amplification (RPA). We searched databases of PubMed, Embase and Web of Science for suitable publications on the isothermal diagnosis of schistosomiasis among clinical specimens collected from human subjects by applying the following terms: “schistosomiasis”, “Schistosoma”, “snail fever”, “schistosomiasis MeSH Terms”, ”isothermal”, “diagnosis/diagnostic”, “detection”, and “assay”. The last searches were performed on 31 December 2021. Publications included after removing duplicates, screening titles and abstracts, reading full-texts, and complementing through reference searches were not restricted by time period, but by the availability of full-texts available in English. Experimental animal studies, investigations on intermediate mollusk hosts, reviews and mathematical models were excluded unless considered highly relevant. Abbreviations: S. = Schistosoma, bp = basepair, min = minutes, EtBr = ethidium bromide, ♂ = male, ♀ = female, PCR = polymerase chain reaction, PPV = positive predictive value, NPV = negative predicitive value, fg = femtogram, ng = nanogram, Bst = Bacillus stearothermophilus, NA = not available, N = sample size, AM = ante merdien, PM = post meridien.
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Figure 4. Articles identified on the isothermal diagnosis of Schistosoma mansoni with details on participants, specimens, assay features, and test evaluation in terms of detection limits, sensitivity, specificity, PPV and NPV versus a comparator test by chronological order of publication date. Articles highlighted in light blue delineate schistosomal diagnosis based on loop-mediated isothermal amplification (LAMP). We searched databases of PubMed, Embase and Web of Science for suitable publications on the isothermal diagnosis of schistosomiasis among clinical specimens collected from human subjects by applying the following terms: “schistosomiasis”, “Schistosoma”, “snail fever”, “schistosomiasis MeSH Terms”, ”isothermal”, “diagnosis/diagnostic”, “detection”, and “assay”. The last searches were performed on 31 December 2021. Publications included after removing duplicates, screening titles and abstracts, reading full-texts, and complementing through reference searches were not restricted by time period, but by the availability of full-texts available in English. Experimental animal studies, investigations on intermediate mollusk hosts, reviews and mathematical models were excluded unless considered highly relevant. Abbreviations: S. = Schistosoma, KK = Kato-Katz, bp = basepair, min = minutes, EtBr = ethidium bromide, ♂ = male, ♀ = female, PCR = polymerase chain reaction, PPV = positive predictive value, NPV = negative predicitive value, fg = femtogram, Bst = Bacillus stearothermophilus, NA = not available.
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Figure 5. Articles identified on the isothermal diagnosis of mixed Schistosoma infections with details on participants, specimens, assay features, and test evaluation in terms of detection limits, sensitivity, specificity, PPV and NPV versus a comparator test by chronological order of publication date. Articles highlighted in light blue delineate schistosomal diagnosis based on loop-mediated isothermal amplification (LAMP). We searched databases of PubMed, Embase and Web of Science for suitable publications on the isothermal diagnosis of schistosomiasis among clinical specimens collected from human subjects by applying the following terms: “schistosomiasis”, “Schistosoma”, “snail fever”, “schistosomiasis MeSH Terms”, ”isothermal”, “diagnosis/diagnostic”, “detection”, and “assay”. The last searches were performed on 31 December 2021. Publications included after removing duplicates, screening titles and abstracts, reading full-texts, and complementing through reference searches were not restricted by time period, but by the availability of full-texts available in English. Experimental animal studies, investigations on intermediate mollusk hosts, reviews and mathematical models were excluded unless considered highly relevant. Abbreviations: S. = Schistosoma, bp = basepair, min = minutes, EtBr = ethidium bromide, PCR = polymerase chain reaction, PPV = positive predictive value, NPV = negative predicitive value, ng = nanogram, Bst = Bacillus stearothermophilus, NA = not available, AM = ante merdien, PM = post meridien.

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