https://orcid.org/0000-0002-3897-5925
Figures
Abstract
Schistosomiasis, a zoonotic parasitic disease caused by schistosome infection, remains a serious public health concern. However, diagnostic methods with both high sensitivity and specificity or early diagnosis for detecting schistosome infection are still lacking. This study aimed to evaluate the diagnostic potential of parasite-derived exosomes miRNAs in Schistosoma japonicum (S. japonicum) infection. Exosomes were isolated and purified from adult worms and eggs of S. japonicum using ultracentrifugation. Nine highly abundant miRNAs were identified in these exosomes through small RNA sequencing. TaqMan probe-based reverse transcription real-time PCR (RT-qPCR) revealed that serum levels of all nine parasite-derived miRNAs were significantly elevated in infected mice at different time points post-infection. Compared with healthy controls, infected patients exhibited significantly elevated serum levels of sja-miR-61 and sja-miR-7-5p. Receiver operating characteristic (ROC) analysis indicated that the area under the curve (AUC) values for serum sja-miR-61 and sja-miR-7-5p were 0.8750 and 0.8375, respectively, surpassing those of the other miRNAs examined in human serum. The sensitivity and specificity for detecting these miRNAs in human serum were 85.00% and 85.00% for sja-miR-61, and 70.00% and 80.00% for sja-miR-7-5p. When both miRNAs were detected in combination, sensitivity increased to 95.00% with a specificity of 75.00%. In mice, the sensitivity and specificity for detecting sja-miR-7-5p at 2 weeks post-infection were 73.33% and 86.67%, improving to 93.33% and 93.33% at 6 weeks. For sja-miR-61, sensitivity and specificity were 93.33% and 80.00% at 2 weeks, reaching 100.00% and 100.00% at 6 weeks. Serum levels of sja-miR-61 and sja-miR-7-5p dropped to undetectable levels two weeks after praziquantel treatment, underscoring their utility in monitoring treatment response. Furthermore, measurement of sja-miR-61 and sja-miR-7-5p in mice with varying infection intensities showed rising serum levels corresponding to increased worm burdens, with a minimum detection threshold equivalent to an infection with 5 cercariae, highlighting the high sensitivity of this approach for detecting low-level infections in mice. Collectively, the detection of these miRNAs in patient or mouse serum using TaqMan RT-qPCR exhibits good sensitivity and specificity, along with considerable value for early diagnosis and treatment monitoring. sja-miR-61 and sja-miR-7-5p represent promising biomarkers for the diagnosis of schistosomiasis japonica.
Author summary
The diagnosis of schistosomiasis japonica remains challenging due to a lack of sensitive and specific tests. We identified that sja-miR-61 and sja-miR-7-5p, derived from parasite exosomes, serve as excellent circulating biomarkers for Schistosoma japonicum (S. japonicum) infection. In human patients, both miRNAs were significantly elevated in serum and served as effective diagnostic biomarkers. Whereas the individual biomarkers sja-miR-61 and sja-miR-7-5p showed 85.00% sensitivity/specificity (AUC = 0.8750) and 70.00% sensitivity/80.00% specificity (AUC = 0.8375) respectively using TaqMan RT-qPCR, a combined test achieved a markedly superior performance, detecting infection with 95.00% sensitivity and 75.00% specificity. In mice, high sensitivity (100%) and specificity (80%) of the combined test were achieved as early as 2 weeks post-infection. Serum miRNA levels became undetectable within two weeks after praziquantel (PZQ) therapy, allowing for rapid assessment of cure. The assay can detect infections with as few as 5 cercariae, addressing a critical need in low-endemic areas. In conclusion, the detection of serum sja-miR-61 and sja-miR-7-5p via TaqMan RT-qPCR provides a sensitive and specific method for diagnosing schistosomiasis japonica, with significant utility for early diagnosis, treatment efficacy monitoring, and surveillance.
Citation: Zhang X-Y, Li J-P, Lu J-L, Mei C-J, Yang Y-Y, Dong P-P, et al. (2026) Parasite exosomes-derived circulating sja-miR-61 and sja-miR-7-5p as Novel biomarkers for the detection of Schistosoma japonicum infection using TaqMan real-time PCR. PLoS Negl Trop Dis 20(5): e0014368. https://doi.org/10.1371/journal.pntd.0014368
Editor: Murtala Isah, Umaru Musa Yar’Adua University, NIGERIA
Received: October 27, 2025; Accepted: May 12, 2026; Published: May 20, 2026
Copyright: © 2026 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated or analyzed during this study are included in this published article and its supplementary information files. The microRNA-seq data have been deposited in the NCBI Sequence Read Archive database [Accession number: PRJNA1342957] (https://www.ncbi.nlm.nih.gov/sra/PRJNA1342957).
Funding: The research was funded by Key projects of Jiangsu Provincial Health Commission (No. K2023042 to LS); Wuxi Municipal Bureau on Science and Technology (No. Y20222028 to LS); the General Foundation of Wuxi Municipal Health Commission of Jiangsu Province, China (No. M202202 to LS); Jiangsu Province Capability Improvement Project through Science, Technology, and Education (No. ZDXYS202207 to LS); Wuxi Municipal Bureau on Science and Technology (Basic Research, No. K20252014 to CM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Schistosomiasis is a major zoonotic parasitic disease endemic in tropical and subtropical regions. Affecting over 70 countries, it places nearly 800 million people at risk and accounts for more than 70 million disability-adjusted life years (DALYs) lost annually [1,2]. The primary species infecting humans include Schistosoma mansoni (S. mansoni), S. japonicum, and S. haematobium. S. japonicum, found mainly in East and Southeast Asia—such as China, Japan, the Philippines, and Indonesia—is notable for its high daily egg output, reaching up to 3000 eggs per adult worm [3]. A substantial proportion of these eggs lodge in the host liver, triggering granulomatous inflammation and fibrosis that may progress to portal hypertension, hepatosplenomegaly, ascites, and in advanced cases, to cirrhosis and liver cancer, with severe clinical consequences [4,5]. As control measures advance and infection rates decline, schistosome infections are increasingly characterized by low intensity, complicating accurate diagnosis. Thus, developing highly sensitive and specific methods for early detection is essential to prevent disease progression and curb transmission.
The current diagnostic gold standard for schistosomiasis japonica relies on parasitological techniques, primarily the Kato-Katz thick smear and miracidium hatching test. However, these methods exhibit limited sensitivity, particularly in early and low-intensity infections, with false-negative rates as high as 40–50%. They are also susceptible to operator error and require experienced personnel [5]. Immunoassays targeting schistosome circulating antigens offer a means to detect active infection [6], yet these antigens are often present at low concentrations in serum, challenging to measure accurately, and may cross-react, leading to reduced specificity. Alternatively, detection of host antibodies against schistosome antigens is widely used [7], but seroconversion occurs relatively late, limiting utility for early diagnosis, and antibodies may persist after cure, complicating the distinction between past and current infection.
Nucleic acid-based methods have gained attention for their high sensitivity and rapid turnaround, contributing significantly to advances in schistosomiasis diagnostics [8–11]. The performance of nucleic acid tests hinges on the selection of specific and reproducible molecular targets. Although several schistosome DNA sequences have been evaluated for this purpose [12,13], research focused on early infection remains limited, and some existing protocols are relatively complex to implement [14]. Therefore, there is a clear need to develop simpler, more sensitive, and specific diagnostic tools capable of detecting early-stage infection.
Exosomes, identified in cultures of lymphocytes and dendritic cells, are small (30–150 nm in diameter), lipid bilayer–enclosed extracellular vesicles secreted by various cell types [15]. They are detectable in multiple body fluids, including blood, urine, lymph, cerebrospinal fluid, bile, and saliva [16]. Exosome‑encapsulated miRNAs have emerged as promising diagnostic biomarkers owing to their high specificity, sensitivity, species specificity [17], and stability under diverse conditions [18]. Specific profiles of circulating exosome miRNAs enable early detection of several cancers, highlighting their potential in tumor diagnosis and prognosis [19,20]. For example, elevated levels of miR‑212‑5p, miR‑1248, and miR‑1250‑5p in hepatocellular carcinoma (HCC), when combined with clinical parameters such as gender and cirrhosis, yield high diagnostic specificity and sensitivity [21]. Similarly, plasma exosome miRNA‑30a and miRNA‑192 aid in diagnosing alcoholic hepatitis [22], while serum exosome miR‑195‑3p shows promise as a biomarker for osteosarcoma [23].
MiRNAs are also abundantly present in parasite‑derived exosomes [24–27]. For instance, next‑generation sequencing of S. mansoni adult worm exosomes identified 143 miRNAs, among which 25 were confirmed by qPCR to be elevated in sera from infected mice [28]. In S. japonicum–infected rabbits, four schistosome‑specific miRNAs (sja‑miR‑3479, sja‑miR‑10, sja‑miR‑3096, and sja‑miR‑8185) were detected in serum [29], and sja‑miR‑277 and sja‑miR‑3479‑3p were identified in infected mice, suggesting a correlation with infection and pathology [30]. However, studies on the diagnostic utility of parasite‑derived exosome miRNAs remain limited, and no highly sensitive and specific parasite miRNA markers have been established for schistosomiasis diagnosis [31].
In this study, exosomes were isolated from secretions and excretions of S. japonicum adult worms and eggs. Small RNA sequencing revealed a set of highly abundant miRNAs derived from these parasite exosomes. Their expression was preliminarily verified in a mouse model using TaqMan probe-based reverse transcription real-time PCR (RT-qPCR). Subsequent evaluation in both patient and mouse sera demonstrated that detection of circulating sja‑miR‑61 and sja‑miR‑7‑5p offers high sensitivity and specificity. Further analysis confirmed the value of these parasite‑derived miRNAs for monitoring treatment efficacy and identified sja‑miR‑61 as a potential biomarker for early detection. This study provides a scientific foundation for the discovery of diagnostic and early‑detection biomarkers for schistosomiasis, and supports future development of field‑applicable diagnostic kits.
Materials and methods
Ethics statement
All animals including mice and rabbits procedures followed the Guidelines for the Ethical Review of Experimental Animal Welfare (GB/T 35892–2018) and were approved by the Ethics Review Committee of Jiangsu Institute of Parasitic Diseases (Approved numbers: JIPD-2023–002). Schistosomiasis patients and healthy individuals with provided informed consent followed the protocols approved by the Ethics Review Committee of Jiangsu Institute of Parasitic Diseases [32].
Study design
The detection efficacy of miRNAs was evaluated in both human patients and mouse models of Schistosoma japonicum infection using the TaqMan RT-qPCR method [33,34] (Fig 1). First, to analyze the dynamics of highly abundant exosome miRNAs during infection, five C57BL/6J mice were percutaneously infected with 15 ± 1 cercariae each [35,36], which were released from schistosome-infected positive snails, provided by the Snail Room of Jiangsu Institute of Parasitic Diseases. Blood was collected from the facial vein before infection (0 weeks, w) and at 2, 4, 6, 8, 10, 12, and 14 w post-infection (Fig 1A). For validation in human subjects, serum samples were obtained from 20 schistosomiasis patients (confirmed by fecal egg positivity) in Hubei Province, China, and from 20 healthy controls provided by the Health Management Center of Jiangsu Institute of Parasitic Diseases. All samples were aliquoted for subsequent detection (Fig 1B). To assess the potential of parasite-derived miRNAs for early diagnosis, serum was collected from an additional 15 mice at 2 w and 6 w post-infection (Fig 1C). In a treatment monitoring experiment, five infected mice were administered PZQ (Sigma, St. Louis, Missouri, USA), dissolved in a 1% carboxymethyl cellulose solution, 250 mg/kg/day for 3 days by oral gavage at 6 w. Blood was drawn before infection, at 2 w and 4 w during infection, and at 0, 2, 4, 6, and 8 w after treatment (corresponding to total time points of 6–14 w), which was kept at 4 °C overnight, and serum, separated at 2,500 × g for 5 minutes, was used for miRNA analysis (Fig 1D). Finally, to evaluate miRNA performance in low-intensity infections, 35 mice were randomly assigned to seven groups (n = 5) and infected with 0, 5 ± 1, 10 ± 1, 15 ± 1, 20 ± 1, 25 ± 1, or 30 ± 1 cercariae. At 6 w post-infection, blood was collected via the facial vein and processed for serum. Adult worms were then recovered by portal vein perfusion with anticoagulant saline (0.3% sodium citrate, 0.7% NaCl) and counted (Fig 1E).
(A) Collection of serum samples from mice at different time points post-S. japonicum infection. (B) Collection of serum samples from S. japonicum-infected patients and healthy individuals. (C) Collection of serum samples from mice with early S. japonicum infection. (D) Collection of serum samples from mice pre- and post-praziquantel treatment. (E) Collection of serum samples from mice with different infection intensities.
Exosome preparation
Six New Zealand White rabbits (1.5–2.5 kg, conventional grade) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Each rabbit was percutaneously infected with 1,500 S. japonicum cercariae, which were shed from infected Oncomelania hupensis snails provided by the Snail Department of the Jiangsu Institute of Parasitic Diseases. At 42 days post-infection, adult worms were perfused from the superior mesenteric vein using anticoagulant saline (0.3% sodium citrate, 0.7% NaCl), and the liver was excised.
The harvested 200 pairs of adult worms were washed three to five times with 200 mL of PBS and cultured in 40 mL of RPMI 1640 medium (VivaCell Biosciences, Shanghai, China) in a 90 mm culture dish supplemented with 10% exosome-depleted fetal bovine serum, 100 U/mL penicillin (Solarbio, Beijing, China), and 100 µg/mL streptomycin (Solarbio, Beijing, China), in a 5% CO₂ incubator for 48 hours. The excised livers were minced and homogenized in pre-chilled 1.2% NaCl solution using an electric homogenizer. The homogenate was diluted approximately 10-fold with additional cold 1.2% NaCl solution and sequentially filtered through 80-, 150-, and 200-mesh sieves. The resulting filtrate was collected in a 300-mesh nylon bag, and after water was removed, the eggs retained in the bag were harvested. The egg pellet was resuspended in 100 mL ice-cold 1.2% NaCl solution and centrifuged at 100 × g for approximately 1 minute. The supernatant and upper white layer were discarded, and the lower yellow layer containing the eggs was collected. The eggs were then digested with 0.05% pre-chilled collagenase IV (Sigma, St. Louis, Missouri, USA) in PBS at 37 °C for 1.5 hours [37], washed with RPMI 1640 medium containing 10% exosome-depleted serum (VivaCell Biosciences, Shanghai, China), and collected by centrifugation at 1,500 × g, then counted under a microscope. Finally, 5 × 104 of eggs were incubated in 40 mL of RPMI 1640 medium in a 90 mm culture dish with 10% exosome-depleted serum in a 5% CO₂ incubator for 48 hours.
All the culture supernatants (40 mL) from both adult worms and eggs (after 48-hour culture in exosome-depleted serum medium) were collected. Exosomes were isolated using sequential ultracentrifugation: first at 300 × g for 10 minutes at 4 °C to remove cells, followed by 1,500 × g for 30 minutes to pellet debris. The supernatant was then subjected to ultracentrifugation at 120,000 × g for 70 minutes. The resulting exosome pellet was collected and stored at –80 °C. Three replicate samples were taken from a single pool of adult and egg exosomes.
Exosome characterization and small RNA sequencing
Collected adult worm and egg-derived exosomes were shipped on dry ice to Shanghai OE Biotech Co., Ltd. Morphology was examined using a HT7700 transmission electron microscope (TEM) at an accelerating voltage of 100 kV. Particle size distribution was determined using a NanoFCM N30E.
Three replicate samples each of adult worm and egg exosomes were sent to Shanghai OE Biotech Co., Ltd. for small RNA sequencing analysis on an Illumina high-throughput next-generation sequencing platform to identify miRNA profiles. Briefly, total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Texas, USA) following the manufacturer’s protocol. RNA quantity and integrity were assessed using a Nanodrop 2000 (Thermo Fisher Scientific, Massachusetts, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA), respectively. For each sample, 1 µg of total RNA was used to construct small RNA libraries using the NEBNext Small RNA Library Prep Set for Illumina (NEB, Massachusetts, USA). Adapters were ligated to both ends of the RNA, followed by reverse transcription to cDNA and PCR amplification. PCR products (~140–160 bp) were size-selected and purified by agarose gel electrophoresis to generate the final small RNA libraries. Library quality was verified using the Agilent 2100 Bioanalyzer before sequencing on the Illumina Novaseq 6000 platform. Raw sequencing data (RawData/RawReads) generated from base calling were processed to remove low-quality reads, reads containing 5’ primer contaminants or poly(A) tails, reads without 3’ adapters or insert tags, and reads shorter than 15 nt or longer than 41 nt, resulting in clean reads. Raw sequencing data were submitted to the NCBI SRA database (Accession number: PRJNA1342957).
For small RNA analysis, clean reads were first analyzed for length distribution against the reference genome. Bowtie software was used to align sequences against the Rfam v10.1 database (http://www.sanger.ac.uk/software/Rfam) to annotate and filter out rRNA, scRNA, cis-regulatory elements, snRNA, tRNA, etc. Subsequent alignments were performed sequentially against cDNA sequences, the Repbase database for repetitive sequences, and the miRBase database (http://www.mirbase.org/) to remove degraded transcript sequences, repeats, and to identify and annotate known miRNAs. Expression patterns of known miRNAs across samples were analyzed. Novel miRNAs were predicted from the remaining unannotated reads using miRDeep2 software, which identifies potential miRNA precursors based on hairpin structures and known miRNAs in miRBase, predicting mature and star sequences.
SYBR Green RT-qPCR
RNA was extracted from 100 mg of cercariae, adult worms, and eggs using a tissue RNA extraction kit (SparkJade, Jinan, China), and 500 ng of RNA was reverse-transcribed into cDNA using a miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The miRNA at the cercariae, adult worm, and egg stages were amplified using miRNA Unimodal SYBR qPCR Master Mix (Vazyme, Nanjing, China), with amplification primers listed in S1 Table. The reaction conditions were: 95 °C 30 s; 95 °C 10 s, 60 °C 30 s, 40 cycles. The relative expression levels were calculated by the 2-ΔΔCt method using U6 as a control [38].
TaqMan RT-qPCR
Total miRNA was extracted from 200 µL of mouse serum or 300 µL of human serum using a Plasma miRNA Extraction Kit (HaiGene, B1804, Harbin, China). Extracted miRNA was reverse transcribed into cDNA using the TaqMan miRNA cDNA Synthesis Kit (Serum/Plasma) (HaiGene, D1803, Harbin, China). Custom-designed HG TaqMan miRNA qPCR Assays (HaiGene, Harbin, China) were used for TaqMan probe-based qPCR amplification of target miRNAs (1,250 ng) from cDNA samples of infected mouse serum, patient serum, and controls. The reaction mixture (20 µL total) contained: 4 µL 5 × Golden HS TaqMan qPCR Mix, 1 µL 10 × miRNA TaqMan Assay, 2.5 µL cDNA template, and 12.5 µL ddH₂O. Thermal cycling conditions were: 95°C for 15 min; 40–45 cycles of 95°C for 10 s and 60°C for 30 s. Amplification and cycle threshold (Ct) calculation were performed using an Applied Biosystems QuantStudio 6 & 7 Pro instrument (Thermo Fisher Scientific, Massachusetts, USA). Samples with Ct values > 36 were excluded. miR-16 was used as the housekeeping gene [39,40]. The amplified products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing to confirm their specificity.
Statistical analysis
Data analysis was performed using GraphPad Prism 9.5. Normally distributed measurement data were expressed as mean ± standard deviation. Group comparisons were conducted using the t-test. To account for inter-plate variability, Ct values for all samples were normalized using the formula: Normalized Ct value of sample miRNA = Raw Ct value of sample miRNA × (Mean Ct value of reference gene in healthy individuals / Mean Ct value of reference gene in the sample). The mean Ct value for each miRNA across all healthy individuals was calculated, and the ΔCt value for each sample was obtained by subtracting the normalized sample Ct value from the mean Ct value of healthy controls. These ΔCt values were used to evaluate the diagnostic performance of each miRNA [31].
Receiver operating characteristic (ROC) curves were constructed using ΔCt values to evaluate the diagnostic performance of microRNAs in discriminating between schistosome-infected patients and healthy controls, as well as between pre- and post-infection mice. The area under the curve (AUC), sensitivity, and specificity were calculated. The optimal cutoff value on each ROC curve was selected as the point yielding the maximum Youden’s index (Youden’s index = Sensitivity + Specificity – 1). In cases where multiple points shared the same Youden’s index, the one with the higher sensitivity was preferred [41,42]. A sample was classified as positive if its ΔCt value was greater than the established cutoff. For combined diagnostic evaluation, a sample was deemed positive if the ΔCt value of either sja-miR-61 or sja-miR-7-5p exceeded its respective cutoff. A P value less than 0.05 was considered statistically significant.
Results
Morphological characteristics and miRNA composition of exosomes derived from S. japonicum adults and eggs
Exosomes from S. japonicum adult worms and eggs were isolated and purified by ultracentrifugation. TEM analysis revealed that both adult worm-derived and egg-derived exosomes exhibited a cup-shaped, spherical morphology with a double-membrane structure, consistent with typical exosome morphology (Fig 2A, B). Nanoparticle tracking analysis showed particle sizes ranging from 50 to 150 nm, with average sizes of 86.98 nm for adult worm exosomes (Fig 2C) and 82.60 nm for egg exosomes (Fig 2D).
A: TEM image of adult worm-derived exosomes; B: TEM image of egg-derived exosomes; C: Size distribution of adult worm-derived exosomes; D: Size distribution of egg-derived exosomes.
Illumina high-throughput next-generation sequencing was used to determine the miRNA profiles in adult worm and egg exosomes. All identified miRNA sequences and their read counts are provided in S2 and S3 Tables. The top 5 most abundant miRNAs (by read count) in adult worm exosomes were sja-miR-125b, sja-miR-71a, sja-miR-125, sja-miR-61, and sja-miR-36-3p. The top 5 most abundant miRNAs in egg exosomes were sja-miR-36-3p, sja-miR-1, sja-miR-71b-5p, sja-bantam, and sja-miR-7-5p. Notably, sja-miR-36-3p was highly abundant in both adult worm and egg exosomes. Sequences, genomic locations, and read counts for these 9 miRNAs are summarized in Table 1. The expression of these miRNAs in schistosome cercariae, adults, and eggs was detected using the SYBR Green RT-qPCR. The results showed that sja-miR-125b, sja-miR-71a, sja-miR-125, and sja-miR-61 were most highly expressed in the adult stage, whereas sja-miR-36-3p, sja-miR-1, sja-miR-71b-5p, sja-bantam, and sja-miR-7-5p were most highly expressed in the egg stage, consistent with the sequencing results. Furthermore, sja-miR-71a, sja-miR-125, sja-miR-61, sja-miR-1, sja-bantam, and sja-miR-7-5p were also expressed at the cercariae stage (S1 Fig).
Levels of high-abundance parasite-derived exosomal miRNAs in mouse sera at different stages of schistosome infection
To evaluate the diagnostic potential of these high-abundance miRNAs, specific TaqMan probes were used in RT-qPCR to measure their levels in mouse sera collected pre-infection (0 w) and at 2, 4, 6, 8, 10, 12, and 14 w. Compared to pre-infection levels, all nine miRNAs were significantly elevated in sera from infected mice. The accuracy of the sequence was verified by sequencing the amplified products. The sequencing results were consistent with the 5’ and central sequences of these miRNAs, with 2–3 base inconsistencies at the 3’ end (S2 Fig), which was related to the presence of a 3’ isomiR of miRNA [43]. Among adult worm exosome-derived miRNAs, sja-miR-125b (Fig 3A), sja-miR-125 (Fig 3C), and sja-miR-61 (Fig 3D) peaked before 6 w, with fluctuating but persistently elevated levels thereafter. sja-miR-71a (Fig 3B) peaked as early as 2 w, suggesting high expression during the schistosomula stage. Egg exosome-derived miRNAs—sja-miR-36-3p (Fig 3E), sja-miR-1 (Fig 3F), sja-miR-71b-5p (Fig 3G), sja-bantam (Fig 3H), and sja-miR-7-5p (Fig 3I)—also increased gradually post-infection but peaked later, between 10 and 14 w. This delayed peak likely correlates with egg production commencing around 3 weeks post-infection and subsequent accumulation. These results demonstrate significantly elevated serum levels of all nine miRNAs from 2 w post-infection onwards compared to pre-infection (all p < 0.05, Fig 3), indicating their potential diagnostic value.
A: sja-miR-125b; B: sja-miR-71a; C: sja-miR-125; D: sja-miR-61; E: sja-miR-36-3p; F: sja-miR-1; G: sja-miR-71b-5p; H: sja-bantam; I: sja-miR-7-5p (Compared with pre-infection, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant).
Diagnostic performance of TaqMan RT-qPCR for detecting parasite-derived exosomal miRNAs in sera of schistosome-infected patients
To validate the diagnostic value of these miRNAs in human infection, their levels were measured in human sera using TaqMan RT-qPCR. sja-miR-125 and sja-miR-125b were not amplified in patient sera. After adding these two miRNAs to human serum samples respectively, the corresponding miRNA was amplified, which confirmed that the absence was true (S3 Fig). The remaining seven miRNAs were detected (Fig 4A-G). Compared to healthy controls, the mean ΔCt values for sja-miR-71a (Fig 4A), sja-miR-61 (Fig 4B), sja-miR-36-3p (Fig 4C), sja-miR-1 (Fig 4D), sja-miR-71b-5p (Fig 4E), and sja-miR-7-5p (Fig 4G) were significantly higher in patient sera (all p < 0.05). The differences in levels (mean ΔCt) for sja-miR-61 and sja-miR-7-5p between patients and controls were more pronounced than for the other miRNAs. In contrast, the level of sja-bantam (Fig 4F) did not differ significantly between groups (p > 0.05).
Comparison of levels between S. japonicum patient and healthy control sera for (A) sja-miR-71a, (B) sja-miR-61, (C) sja-miR-36-3p, (D) sja-miR-1, (E) sja-miR-71b-5p, (F) sja-bantam, (G) sja-miR-7-5p. Receiver operating characteristic (ROC) curves for the detection of the 7 miRNAs in patient vs. healthy control sera: (H) sja-miR-71a, (I) sja-miR-61, (J) sja-miR-36-3p, (K) sja-miR-1, (L) sja-miR-71b-5p, (M) sja-bantam, (N) sja-miR-7-5p (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant).
Diagnostic performance was assessed using ROC curves and AUC analysis. The AUC indicates the ability of a miRNA to discriminate between diseased and healthy populations, with values closer to 1.0 indicating better performance. Analysis showed that sja-miR-61 and sja-miR-7-5p had the highest AUC values, 0.8750 and 0.8375, respectively (Fig 4H-N). Detecting sja-miR-61 in patient serum yielded 17 positives out of 20 (85.00% positivity rate); among 20 healthy controls, 3 were positive (15.00% false positive rate), resulting in 85.00% sensitivity and 85% specificity. For sja-miR-7-5p, 14 out of 20 patients were positive (70.00% positivity rate), and 4 out of 20 controls were positive (20.00% false positive rate), yielding 70.00% sensitivity and 80.00% specificity (Table 2). Combined detection of both miRNAs increased sensitivity to 95.00% with a specificity of 75.00% (Table 2). These findings suggest that sja-miR-61 and sja-miR-7-5p offer good sensitivity and specificity for diagnosing human schistosome infection, and their combined use enhances detection sensitivity.
Detection of sja-miR-61 and sja-miR-7-5p in sera of mice at 2 and 6 weeks post-infection demonstrates value for early diagnosis
Given the promising sensitivity and specificity observed in human studies, we further investigated the potential of sja-miR-61 and sja-miR-7-5p for early diagnosis. Their levels were measured in mouse sera pre-infection and at 2 and 6 w using TaqMan RT-qPCR. ROC curve analysis based on ΔCt values was used to evaluate diagnostic performance.
At 2 weeks post-infection, detection of sja-miR-61 yielded 14 positives out of 15 infected mice (93.33% positivity rate), while sja-miR-7-5p detection yielded 11 positives (73.33% positivity rate) (Table 3). At 6 weeks post-infection, sja-miR-61 was detected in all 15 infected mice (100.00% positivity), and sja-miR-7-5p was detected in 14 out of 15 mice (93.33% positivity) (Table 4).
ROC analysis of the 2-week samples yielded an AUC of 0.9156 (95% CI: 0.8187-1.000, p < 0.0001) for sja-miR-61, with an optimal cut-off of 0.6389 demonstrating 93.33% sensitivity and 80.00% specificity. In contrast, sja-miR-7-5p showed an AUC of 0.8778 (95% CI: 0.7516-1.000, p ＜ 0.01), for which a cut-off of 0.4900 provided 73.33% sensitivity and 86.67% specificity (Table 3). At the 6-week time point, sja-miR-61 achieved a perfect AUC of 1.000 (95% CI: 1.000-1.000, p ＜ 0.0001) with a cut-off of 0.9855, resulting in 100.00% sensitivity and specificity. Similarly, sja-miR-7-5p exhibited an AUC of 0.9778 (95% CI: 0.9349-1.000, p ＜ 0.0001), and a cut-off of 0.8000 yielded 93.33% for both sensitivity and specificity (Fig 5) (Table 4).
(A) ROC curve for sja-miR-61 detection at 2 weeks post infection; (B) ROC curve for sja-miR-7-5p detection at 2 weeks post infection; (C) Comparison of ROC curves for combined detection of sja-miR-7-5p and sja-miR-61 at 2 weeks post infection; (D) ROC curve for sja-miR-61 detection at 6 weeks post infection; (E) ROC curve for sja-miR-7-5p detection at 6 weeks post infection; (F) Comparison of ROC curves for combined detection of sja-miR-7-5p and sja-miR-61 at 6 weeks post infection.
These results establish sja-miR-61 as a valuable biomarker for the early diagnosis of schistosome infection, demonstrating robust sensitivity and specificity as early as 2 weeks post-infection. Furthermore, a combined detection approach utilizing both miRNAs increased the sensitivity to 100.00% (with a specificity of 80.00%) for 2-week samples and to 100.00% (with a specificity of 93.33%) for 6-week samples, thereby confirming that a dual-miRNA strategy can significantly enhance diagnostic sensitivity.
Value of Detecting Serum sja-miR-61 and sja-miR-7-5p Levels for Assessing PZQ Treatment Efficacy in Infected Mice
Levels of sja-miR-61 and sja-miR-7-5p were measured by TaqMan RT-qPCR in sera from mice collected pre- and post-PZQ treatment (starting at 6 w). Using the ΔCt cut-off values established for 6 w pre-treatment samples as the positivity standard, results showed that both miRNA levels dropped to negative levels by 2 weeks post-treatment. The negative conversion rate was 100.00% for both sja-miR-61 and sja-miR-7-5p. In contrast, miRNA levels remained high in untreated control mice, with statistically significant differences persisting until the end of the observation period at 14 w (Fig 6A-B). These results suggest that detecting serum levels of these schistosome exosome-derived miRNAs holds promise for evaluating the efficacy of PZQ treatment.
Mice were treated with praziquantel at 6 weeks post infection in PZQ group. A: sja-miR-61; B: sja-miR-7-5p (Infection compared with PZQ group, ** p ＜ 0.01, *** p ＜ 0.001, **** p ＜ 0.0001, ns, not significant).
Circulating serum sja-miR-61 and sja-miR-7-5p as biomarkers for low-intensity schistosome infection in mice
To further validate the diagnostic value of sja-miR-61 and sja-miR-7-5p for low-intensity infections, mice were infected with 5 ± 1, 10 ± 1, 15 ± 1, 20 ± 1, 25 ± 1, or 30 ± 1 cercariae. Sera were collected at 6 weeks post-infection, followed by perfusion of adult worms. The mean adult worm counts were 2.0 ± 2.1, 4.4 ± 1.5, 6.3 ± 2.7, 10.4 ± 2.9, 16.8 ± 4.5, and 21.5 ± 2.5 worms, respectively (Fig 7A). TaqMan qPCR analysis revealed gradually increasing serum levels of both sja-miR-61 and sja-miR-7-5p with higher infection intensities. Applying the ΔCt cut-off values determined for 6 weeks post-infection, at the lowest infection level (5 cercariae), detection of sja-miR-7-5p and sja-miR-61 each yielded 3 positives out of 5 mice (60.00% positivity rate). In the 10-cercariae group, sja-miR-7-5p was detected in 4 out of 5 mice (80.00% positivity), while sja-miR-61 was detected in all 5 mice (100.00% positivity). With the exception of one mouse in the 25-cercariae group testing negative for sja-miR-7-5p and one mouse in the 15-cercariae group testing negative for sja-miR-61, all other mice in the 15, 20, 25, and 30 cercariae groups tested positive for both miRNAs. Circulating sja-miR-7-5p and sja-miR-61 were detectable in mice infected with just 5 cercariae, with levels significantly different from pre-infection levels (p < 0.05) (Fig 7B, C). This indicates that sja-miR-61 and sja-miR-7-5p can serve as nucleic acid biomarkers for diagnosing low-intensity schistosome infections in mice. However, when the infection level observed in mice is translated to humans based on worm biomass per millilitre of blood, it corresponds to a moderate-to-heavy rather than a low-level infection. This underscores the need for caution when extrapolating data across species.
(A) Adult worm burdens in mice 6 weeks post-infection with 0, 5, 10, 15, 20, 25, or 30 cercariae. (B) Levels of sja-miR-61 in sera of mice with different infection intensities. (C) Levels of sja-miR-7-5p in sera of mice with different infection intensities. (Compared with pre-infection, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant).
Discussion
Current challenges in the diagnosis of schistosome infection primarily involve the detection of low-intensity infections and achieving timely diagnosis [44]. Parasite-derived exosome miRNAs present benefits including species specificity and stability [45], while nucleic acid detection methods are recognized for high sensitivity and rapid turnaround [46]. Consequently, exosome miRNAs are increasingly being investigated as molecular biomarkers for disease diagnosis, including early detection. Our study screened high-abundance miRNAs in exosomes derived from adult worms and eggs of S. japonicum, identifying parasite-derived exosome sja-miR-61 and sja-miR-7-5p as molecular markers for diagnosing schistosomiasis and monitoring drug treatment efficacy, with sja-miR-61 showing particular promise for early diagnosis.
Schistosomes reside within the host’s circulatory system. MiRNAs expressed by adult worms or eggs can be packaged into exosomes, secreted into the host bloodstream, and circulate systemically, rendering them detectable and potentially effective molecular targets for diagnosis. In this study, exosomes were isolated from S. japonicum adults and eggs. Small RNA sequencing identified five highly abundant miRNAs in adult worm-derived exosomes (Table 1). Consistent with findings by Lihui Zhu et al. [47], sja-miR-125b and sja-miR-61 were highly expressed in adult worm exosomes; additionally, sja-miR-71a, sja-miR-125, and sja-miR-36-3p were also present at high levels. In egg-derived exosomes, sja-miR-36-3p, sja-miR-1, sja-miR-71b-5p, sja-bantam, and sja-miR-7-5p were highly abundant. To evaluate the diagnostic utility of these miRNAs, specific TaqMan probes were designed and synthesized for all nine candidates. A TaqMan RT-qPCR assay targeting these S. japonicum adult and egg exosome-derived miRNAs was established. TaqMan Real-time PCR amplification of serum samples from infected mice revealed significantly elevated levels of all nine high-abundance miRNAs starting from 2 weeks post-infection. However, the time to peak serum levels varied among these miRNAs. miRNAs highly abundant in egg exosomes peaked later than those derived from adult worm exosomes. This is likely because after cercariae infect the host, they migrate and mature into adults residing in the mesenteric veins, a process taking 4–6 weeks [48,49]. Adult worms then begin egg production. Animal studies indicate that fecal egg counts in C57BL/6 mice infected with S. japonicum start to rise around 5 weeks, stabilizing at a high level after 8 weeks [50], with peak fecal egg output typically reached at 7–8 weeks [51], and total liver egg burden peaking around 10 weeks [52]. This suggests that levels of egg-derived miRNAs in the host correlate with the timing and quantity of egg production. Minor discrepancies in trends observed in our data may be attributed to miRNA degradation in serum or individual variations among mice. Collectively, these results support the potential diagnostic value of detecting miRNAs derived from adult worm or egg exosomes for schistosome infection.
ROC curve analysis was performed to evaluate the diagnostic potential of these miRNAs in patient sera. ROC curves illustrate the relationship between the true positive rate (sensitivity) and the false positive rate (1 − specificity) across a range of decision thresholds and serve as a fundamental tool for assessing diagnostic accuracy. The selection of an appropriate threshold is critical for optimizing the balance between sensitivity and specificity [53]. In this study, the point on the ROC curve corresponding to the maximum likelihood ratio yielded low sensitivity (<60.00%), rendering it unsuitable as a screening cut-off. Therefore, an alternative threshold was chosen based on a relatively high likelihood ratio and the maximum Youden’s index, with priority given to sensitivity in cases where Youden’s index was equal [41,42,54]. Using this criterion, sja-miR-7-5p demonstrated a sensitivity of 70.00% and specificity of 80.00%, whereas sja-miR-61 achieved both sensitivity and specificity of 85.00%. These values are higher than those reported for sja-miR-2b-5p and sja-miR-2c-5p in human schistosome infection (66%/68% and 55%/80%, respectively) [55]. Combined detection of sja-miR-7-5p and sja-miR-61 resulted in 95.00% sensitivity and 75.00% specificity for diagnosing human infection, indicating that a multi-miRNA strategy can enhance sensitivity, though further improvement in specificity is needed.
Schistosome infections involve numerous reservoir hosts, including large livestock such as cattle and sheep, as well as over 40 mammalian species including rodents. With ecological restoration, wildlife populations—particularly wild rodents—are recovering or increasing [56,57]. The persistence of schistosomiasis in natural foci is complicated by the presence of reservoir hosts. Wildlife and intermediate host snails can become key sources of transmission, underscoring the importance of monitoring schistosome infection in wild animals, especially rodents, for early identification of reservoir hosts and implementation of targeted control measures. Such efforts are essential for the overall containment of schistosomiasis transmission. Few studies have evaluated the diagnostic performance of parasite-derived miRNAs for detecting schistosome infection in mice. In this study, at this early time point of 2 weeks post-infection, sja-miR-61 exhibited high sensitivity (93.33%). Combined detection of both serum sja-miR-61 and sja-miR-7-5p further improved diagnostic sensitivity to 100.00%. By 6 weeks post-infection, both sja-miR-61 and sja-miR-7-5p reached sensitivities and specificities between 90.00% and 100.00%, indicating even stronger diagnostic performance in rodents. Notably, sja-miR-61 showed high sensitivity as early as 2 weeks post-infection, highlighting its promise as an early diagnostic marker in mice. However, the diagnostic value in humans at an early stage still needs to be confirmed among individuals infected at this stage.
Immunological assays such as ELISA, which detect antibodies against antigens like KLH or SEA, can achieve 90.00%–100.00% sensitivity but often suffer from lower specificity [52,58–61]. Moreover, serological detection of anti-schistosome antibodies cannot differentiate between current and past infections [62]. In contrast, detection of circulating parasite-derived miRNAs in serum offers higher sensitivity and specificity. DNA-based methods also demonstrate high diagnostic performance; for example, real-time PCR detection in goat serum reported 98.74% sensitivity and 100% specificity [63–65]. Our findings were consistent with these results and underscore the advantages of nucleic acid-based diagnostics for schistosomiasis.
PZQ remains the primary therapeutic agent for schistosomiasis. However, after decades of widespread use, reduced susceptibility to PZQ has been documented in S. mansoni [66]. Although confirmed PZQ resistance has not yet been reported in S. japonicum, monitoring therapeutic efficacy remains essential. Previous study has shown that schistosome-derived miRNAs in serum exosomes of infected individuals become undetectable by 32 weeks post-treatment [31]. Similarly, our study observed a decline in serum levels of sja-miR-7-5p and sja-miR-61 following praziquantel administration, suggesting that schistosome-derived miRNAs in serum can serve as an indicator of the efficacy of PZQ treatment. However, there are some differences. The previous study reported that the miRNAs decline slowly after PZQ treatment, remaining low positive at 11 weeks after treatment and turning negative after approximately 32 weeks [31]. Our study, however, found that sja-miR-61 and sja-miR-7-5p declined rapidly after PZQ treatment, turning negative within two weeks. One possible reason for this discrepancy is that the previous study involved treating human-infected individuals with 60 mg/kg of PZQ, whereas this study used a mouse model treated with 250 mg/kg of PZQ. Compared to human studies, the mouse model allows for a controllable worm burden, consistent infection timing and more thorough clearance after treatment.
Notably, in our study, while the egg-derived sja-miR-7-5p also turned negative after PZQ treatment, its levels frequently hovered near the cut-off value, in contrast to the more pronounced and sustained decrease observed for the adult worm-derived sja-miR-61. This discrepancy may be attributed to the fact that PZQ is primarily effective against adult worms but not less mature eggs. Although eggs may persist in host tissues after treatment, deceased adult worms cease miRNA production. The remaining entrapped eggs might continue to release exosome miRNAs for a limited time. Consequently, following PZQ treatment, serum levels of adult exosome-derived sja-miR-61 decline sharply, whereas egg exosome-derived sja-miR-7-5p, though decreased, may more frequently approach the cut-off, potentially leading to ambiguous results. These findings suggest that parasite exosome-derived sja-miR-61 holds greater promise for monitoring treatment response.
Accurate diagnosis of low-intensity schistosome infections is crucial, particularly among travelers from endemic regions who may act as potential sources of transmission if undetected [67]. Thus, developing sensitive diagnostic methods for low-level infections is a research priority. Studies by Jing Xu et al. successfully detected S. japonicum DNA in sera of rabbits with low-intensity infections (defined as fewer than 10 eggs per gram of feces) and reported high sensitivity (95.1%) in human low-infection cohorts [68], supporting the utility of nucleic acid-based tests in such settings. RT-PCR can detect specific miRNAs at concentrations as low as 0.1–10 fmol [69]. In our study, both sja-miR-61 and sja-miR-7-5p were detectable in mice infected with only five cercariae (mean worm burden: 2.0 ± 2.1). At this minimal infection level, three out of five mice tested positive for each miRNA, corresponding to a sensitivity of 60%. This confirms the diagnostic relevance of both miRNAs even in very low-intensity infections. Although the infection level is speculative based on data from mice, it provides a useful guideline for human low-infection detection. In the future, it will be necessary to verify the low-level infection in the human population.
The limitations of our study are the relatively small number of patient samples and the long storage duration of human serum samples (up to 10 years), which may have contributed to the lower sensitivity of circulating miRNA detection in human subjects compared to the mouse model. As China has achieved interruption of schistosomiasis transmission and progresses toward elimination, acquiring fresh patient serum samples has become increasingly challenging. Furthermore, the performance of the assay for diagnosing low-intensity and early infections in human populations could not be comprehensively validated. Additionally, the precision of the sensitivity and specificity estimates was constrained by the limited number of cases. Accordingly, the 95% confidence intervals for the sensitivity and specificity of the miRNAs ranged from approximately 30% to 40%. Therefore, larger and independent cohorts are required to validate these findings and to provide more precise estimates of diagnostic accuracy to support clinical translation.
Future studies should aim to collect more fresh serum samples from infected patients, as well as from wildlife reservoir hosts and individuals with other parasitic infections, to further validate the diagnostic utility of sja-miR-61 and sja-miR-7-5p in both human and animal schistosome infections. In this study, specificity was evaluated solely using healthy controls. Since serum samples from individuals infected with other helminths (e.g., Ascaris, Strongyloides, or filarial nematodes) were not tested, the true clinical specificity of the assay remains to be determined. Future studies incorporating a broader panel of pathogen-specific controls are needed to fully establish the specificity of these schistosomal miRNA markers.
Overall, our study suggests that miRNAs derived from parasite exosomes, serve both as circulating biomarkers for parasites infection, even in low-level infection, and evaluation of drug efficacy in treatment. The results of our study will provide an effective diagnostic method for patients and animals with schistosome infection.
Supporting information
S1 Table. The primer sequences of the top 9 miRNAs and housekeeping gene U6 expressed in S. japonicum cercaria, adults and eggs.
https://doi.org/10.1371/journal.pntd.0014368.s001
(DOCX)
S2 Table. miRNAs obtained from S. japonicum worms by sequencing.
https://doi.org/10.1371/journal.pntd.0014368.s002
(XLS)
S3 Table. miRNAs obtained from S. japonicum eggs by sequencing.
https://doi.org/10.1371/journal.pntd.0014368.s003
(XLS)
S1 Fig. The relative expression of miRNAs in the cercariae, adult worms, and egg stages of schistosome.
https://doi.org/10.1371/journal.pntd.0014368.s004
(DOCX)
S2 Fig. The sequencing results of the miRNA amplification products from infected mice serum.
https://doi.org/10.1371/journal.pntd.0014368.s005
(DOCX)
S3 Fig. The amplification curve after adding miRNA to human serum.
https://doi.org/10.1371/journal.pntd.0014368.s006
(DOCX)
References
- 1. Deol AK, Fleming FM, Calvo-Urbano B, Walker M, Bucumi V, Gnandou I, et al. Schistosomiasis - Assessing Progress toward the 2020 and 2025 Global Goals. N Engl J Med. 2019;381(26):2519–28. pmid:31881138
- 2. Alibrahim AOE, Elkholy WA, El-Derbawy MM, Zahran NF, Alexiou A, Papadakis M, et al. Schistosomiasis Chemotherapy, Chemoprevention, and Vaccines: History, Progress, and Priorities. Immun Inflamm Dis. 2024;12(11):e70054.
- 3. Mawa PA, Kincaid-Smith J, Tukahebwa EM, Webster JP, Wilson S. Schistosomiasis Morbidity Hotspots: Roles of the Human Host, the Parasite and Their Interface in the Development of Severe Morbidity. Front Immunol. 2021;12:635869.
- 4. Liu Z, Zhang L, Liang Y, Lu L. Pathology and molecular mechanisms of Schistosoma japonicum-associated liver fibrosis. Front Cell Infect Microbiol. 2022;12:1035765. pmid:36389166
- 5. Chatterji T, Khanna N, Alghamdi S, Bhagat T, Gupta N, Alkurbi MO, et al. A Recent Advance in the Diagnosis, Treatment, and Vaccine Development for Human Schistosomiasis. Trop Med Infect Dis. 2024;9(10):243. pmid:39453270
- 6. Wang C, Chen L, Yin X, Hua W, Hou M, Ji M, et al. Application of DNA-based diagnostics in detection of schistosomal DNA in early infection and after drug treatment. Parasit Vectors. 2011;4:164. pmid:21864384
- 7. Jie W, Chuan-Xin Y, Di X, Fei Z, Xu-Ren Y, Li-Jun S. Patterns and diagnostic value of the antibody response to fructose-1,6-bisphosphate aldolase of Schistosoma japonicum. J Pathog Biol. 2015.
- 8. Driscoll AJ, Kyle JL, Remais J. Development of a novel PCR assay capable of detecting a single Schistosoma japonicum cercaria recovered from Oncomelania hupensis. Parasitology. 2005;131(Pt 4):497–500. pmid:16174414
- 9. He P, Song L-G, Xie H, Liang J-Y, Yuan D-Y, Wu Z-D, et al. Nucleic acid detection in the diagnosis and prevention of schistosomiasis. Infect Dis Poverty. 2016;5:25. pmid:27025210
- 10. Poulton K, Webster B. Development of a lateral flow recombinase polymerase assay for the diagnosis of Schistosoma mansoni infections. Anal Biochem. 2018;546:65–71. pmid:29425749
- 11. Ullah H, Qadeer A, Rashid M, Rashid MI, Cheng G. Recent advances in nucleic acid-based methods for detection of helminth infections and the perspective of biosensors for future development. Parasitology. 2020;147(4):383–92. pmid:31840627
- 12. Gava SG, de Carvalho IR, Sato MO, Sato M, Fava N de MN, Parreiras PM, et al. Advancing schistosomiasis surveillance: standardization and application of an environmental DNA (eDNA)-based approach for detecting Schistosoma mansoni in Brazil. BMC Infect Dis. 2025;25(1):703. pmid:40375164
- 13. Wu S, Du C, Cao C, Yang J, Luo F, Ma X, et al. Molecular diagnosis of suspected intestinal schistosomiasis in a non-endemic area of Yunnan Province, China. Infect Dis Poverty. 2025;14(1):99. pmid:41068848
- 14. Guo J-J, Zheng H-J, Xu J, Zhu X-Q, Wang S-Y, Xia C-M. Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis. PLoS Negl Trop Dis. 2012;6(3):e1579. pmid:22479661
- 15. Farooqi AA, Desai NN, Qureshi MZ, Librelotto DRN, Gasparri ML, Bishayee A, et al. Exosome biogenesis, bioactivities and functions as new delivery systems of natural compounds. Biotechnol Adv. 2018;36(1):328–34. pmid:29248680
- 16. Zhou B, Xu K, Zheng X, Chen T, Wang J, Song Y, et al. Application of exosomes as liquid biopsy in clinical diagnosis. Signal Transduct Target Ther. 2020;5(1):144. pmid:32747657
- 17. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008;36(Database issue):D154-8. pmid:17991681
- 18. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105(30):10513–8. pmid:18663219
- 19. El Hayek T, Alnaser-Almusa OA, Alsalameh SM, Alhalabi MT, Sabbah AN, Alshehri EA, et al. Emerging role of exosomal microRNA in liver cancer in the era of precision medicine; potential and challenges. Front Mol Biosci. 2024;11:1381789. pmid:38993840
- 20. Wang H-C, Yin W-X, Jiang M, Han J-Y, Kuai X-W, Sun R, et al. Function and biomedical implications of exosomal microRNAs delivered by parenchymal and nonparenchymal cells in hepatocellular carcinoma. World J Gastroenterol. 2023;29(39):5435–51. pmid:37900996
- 21. Hu X, Huang F, Yao J, Lv J, Mai J, Li N, et al. Cross-sectional study on the diagnostic significance of plasma exosomal miRNAs in HBV-related hepatocellular carcinoma. J Transl Med. 2024;22(1):1006. pmid:39511689
- 22. Momen-Heravi F, Saha B, Kodys K, Catalano D, Satishchandran A, Szabo G. Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis. J Transl Med. 2015;13:261. pmid:26264599
- 23. Ye Z, Zheng Z, Peng L. MicroRNA profiling of serum exosomes in patients with osteosarcoma by high-throughput sequencing. J Investig Med. 2020;68(4):893–901. pmid:32060049
- 24. Copeland CS, Marz M, Rose D, Hertel J, Brindley PJ, Santana CB, et al. Homology-based annotation of non-coding RNAs in the genomes of Schistosoma mansoni and Schistosoma japonicum. BMC Genomics. 2009;10:464. pmid:19814823
- 25. Huang J, Hao P, Chen H, Hu W, Yan Q, Liu F, et al. Genome-wide identification of Schistosoma japonicum microRNAs using a deep-sequencing approach. PLoS One. 2009;4(12):e8206. pmid:19997615
- 26. Liu Q, Tuo W, Gao H, Zhu X-Q. MicroRNAs of parasites: current status and future perspectives. Parasitol Res. 2010;107(3):501–7. pmid:20532562
- 27. Wang Z, Xue X, Sun J, Luo R, Xu X, Jiang Y, et al. An “in-depth” description of the small non-coding RNA population of Schistosoma japonicum schistosomulum. PLoS Negl Trop Dis. 2010;4(2):e596. pmid:20161724
- 28. Samoil V, Dagenais M, Ganapathy V, Aldridge J, Glebov A, Jardim A, et al. Vesicle-based secretion in schistosomes: Analysis of protein and microRNA (miRNA) content of exosome-like vesicles derived from Schistosoma mansoni. Sci Rep. 2018;8(1):3286. pmid:29459722
- 29. Cheng G, Luo R, Hu C, Cao J, Jin Y. Deep sequencing-based identification of pathogen-specific microRNAs in the plasma of rabbits infected with Schistosoma japonicum. Parasitology. 2013;140(14):1751–61. pmid:23942009
- 30. Cai P, Gobert GN, You H, Duke M, McManus DP. Circulating miRNAs: Potential Novel Biomarkers for Hepatopathology Progression and Diagnosis of Schistosomiasis Japonica in Two Murine Models. PLoS Negl Trop Dis. 2015;9(7):e0003965. pmid:26230095
- 31. Meningher T, Lerman G, Regev-Rudzki N, Gold D, Ben-Dov IZ, Sidi Y, et al. Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis. J Infect Dis. 2017;215(3):378–86. pmid:28362903
- 32. Gao H, Xiao D, Song L, Zhang W, Shen S, Yin X, et al. Assessment of the diagnostic efficacy of enolase as an indication of active infection of Schistosoma japonicum. Parasitol Res. 2016;115(1):151–64. pmid:26420425
- 33. Ravishankar H, Mangani AS, Phoebe Moses GL, Mani SP, Parameswaran S, Khetan V, et al. Serum exosomal miRNA as biomarkers for Retinoblastoma. Exp Eye Res. 2020;199:108184. pmid:32795526
- 34. Abdel-Al A, El-Ahwany E, Zoheiry M, Hassan M, Ouf A, Abu-Taleb H, et al. miRNA-221 and miRNA-222 are promising biomarkers for progression of liver fibrosis in HCV Egyptian patients. Virus Res. 2018;253:135–9. pmid:29932949
- 35. Francisco JS, Terra MABL, Klein GCT, Dias de Oliveira BCEP, Pelajo-Machado M. The hepatic extramedullary hematopoiesis during experimental murine Schistosomiasis mansoni. Front Immunol. 2022;13:955034. pmid:36091027
- 36. Gao Y, Zhang X, Jiang T, Zhou H, Liu H, Hu Y, et al. Inhibition of hepatic natural killer cell function via the TIGIT receptor in schistosomiasis-induced liver fibrosis. PLoS Pathog. 2023;19(3):e1011242. pmid:36930687
- 37. Wang Y, Gong W, Zhou H, Hu Y, Wang L, Shen Y, et al. A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis. Front Immunol. 2022;13:860807. pmid:35572578
- 38. Duan Z-Y, Cai G-Y, Li J-J, Bu R, Wang N, Yin P, et al. U6 can be used as a housekeeping gene for urinary sediment miRNA studies of IgA nephropathy. Sci Rep. 2018;8(1):10875. pmid:30022109
- 39. Lange T, Stracke S, Rettig R, Lendeckel U, Kuhn J, Schlüter R, et al. Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients. PLoS One. 2017;12(8):e0183435. pmid:28859135
- 40. Xiang M, Zeng Y, Yang R, Xu H, Chen Z, Zhong J, et al. U6 is not a suitable endogenous control for the quantification of circulating microRNAs. Biochem Biophys Res Commun. 2014;454(1):210–4. pmid:25450382
- 41. Zou KH, Yu C-R, Liu K, Carlsson MO, Cabrera J. Optimal thresholds by maximizing or minimizing various metrics via ROC-type analysis. Acad Radiol. 2013;20(7):807–15. pmid:23582776
- 42. Ruopp MD, Perkins NJ, Whitcomb BW, Schisterman EF. Youden Index and optimal cut-point estimated from observations affected by a lower limit of detection. Biom J. 2008;50(3):419–30. pmid:18435502
- 43. Ferre A, Santiago L, Sánchez-Herrero JF, López-Rodrigo O, Sánchez-Curbelo J, Sumoy L, et al. 3’IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation. Int J Mol Sci. 2023;24(20):15436. pmid:37895116
- 44. Yan Z, Hui-Fen D, Ming-Sen J, Qin-Ping Z. Progress of molecular detection of Schistosoma. Parasitology. 2017;29:798–801.
- 45. Zhu L, Liu J, Cheng G. Role of microRNAs in schistosomes and schistosomiasis. Front Cell Infect Microbiol. 2014;4:165. pmid:25426450
- 46. Ally O, Kanoi BN, Ochola L, Nyanjom SG, Shiluli C, Misinzo G, et al. Schistosomiasis diagnosis: Challenges and opportunities for elimination. PLoS Negl Trop Dis. 2024;18(7):e0012282. pmid:38990839
- 47. Zhu L, Liu J, Dao J, Lu K, Li H, Gu H, et al. Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions. Sci Rep. 2016;6:25885. pmid:27172881
- 48. Ross AG, Sleigh AC, Li Y, Davis GM, Williams GM, Jiang Z, et al. Schistosomiasis in the People’s Republic of China: prospects and challenges for the 21st century. Clin Microbiol Rev. 2001;14(2):270–95. pmid:11292639
- 49. Ling-Ling D, Xiang-Qian Z, Xin-Gang F, Ying-Ying J, Jing-Xiu XU, Yong-Hong S. Ultrastructural comparison of Schistosoma japonicum at different developmental stages and genders. Chin J Anim Infect Dis. 2016;24(2):42–6.
- 50. Kai C, Yun L, Xiao-Ming TU, Min H, Hai-Wei WU. Characteristics of fecal egg distribution in mouse models of Schistosoma japonicum (Chinese strain) infection. J Pathog Biol. 2010;5:430–3.
- 51. Ichii S, Irie Y, Yasuraoka K. Growth and fecundity of Schistosoma japonicum in mice maintained at different environmental temperatures. Jpn J Exp Med. 1990;60(1):39–44. pmid:2112654
- 52. Cheever AW, Macedonia JG, Mosimann JE, Cheever EA. Kinetics of egg production and egg excretion by Schistosoma mansoni and S. japonicum in mice infected with a single pair of worms. Am J Trop Med Hyg. 1994;50(3):281–95. pmid:8147487
- 53. Bosqui LR, Gonçalves ALR, Gonçalves-Pires M do RF, Custodio LA, de Menezes MCND, Murad VA, et al. Detection of parasite-specific IgG and IgA in paired serum and saliva samples for diagnosis of human strongyloidiasis in northern Paraná state, Brazil. Acta Trop. 2015;150:190–5. pmid:26238982
- 54. Weiss HL, Niwas S, Grizzle WE, Piyathilake C. Receiver operating characteristic (ROC) to determine cut-off points of biomarkers in lung cancer patients. Dis Markers. 2003;19(6):273–8. pmid:15258328
- 55. Mu Y, Cai P, Olveda RM, Ross AG, Olveda DU, McManus DP. Parasite-derived circulating microRNAs as biomarkers for the detection of human Schistosoma japonicum infection. Parasitology. 2020;147(8):889–96. pmid:31840631
- 56. Jiang G, Liu J, Xu L, Yu G, He H, Zhang Z. Climate warming increases biodiversity of small rodents by favoring rare or less abundant species in a grassland ecosystem. Integr Zool. 2013;8(2):162–74. pmid:23731812
- 57. Lü S, Lü C, Li YL, Xu J, Hong QB, Zhou J, et al. Expert consensus on the strategy and measures to interrupt the transmission of schistosomiasis in China. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2021;33(1):10–4. pmid:33660468
- 58. Li Y, Ross AG, Li Y, He YK, Luo XS, McManus DP. Serological diagnosis of Schistosoma japonicum infections in China. Trans R Soc Trop Med Hyg. 1997;91(1):19–21. pmid:9093618
- 59. Rahman MO, Sassa M, Parvin N, Islam MR, Yajima A, Ota E. Diagnostic test accuracy for detecting Schistosoma japonicum and S. mekongi in humans: A systematic review and meta-analysis. PLoS Negl Trop Dis. 2021;15(3):e0009244. pmid:33730048
- 60. Mei Z, Lv S, Tian L, Wang W, Jia T. The Efficiency of Commercial Immunodiagnostic Assays for the Field Detection of Schistosoma japonicum Human Infections: A Meta-Analysis. Pathogens. 2022;11(7):791. pmid:35890035
- 61. Cai P, Weerakoon KG, Mu Y, Olveda RM, Ross AG, Olveda DU, et al. Comparison of Kato Katz, antibody-based ELISA and droplet digital PCR diagnosis of schistosomiasis japonica: Lessons learnt from a setting of low infection intensity. PLoS Negl Trop Dis. 2019;13(3):e0007228. pmid:30830925
- 62. Chala B. Advances in Diagnosis of Schistosomiasis: Focus on Challenges and Future Approaches. Int J Gen Med. 2023;16:983–95. pmid:36967838
- 63. Zhou L, Tang J, Zhao Y, Gong R, Lu X, Gong L, et al. A highly sensitive TaqMan real-time PCR assay for early detection of Schistosoma species. Acta Trop. 2011;120(1–2):88–94. pmid:21763257
- 64. Guo Q, Chen C, Zhou K, Li Y, Tong L, Yue Y, et al. Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat. Parasit Vectors. 2020;13(1):535. pmid:33109260
- 65. Zhang X, He C-C, Liu J-M, Li H, Lu K, Fu Z-Q, et al. Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals. Infect Dis Poverty. 2017;6(1):86. pmid:28407808
- 66. Pinto-Almeida A, Mendes T, Armada A, Belo S, Carrilho E, Viveiros M, et al. The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype. PLoS One. 2015;10(10):e0140147. pmid:26445012
- 67. Bergquist R, Yang G-J, Knopp S, Utzinger J, Tanner M. Surveillance and response: Tools and approaches for the elimination stage of neglected tropical diseases. Acta Trop. 2015;141(Pt B):229–34. pmid:25301340
- 68. Xu J, Guan Z-X, Zhao B, Wang Y-Y, Cao Y, Zhang H-Q, et al. DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans. PLoS Negl Trop Dis. 2015;9(4):e0003668. pmid:25874964
- 69. Yan J, Zhang N, Qi C, Liu X, Shangguan D. One-step real time RT-PCR for detection of microRNAs. Talanta. 2013;110:190–5. pmid:23618193