Meta-analysis of variable-temperature PCR technique performance for diagnosising Schistosoma japonicum infections in humans in endemic areas

Background As China is moving onto schistosomiasis elimination/eradication, diagnostic methods with both high sensitivity and specificity for Schistosoma japonicum infections in humans are urgently needed. Microscopic identification of eggs in stool is proven to have poor sensitivity in low endemic regions, and antibody tests are unable to distinguish between current and previous infections. Polymerase chain reaction (PCR) technologies for the detection of parasite DNA have been theoretically assumed to show high diagnostic sensitivity and specificity. However, the reported performance of PCR for detecting S. japonicum infection varied greatly among studies. Therefore, we performed a meta-analysis to evaluate the overall diagnostic performance of variable-temperature PCR technologies, based on stool or blood, for detecting S. japonicum infections in humans from endemic areas. Methods We searched literatures in eight electronic databases, published up to 20 January 2021. The heterogeneity and publication bias of included studies were assessed statistically. The risk of bias and applicability of each eligible study were assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool (QUADAS-2). The bivariate mixed-effects model was applied to obtain the summary estimates of diagnostic performance. The hierarchical summary receiver operating characteristic (HSROC) curve was applied to visually display the results. Subgroup analyses and multivariate regression were performed to explore the source of heterogeneity. This research was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines and was registered prospectively in PROSPERO (CRD42021233165). Results A total of 2791 papers were retrieved. After assessing for duplications and eligilibity a total of thirteen publications were retained for inclusion. These included eligible data from 4268 participants across sixteen studies. High heterogeneity existed among studies, but no publication bias was found. The pooled analyses of PCR data from all included studies resulted in a sensitivity of 0.91 (95% CI: 0.83 to 0.96), specificity of 0.85 (95% CI: 0.65 to 0.94), positive likelihood ratio of 5.90 (95% CI: 2.40 to 14.60), negative likelihood ratio of 0.10 (95% CI: 0.05 to 0.20) and a diagnostics odds ratio of 58 (95% CI: 19 to 179). Case-control studies showed significantly better performances for PCR diagnostics than cross-sectional studies. This was further evidenced by multivariate analyses. The four types of PCR approaches identified (convention PCR, qPCR, Digital droplet PCR and nested PCR) differed significantly, with nested PCRs showing the best performance. Conclusions Variable-temperature PCR has a satisfactory performance for diagnosing S. japonicum infections in humans in endemic areas. More high quality studies on S. japonicum diagnostic techniques, especially in low endemic areas and for the detection of dual-sex and single-sex infections are required. These will likely need to optimise a nested PCR alongside a highly sensitive gene target. They will contribute to successfully monitoring endemic areas as they move towards the WHO 2030 targets, as well as ultimately helping areas to achieve these goals.

Comparative transcriptome profiles of Schistosoma japonicum larval stages: Implications for parasite biology and host invasion

Schistosoma japonicum is prevalent in Asia with a wide mammalian host range, which leads to highly harmful zoonotic parasitic diseases. Most previous transcriptomic studies have been performed on this parasite, but mainly focus on stages inside the mammalian host. Moreover, few larval transcriptomic data are available in public databases. Here we mapped the detailed transcriptome profiles of four S. japonicum larval stages including eggs, miracidia, sporocysts and cercariae, providing a comprehensive development picture outside of the mammalian host. By analyzing the stage-specific/enriched genes, we identified functional genes associated with the biological characteristic at each stage: e.g. we observed enrichment of genes necessary for DNA replication only in sporocysts, while those involved in proteolysis were upregulated in sporocysts and/or cercariae. This data indicated that miracidia might use leishmanolysin and neprilysin to penetrate the snail, while elastase (SjCE2b) and leishmanolysin might contribute to the cercariae invasion. The expression profile of stem cell markers revealed potential germinal cell conversion during larval development. Additionally, our analysis indicated that tandem duplications had driven the expansion of the papain family in S. japonicum. Notably, all the duplicated cathepsin B-like proteases were highly expressed in cercariae. Utilizing our 3rd version of S. japonicum genome, we further characterized the alternative splicing profiles throughout these four stages. Taken together, the present study provides compressive gene expression profiles of S. japonicum larval stages and identifies a set of genes that might be involved in intermediate and definitive host invasion.

Proteomics Investigations of Potential Protein Biomarkers in Sera of Rabbits Infected With Schistosoma japonicum

Schistosomiasis is a chronic parasitic disease that continues to be a pressing public health problem in many developing countries. The primary pathological damage from the disease is granuloma and fibrosis caused by egg aggregation, and early treatment can effectively prevent the occurrence of liver fibrosis. Therefore, it is very important to identify biomarkers that can be used for early diagnosis of Schistosoma japonicum infection. In this study, a label-free proteomics method was performed to observe the alteration of proteins before infection, 1 and 6 weeks after infection, and 5 and 7 weeks after treatment. A total of 10 proteins derived from S. japonicum and 242 host-derived proteins were identified and quantified as significantly changed. Temporal analysis was carried out to further analyze potential biomarkers with coherent changes during infection and treatment. The results revealed biological process changes in serum proteins compared to infection and treatment groups, which implicated receptor-mediated endocytosis, inflammatory response, and acute-phase response such as mannan-binding lectin serine peptidase 1, immunoglobulin, and collagen. These findings offer guidance for the in-depth analysis of potential biomarkers of schistosomiasis, host protein, and early diagnosis of S. japonicum and its pathogenesis. Data are available via ProteomeXchange with identifier PXD029635.

Therapeutic Effect of Schistosoma japonicum Cystatin on Atherosclerotic Renal Damage

Atherosclerosis is a chronic inflammation of the arterial vessel wall driven by lipid metabolism disorders. Although helminthic infection and their derivatives have been identified to attenuate the chronic inflammatory diseases, the immunomodulatory effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) on metabolic diseases and atherosclerosis has not been reported. In this study, we investigated the therapeutic efficacy of rSj-Cys on atherosclerotic renal damage and explored the related immunological mechanism. The results demonstrated that treatment with rSj-Cys significantly reduced body weight gain, hyperlipidemia, and atherosclerosis induced by the high-fat diet in apoE–/– mice. The treatment of rSj-Cys also significantly improved kidney functions through promoting macrophage polarization from M1 to M2, therefore inhibiting M1 macrophage–induced inflammation. The possible mechanism underlying the regulatory effect of rSj-Cys on reducing atherosclerosis and atherosclerotic renal damage is that rSj-Cys stimulates regulatory T cell and M2 macrophage polarization that produce regulatory cytokines, such as interleukin 10 and transforming growth factor β. The therapeutic effect of rSj-Cys on atherosclerotic renal damage is possibly through inhibiting the activation of TLR2/Myd88 signaling pathway. The results in this study provide evidence for the first time that Schistosoma-derived cystatin could be developed as a therapeutic agent to treat lipid metabolism disorder and atherosclerosis that threats million lives around the world.

Bacillus subtilis Attenuates Hepatic and Intestinal Injuries and Modulates Gut Microbiota and Gene Expression Profiles in Mice Infected with Schistosoma japonicum

Parasitic infection can induce pathological injuries and impact the gut microbiota diversity and composition of the host. Bacillus subtilis is a nonpathogenic and noninvasive probiotic bacterium for humans and other animals, playing an important role in improving the host immune system’s ability to respond to intestinal and liver diseases and modulating gut microbiota. However, whether B. subtilis can impact biological functions in Schistosoma japonicum–infected mice is unclear. This study used oral administration (OA) of B. subtilis to treat mice infected with S. japonicum. We evaluated changes in the gut microbiota of infected mice using 16 S rRNA gene sequencing and differentially expressed gene profiles using transcriptome sequencing after OA B. subtilis. We found that OA B. subtilis significantly attenuated hepatic and intestinal pathological injuries in infected mice. The gut microbiota of mice were significantly altered after S. japonicum infection, while OA B. subtilis remodel the diversity and composition of gut microbiomes of infected mice. We found that the S. japonicum–infected mice with OA B. subtilis had an overabundance of the most prevalent bacterial genera, including Bacteroides, Enterococcus, Lactobacillus, Blautia, Lachnoclostridium, Ruminiclostridium, and Enterobacter. Transcriptomic analysis of intestinal tissues revealed that OA B. subtilis shaped the intestinal microenvironment of the host responding to S. japonicum infection. Differentially expressed genes were classified into KEGG pathways between S. japonicum–infected mice and those without included cell adhesion molecules, intestinal immune network for IgA production, hematopoietic cell lineage, Fc epsilon RI signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, calcium signaling pathway, Fc gamma R-mediated phagocytosis, chemokine signaling pathway, phospholipase D signaling pathway, NF-kappa B signaling pathway, B cell receptor signaling pathway, pancreatic secretion, and phagosome. In conclusion, our findings showed that OA B. subtilis alleviates pathological injuries and regulates gene expression, implying that B. subtilis supplementation may be a potential therapeutic strategy for schistosomiasis. Our study may highlight the value of probiotics as a beneficial supplementary therapy during human schistosomiasis, but further studies are needed.

rSjP40 Inhibited the Activity of Collagen Type I Promoter via Ets-1 in HSCs

Liver fibrosis is a severe disease characterized by excessive deposition of extracellular matrix (ECM) components in the liver. Activated hepatic stellate cells (HSCs) are a major source of ECM and a key regulator of liver fibrosis. Collagen type I alpha I (COL1A1) is one of the main components of ECM and is a major component in fibrotic tissues. Previously, we demonstrated that soluble egg antigen from Schistosoma japonicum could inhibit the expression of COL1A1 in activated HSCs. In addition, studies have found that Ets proto-oncogene 1 (Ets-1) suppresses the production of ECM by down-regulating matrix related genes such as COL1A1 induced by transforming growth factor β, and ultimately inhibits liver fibrosis. In this study, the major aim was to investigate the effect and mechanism of Ets-1 on inhibiting COL1A1 gene promoter activity in HSCs by recombinant Schistosoma japonicum protein P40 (rSjP40). We observed the rSjP40 inhibited the expression of COL1A1 by inhibiting the activity of the COL1A1 promoter, and the core region of rSjP40 acting on COL1A1 promoter was located at -1,722/-1,592. In addition, we also demonstrated that rSjP40 could promote the expression of Ets-1, and Ets-1 has a negative regulation effect on the COL1A1 promoter in human LX-2 cells. These data suggest that rSjP40 might inhibit the activity of COL1A1 promoter and inhibit the activation of HSCs by increasing the expression of transcription factor Ets-1, which will provide a new experimental basis for the prevention and treatment of liver fibrosis.

Chromosome-level genome assembly reveals female-biased genes for sex determination and differentiation in the human blood fluke Schistosoma japonicum

Schistosomiasis is a neglected tropical disease of humans caused by blood flukes of the genus Schistosoma – the only dioecious parasitic flatworms. Although aspects of sex determination, differentiation and reproduction have been studied in some Schistosoma species, almost nothing is understood for Schistosoma japonicum - the causative agent of schistosomiasis japonica. This relates mainly to a lack of high-quality genomic and transcriptomic resources for this species. As current draft genomes for S. japonicum are highly fragmented, we assembled here a chromosome-level reference genome (seven autosomes, the Z-chromosome and partial W-chromosome), achieving a substantially enhanced gene annotation. Utilising this genome, we discovered that the sex chromosomes of S. japonicum and its congener S. mansoni independently suppressed recombination during evolution, forming four and two ‘strata’, respectively. By exploring the W-chromosome and sex-specific transcriptomes, we identified 35 W-linked genes and 257 female-preferentially transcribed genes (FTGs) and identified a signature for sex determination and differentiation in S. japonicum. These FTGs cluster within autosomes or the Z-chromosome and exhibit a highly dynamic transcription profile during the pairing of female and male schistosomules (advanced juveniles), representing a critical phase for the maturation of the female worms, suggesting distinct layers of regulatory control of gene transcription at this stage of development. Collectively, these data provide a valuable resource for further functional genomic characterisation of S. japonicum, shed light on the evolution of sex chromosomes in this highly virulent human blood fluke and provide a pathway to identify novel targets for development of intervention tools against schistosomiasis.