摘 要

背景

棘球蚴病是由棘球属绦虫的蚴虫寄生在人或动物体内而引起的人畜共患寄生虫病。该病在全球范围内分布广泛，尤其是在牧区和畜牧业发达的地区流行，对社会经济和人民健康造成重大负担。棘球蚴病具有起病隐匿，潜伏期长的特点，早期感染无明显临床症状或临床症状不具有特异性，使得棘球蚴病的诊断尤为困难。目前临床诊断主要依赖于影像学检查和血清学检测，但存在早期灵敏度不足、交叉反应等问题，亟需开发有效的病原学诊断和早期诊断方法。

目的

本研究（1）通过对棘球蚴病患者术前和术后外泌体RNAs的差异表达分析，以期发现分子病原学诊断标记物；（2）基于纳米材料建立高灵敏度棘球蚴病血清学检测方法，并通过动物模型和临床样本评价其诊断灵敏度和特异性，为本病的早期诊断提供技术支持。

方法

（1）本研究通过排阻和超滤的方法对患者术前和术后血清中的外泌体进行分离及RNA测序，并利用Utest和HISAT2对RNA进行比对和表达量分析，用edgeR对患者术前和术后的外泌体miRNA进行差异表达分析。随后设计分子探针，利用qRT-PCR分别评价表达丰度较高的虫源差异miRNA和人源差异miRNA在诊断中的应用潜力。（2）本研究制备抗原，采用二抗和HRP标记AuNPs制备探针并使用抗原包被NHS磁珠，利用NHS磁珠的磁性和AuNP探针的信号放大作用建立了高灵敏的Nano-ELISA检测系统，以市售试剂盒为对照，利用小鼠模型对其早期诊断潜力进行验证，并利用临床血浆样本对其的灵敏度和特异性进行评价。

结果

（1）对棘球蚴病患者术前术后血清中的外泌体RNA差异分析显示，差异主要为人来源的外泌体miRNA，虫源miRNA在血清中的表达量较低。在棘球蚴病术前血清样本的外泌体中共发现了117个虫源miRNA，其中有94个为已知，23个为新鉴定序列。利用48例B超检查有典型病灶的阳性血清样本和39例阴性血清样本对初步验证结果较好的3个miRNA（emu-miR-71-5p和emu-miR-10-5p和emu-let-7-5p）进行进一步验证，结果显示虫源miRNA在血清中的拷贝数较低，难以作为理想的诊断标志物。对人来源的外泌体miRNA进行研究，根据表达丰度选取5个miRNA进行验证，发现无鉴别诊断潜力，仅3个miRNA（hsa-miR-183-5p，hsa-miR-222-3p和hsa-miR-196a-5p）在细粒棘球蚴患者阳性血清中的表达量显著高于阴性样本，显示出一定的诊断价值。

（2）利用抗体和信号分子标记AuNPs制成AuNP探针，利用磁珠和AuNP探针建立了Nano-ELISA检测系统，并在AuNP探针和磁珠浓度、孵育时间、血清稀释倍数方面对其进行优化；随后构建多房棘球蚴病小鼠模型，利用感染后3-13天的小鼠血清对该检测方法进行评价。结果显示，与常规ELISA相比，该方法能够提早3天检测到小鼠血清中抗体，具有早期诊断潜力。利用临床血清样本对Nano-ELISA检测系统的灵敏度和特异性进行评价，结果显示，与市售的棘球蚴病ELISA试剂盒的相比，该方法的灵敏度为94.74%，特异性为90.48%，显著高于市售试剂盒（分别为84.21%和80.95%）。

结论

本研究结果表明，棘球绦虫来源的外泌体miRNA因在血清中痕量表达，作为棘球蚴病的诊断标记物仍存在局限性，但人来源的3个外泌体miRNA在细粒棘球蚴病诊断中具有一定的研究价值。本研究构建的Nano-ELIA检测系统在小鼠棘球蚴病的早期诊断中展现出较好的效果，不仅拓展了纳米材料在寄生虫诊断领域的应用，还为人类棘球蚴病的早期诊断提供了新的技术选择。

Abstract

Background

Echinococcosis is a zoonotic parasitic disease caused by larval stage Echinococcus species, which can parasitize both humans and animals. This disease is distributed globally, particularly prevalent in pastoralist and animal husbandry areas, posing a significant burden on public health and socio-economic. Echinococcosis is characterized by insidious onset and a prolonged incubation period, with early infection often presenting no clinical symptoms or non-specific symptoms, which makes the diagnosis of echinococcosis particularly challenging. Currently, clinical diagnosis primarily relies on imaging examinations and serological tests, which face issues such as insufficient early sensitivity and cross-reactivity. Therefore, there is an urgent need to develop effective pathogenetic diagnosis and early detection techniques.

Objectives

The aims of this study were (1) to evaluate the potential of exosomal RNA in the diagnosis of echinococcosis by analysing the differential expression of preoperative and postoperative exosomal RNAs in patients with echinococcosis, thus providing new molecular biomarkers, and (2) to establish a highly sensitive echinococcosis serological detection approach based on nano-materials, and to evaluate its diagnostic sensitivity and specificity by animal models and clinical samples, thereby providing technical support for the early diagnosis of echinococcosis.

Methods

Exosomes were isolated from the serum of echinococcosis patients before and after surgery using exclusion and ultrafiltration methods, followed by RNA sequencing. The RNAs were aligned and expression levels analysed using Utest and HISAT2 software; differential expression analysis of exosomal miRNAs from preoperative and postoperative patients was performed by edgeR software. Subsequently, qRT-PCR was used to evaluate the potentials of differential miRNAs, both echinococcal and human-derived, with higher expression abundance in the diagnosis of echinococcosis. Additionally, we used secondary antibody and HRP-labelled AuNPs as probes, with antigens coated on NHS magnetic beads to establish a highly sensitive Nano-ELISA detection system, based on the magnetic properties of the NHS magnetic beads and the signal amplification of the AuNP probes. Early diagnostic validation and evaluation were conducted in mouse models. The sensitivity and specificity of this method were assessed by comparing it with commercially available ELISA kits using clinical plasma samples.

Results

(1) The differential expression level analysis of exosomal RNAs in the serum of echinococcosis patients before and after surgery analysis showed that these differences mainly originated from human-derived exosomal RNAs, while the expression levels of echinococcal-derived miRNAs in serum were low. A total of 117 echinococcal-derived miRNAs were identified in exosomes from preoperative serum samples, of which 94 were known and 23 were newly identified sequences. Subsequent validation of three promising miRNAs (emu-miR-71-5p, emu-miR-10-5p, and emu-let-7-5p) using 48 positive serum samples with typical lesions from ultrasound examinations and 39 negative serum samples showed that echinococcal-derived miRNAs expressed at very low levels in serum, making them less ideal as diagnostic markers. We further investigated human-derived exosomal miRNAs in serum samples and identified eight differentially expressed miRNAs, five of which were selected for evaluation based on expression abundance. As a result, three miRNAs (hsa-miR-183-5p, hsa-miR-222-3p, and hsa-miR-196a-5p) demonstrated significantly higher expression levels in the positive serum for cystic echinococcosis, indicating certain diagnostic values.

(2) AuNP probes were prepared using labeled AuNPs with antibodies and signaling molecules, successfully establishing a highly sensitive Nano-ELISA method with magnetic beads and AuNP probes. This approach was optimized in terms of the concentration of AuNP probes and magnetic beads, the incubation time, and the serum dilution factors. The mouse model infected by Echinococcus multilocularis was constructed and serum samples were collected from mice at days 3-13 post-infection to evaluate the early diagnostic capability of this Nano-ELISA method. The results showed that compared to the conventional ELISA, the Nano-ELISA was able to detect antibodies in the serum of the mice three days earlier. Subsequently, we evaluated the sensitivity and specificity of the Nano-ELISA system using clinical serum samples, revealing that its sensitivity increased from 84.21% to 94.74% and specificity from 80.95% to 90.48% compared to the commercially available ELISA kit.

Conclusion

The results of this study indicate that echinococcal-derive exosomal miRNAs have limitations as diagnostic markers due to their trace expression in serum. However, three human-derived exosomal miRNAs exhibit certain research value in the diagnosis of cystic echinococcosis. Additionally, the Nano-ELIA detection system constructed in this study hsa demonstrated promising performance in the early diagnosis of echinococcosis in mice, thereby expanding the application of nanomaterials in the field of parasitic diagnostics and providing new technological options for the early diagnosis of human echinococcosis.