Wang Cunmin, Yin Jigang, Shi Zhanpeng, Xu Yijia, Chen Junhong, Yan Yueyang, Zhu Guan, Zhao Jixue
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, China.
Department of Pediatric Surgery, The First Hospital of Jilin University, Changchun, 130021, Jilin, China.
Parasit Vectors. 2025 Sep 24;18(1):376. doi: 10.1186/s13071-025-07043-z.
Intestinal protozoa and helminths remain an under‑recognized cause of gastrointestinal morbidity in China. Molecular high‑throughput tools offer the chance to survey their diversity comprehensively, yet their application in clinical settings has been limited.
We pooled leftover fecal samples from 360 hospital patients in Changchun (36 pools; 12 demographic/seasonal groups) and enriched them by sucrose flotation. Three primer pairs targeting 18S V4‑V5, 18S V9 and 28S D3‑D4 rRNA regions were amplified, and paired‑end libraries (100-140 k reads per amplicon) were sequenced on Illumina platforms. Taxa were assigned with QIIME2 against SILVA, and true prevalences were estimated from pooled‑sample data using a binomial model with profile‑likelihood confidential intervals. Selected positives were confirmed by qPCR, nested PCR, gp60 subtyping and immunofluorescence assay.
From 6.1 million quality‑filtered reads, only 1.65% mapped to parasites; fungal reads dominated (98.35%), underscoring primer bias. Four eukaryotic parasites were detected across 12/36 pools. Cryptosporidium parvum was most frequent (7 pools, true prevalence = 2.14%, 95% CI 0.92-4.10), and all gp60‑typed isolates belonged to subtype IIdA19G1. Blastocystis hominis occurred in five pools (1.48%, 0.53-3.17), predominantly ST1, with single detections of ST3 and ST6. Entamoeba hartmanni appeared in one pool (0.28%, 0.02-1.23). Reads assignable only to Opisthorchiidae suggested liver‑fluke carriage in four adult pools (1.17%, 0.36-2.70). No statistically significant associations were found between infection status and age, sex, season or diarrhea. Amplification success differed markedly between primer sets, limiting quantitative comparisons.
Metabarcoding of rDNA amplicons provides a feasible snapshot of human intestinal‑parasite communities in Northeast China, revealing C. parvum IIdA19G1 as an emerging zoonotic threat and highlighting ongoing food‑borne trematodiasis. However, the overwhelming amplification of fungal templates and inter‑primer bias call for primer redesign and complementary diagnostics before routine clinical adoption.
肠道原生动物和蠕虫仍是中国胃肠道疾病未得到充分认识的病因。分子高通量工具为全面调查它们的多样性提供了机会，但其在临床环境中的应用一直有限。
我们收集了长春360名医院患者的剩余粪便样本（36个样本池；12个人口统计学/季节组），并通过蔗糖浮选法进行富集。针对18S V4-V5、18S V9和28S D3-D4 rRNA区域的三对引物进行扩增，并在Illumina平台上对配对末端文库（每个扩增子100-140k条 reads）进行测序。使用QIIME2根据SILVA对分类群进行分类，并使用具有轮廓似然置信区间的二项式模型从混合样本数据中估计真实患病率。通过qPCR、巢式PCR、gp60亚型分型和免疫荧光试验对选定的阳性样本进行确认。
在610万条经过质量过滤的reads中，只有1.65%与寄生虫匹配；真菌reads占主导（98.35%），突出了引物偏差。在12/36个样本池中检测到四种真核寄生虫。微小隐孢子虫最为常见（7个样本池，真实患病率=2.14%，95%CI 0.92-4.10），所有gp60分型的分离株均属于IIdA19G1亚型。人芽囊原虫出现在5个样本池中（1.48%，0.53-3.17），主要为ST1型，同时检测到单个ST3和ST6型。哈氏内阿米巴出现在1个样本池中（0.28%，0.02-1.23）。仅可归因于后睾科的reads表明，在4个成人样本池中存在肝吸虫感染（1.17%，0.36-2.70）。未发现感染状态与年龄、性别、季节或腹泻之间存在统计学上的显著关联。不同引物组之间的扩增成功率差异显著，限制了定量比较。
rDNA扩增子的元条形码分析为中国东北地区人类肠道寄生虫群落提供了一个可行的概况，揭示了微小隐孢子虫IIdA19G1是一种新出现的人畜共患病威胁，并突出了正在流行的食源性吸虫病。然而，真菌模板的大量扩增和引物间偏差要求在常规临床应用前重新设计引物并采用补充诊断方法。
