ArticleParasites & vectors2025
18S/28S rDNA metabarcoding identifies Cryptosporidium parvum and Blastocystis ST1 as the predominant intestinal protozoa in hospital patients from Changchun, Northeast China.
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Abstract
backgroundIntestinal 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.
methodsWe 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.
resultsFrom 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.
conclusionsMetabarcoding 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.
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