Di Pietra Giuseppe, Gargiulo Raffaele, Ortalli Margherita, Petrullo Luciana, Oliva Ester, Raglio Annibale, Frigo Annachiara, Castagliuolo Ignazio, Besutti Valeria
Laboratory Medicine , ULSS 7 Pedemontana, Bassano del Grappa, Italy.
Department of Molecular Medicine, University of Padua, Padua, Italy.
Parasit Vectors. 2025 Aug 1;18(1):320. doi: 10.1186/s13071-025-06879-9.
Pathogenic intestinal protozoa exhibit a global distribution and are significant causes of diarrhea, estimated to affect approximately 3.5 billion individuals annually. These intestinal infections continue to pose formidable diagnostic challenges. Microscopy remains the reference diagnostic method for intestinal protozoa, but is limited in terms of sensitivity, specificity and the ability to differentiate closely related species. Additionally, microscopy requires an experienced microbiologist. Emerging diagnostic methods, such as immunochromatography and enzyme-linked immunosorbent assay (ELISA), are regarded as suitable techniques for rapid screening. Molecular diagnostic technologies, particularly real-time PCR (RT-PCR), are gaining traction in non-endemic areas characterised by low parasitic prevalence owing to their enhanced sensitivity and specificity, although these techniques still face various technical challenges.
In this multicentre study involving 18 Italian laboratories, we compared the performance of a commercial RT-PCR test (AusDiagnostics) and an in-house RT-PCR assay against traditional microscopy for identifying infections with Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis.
The study analysed 355 stool samples, of which 230 samples were freshly collected and 125 had been stored in preservation media. The data from our analyses show complete agreement between the AusDiagnostics and in-house PCR methods for the detection of G. duodenalis, with both methods demonstrating high sensitivity and specificity, similar to those of conventional microscopy. For Cryptosporidium spp. and D. fragilis detection, both methods showed high specificity but limited sensitivity, likely due to inadequate DNA extraction from the parasite. Molecular assays seem to be critical for the accurate diagnosis of E. histolytica. Overall, PCR results from preserved stool samples were better than those from fresh samples, likely due to better DNA preservation in the former.
Molecular methods show promise for the diagnosis of intestinal protozoan infections. The molecular assays tested in this investigation performed well for G. duodenalis and Cryptosporidium spp. in fixed faecal specimens, while D. fragilis detection was inconsistent. These results suggest that although PCR techniques are promising in terms of reliable and cost-effective parasite identification, further standardisation of sample collection, storage and DNA extraction procedures is necessary for consistent results.
致病性肠道原生动物在全球范围内分布，是腹泻的重要病因，据估计每年约影响35亿人。这些肠道感染继续构成巨大的诊断挑战。显微镜检查仍然是肠道原生动物的参考诊断方法，但在敏感性、特异性以及区分密切相关物种的能力方面存在局限性。此外，显微镜检查需要经验丰富的微生物学家。新兴的诊断方法，如免疫层析法和酶联免疫吸附测定（ELISA），被认为是适合快速筛查的技术。分子诊断技术，特别是实时荧光定量聚合酶链反应（RT-PCR），由于其更高的敏感性和特异性，在寄生虫感染率较低的非流行地区越来越受到关注，尽管这些技术仍然面临各种技术挑战。
在这项涉及18个意大利实验室的多中心研究中，我们比较了一种商业RT-PCR检测方法（AusDiagnostics）和一种内部RT-PCR检测方法与传统显微镜检查在鉴定十二指肠贾第虫、隐孢子虫属、溶组织内阿米巴和脆弱双核阿米巴感染方面的性能。
该研究分析了355份粪便样本，其中230份样本是新鲜采集的，125份样本保存在保存介质中。我们的分析数据显示，AusDiagnostics和内部PCR方法在检测十二指肠贾第虫方面完全一致，两种方法均显示出高敏感性和特异性，与传统显微镜检查相似。对于隐孢子虫属和脆弱双核阿米巴的检测，两种方法均显示出高特异性，但敏感性有限，这可能是由于从寄生虫中提取的DNA不足。分子检测似乎对溶组织内阿米巴的准确诊断至关重要。总体而言，保存在粪便样本中的PCR结果优于新鲜样本，这可能是因为前者的DNA保存更好。
分子方法在肠道原生动物感染的诊断中显示出前景。本研究中测试的分子检测方法在固定粪便标本中对十二指肠贾第虫和隐孢子虫属的检测效果良好，而对脆弱双核阿米巴的检测结果不一致。这些结果表明，尽管PCR技术在可靠且经济高效的寄生虫鉴定方面很有前景，但为了获得一致的结果，样本采集、储存和DNA提取程序需要进一步标准化。
