Ceballos-Caro Ángela, Antón-Berenguer Víctor, Lanza Marta, Renelies-Hamilton Justinn, Barciela Amanda, Köster Pamela C, Carmena David, Flores-Chávez María, Chanove Emeline, Rubio José Miguel
Parasitology Reference and Research Laboratory, National Microbiology Centre, Instituto de Salud Carlos III, 28220 Majadahonda, Spain.
Department of Microbiology and Parasitology, Hospital Universitario Severo Ochoa, 28914 Leganés, Spain.
Diagnostics (Basel). 2025 Mar 4;15(5):620. doi: 10.3390/diagnostics15050620.
: Many tropical diseases such as malaria, Chagas, human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognised as major global vector-borne diseases. Tackling this disease requires an accurate diagnosis that is sensitive, specific, and rapid. This study aimed to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test. : A total of 230 human blood samples were assessed retrospectively for parasite characterisation, as well as 58 stool samples from non-human primates. Primers and probes were designed in the small subunit ribosomal RNA gene, except for spp., for which the novel target was Cytochrome Oxidase Subunit 1. : The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 and 3.01 parasites/µL for species, 1.8 parasites/mL for Trypanosomatidae, and 2 microfilariae/mL in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for Filariae, whose sensitivity dropped to 93.9% and whose negative predicted value dropped to 89.5%. The operational features described a turnaround and a hands-on time shorter than the compared methods with a lower cost per essay. : This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and coinfections.
许多热带疾病，如疟疾、恰加斯病、人类非洲锥虫病和淋巴丝虫病，在流行国家同时存在，影响着全球超过10亿人，被公认为主要的全球媒介传播疾病。应对这些疾病需要一种敏感、特异且快速的准确诊断方法。本研究旨在描述和验证一种新的高灵敏度和特异性的多重分析系统，该系统能够在一次检测中有效检测多种病原体。
共对230份人类血液样本进行回顾性评估以进行寄生虫特征分析，以及对58份来自非人类灵长类动物的粪便样本进行分析。除 属外，引物和探针均设计在小亚基核糖体RNA基因上，对于 属，新的靶标是细胞色素氧化酶亚基1。
所提出方法的分析特异性为100%，与其他血液寄生虫病无非特异性扩增或交叉反应。对于 种，获得的检测限在0.6至3.01个寄生虫/微升之间，对于锥虫科为1.8个寄生虫/毫升，对于丝虫病为2条微丝蚴/毫升。除丝虫病外，灵敏度、特异性、预测值和kappa系数几乎达到100%，丝虫病的灵敏度降至93.9%，阴性预测值降至89.5%。操作特征表明周转时间和实际操作时间比所比较的方法短，每次检测成本更低。
这项工作展示了一种经济高效且高灵敏度的多重工具（RT-PCR-bp），能够使用特异性荧光探针同时检测血液寄生虫病，能够诊断低寄生虫载量和混合感染。
