Castanheira Gabriel Victor, Amato Valdir Sabbaga, Tuon Felipe Francisco, Dantas Letícia Ramos, de Carvalho Silvio Fernando Guimarães, Vieira Thallyta Maria, Moura Ana Paula Venuto, de Sousa Lima Lopes Jackeline Maria, Johanson Gustavo Henrique, Emori Christini Takemi, de Souza Regina Maia
Faculdade de Medicina, Departamento de Infectologia E Medicina Tropical, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
Laboratório de Doenças Infecciosas E Emergentes, Pontifícia Universidade Católica Do Paraná, Rua Imaculada Conceição 1155, Curitiba, PR, 80215-901, Brazil.
Eur J Clin Microbiol Infect Dis. 2025 Mar;44(3):717-728. doi: 10.1007/s10096-025-05037-4. Epub 2025 Jan 14.
This study aimed to standardize qPCR techniques using these molecular markers kDNA and 18S rDNA across three sample types: peripheral blood, guanidine-treated blood, and tissue. The secondary objective is to evaluate the performance of 18S rDNA target in samples from 46 patients with confirmed tegumentary leishmaniasis. After obtaining the standard curve from reference strains with Leishmania, qPCR curves were standardizations and the Cts results of the patient samples were described using abstract measures. Specific specification equations (EEG) with normal distribution and identity link function were constructed to compare each type of clinical sample. To identify the differences among samples and techniques, multiple comparisons with Bonferroni post-test was performed. The kDNA and 18S rDNA demonstrated high sensitivity, detecting as few as 10⁻ parasites/mL. However, 18S rDNA showed limited species discrimination. qPCR performance was evaluated using blood and tissue samples, showing a sensitivity of 54.2% in blood, 12.5% in guanidine-treated blood, and 86.4% in tissue. qPCR agreement with the 18S rDNA target with the three types of samples, positive and negative, in relation to screening were 56.2% in blood, 31.8% in guanidine- blood and tissue 78.6%. As for true positives (PPV), tissue samples presented a probability percentage of individuals being sick of 86.4%, while in blood it was 81.3%. The results underscore the importance of molecular diagnostics in blood samples, improving the accuracy and monitoring of tegumentary leishmaniasis.
本研究旨在使用这些分子标记物kDNA和18S rDNA，对三种样本类型（外周血、胍处理血和组织）的qPCR技术进行标准化。次要目标是评估18S rDNA靶点在46例确诊皮肤利什曼病患者样本中的性能。在用利什曼原虫参考菌株获得标准曲线后，对qPCR曲线进行标准化，并使用抽象测量方法描述患者样本的Cts结果。构建具有正态分布和恒等链接函数的特定规格方程（EEG），以比较每种临床样本类型。为了确定样本和技术之间的差异，进行了Bonferroni事后检验的多重比较。kDNA和18S rDNA显示出高灵敏度，能检测低至10⁻寄生虫/mL。然而，18S rDNA的物种鉴别能力有限。使用血液和组织样本评估qPCR性能，结果显示血液中的灵敏度为54.2%，胍处理血中的灵敏度为12.5%，组织中的灵敏度为86.4%。qPCR与18S rDNA靶点在三种类型样本（阳性和阴性）筛查方面的一致性，血液中为56.2%，胍处理血中为31.8%，组织中为78.6%。至于真阳性（PPV），组织样本中个体患病的概率百分比为86.4%，而血液中为81.3%。结果强调了分子诊断在血液样本中的重要性，提高了皮肤利什曼病的准确性和监测水平。
