de Luna Rafaela Lira Nogueira, Sales Kamila Gaudêncio da Silva, Bonifácio Lucas Lisboa Nunes, Figueredo Luciana Aguiar, Shelite Thomas R, Nogueira Fábio Dos Santos, Otranto Domenico, Dantas-Torres Filipe
Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz), Recife, Brazil.
Department of Biosafety, University of Texas Medical Branch, Galveston, USA.
Parasit Vectors. 2025 Jan 7;18(1):2. doi: 10.1186/s13071-024-06633-7.
We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA).
The RPA-LF assays were tested at different temperatures and reaction times, using DNA from cultured L. braziliensis and L. infantum. The L. infantum RPA-LF was also tested using clinical samples (bone marrow and skin) from infected and uninfected dogs.
The detection limits (analytical sensitivity) of the assays were 0.04 pg/μl and 0.04 ng/μl for L. braziliensis and L. infantum kDNA, respectively. Using clinical samples, the L. infantum RPA-LF successfully detected the parasite kDNA in bone marrow (21/30; 70.0%) and skin samples (23/30, 76.6%) from naturally infected dogs. We found an almost perfect agreement (kappa = 0.807) between RPA-LF for L. infantum and our reference quantitative real-time polymerase chain reaction (qPCR), considering clinical samples with a quantification cycle (C) < 30, whereas the agreement with samples with a C > 30 (lower parasite loads) was moderate (kappa = 0.440).
The RPA-LF assays developed here may be promising diagnostic tools for point-of-care diagnosis of L. infantum and L. braziliensis infection in dogs, particularly in remote rural areas lacking laboratory infrastructure.
我们对两种重组酶聚合酶扩增（RPA）检测方法与侧向流动（LF）试纸条进行了标准化，用于检测巴西利什曼原虫和婴儿利什曼原虫的动基体DNA（kDNA）。
使用来自培养的巴西利什曼原虫和婴儿利什曼原虫的DNA，在不同温度和反应时间下对RPA-LF检测方法进行测试。还使用感染和未感染犬的临床样本（骨髓和皮肤）对婴儿利什曼原虫RPA-LF进行了测试。
该检测方法对巴西利什曼原虫和婴儿利什曼原虫kDNA的检测限（分析灵敏度）分别为0.04 pg/μl和0.04 ng/μl。使用临床样本，婴儿利什曼原虫RPA-LF成功检测到自然感染犬骨髓（21/30；70.0%）和皮肤样本（23/30，76.6%）中的寄生虫kDNA。对于定量循环（C）<30的临床样本，我们发现婴儿利什曼原虫RPA-LF与我们的参考定量实时聚合酶链反应（qPCR）之间几乎完全一致（kappa = 0.807），而与C>30（较低寄生虫载量）的样本之间的一致性为中等（kappa = 0.440）。
此处开发的RPA-LF检测方法可能是用于犬类婴儿利什曼原虫和巴西利什曼原虫感染即时诊断的有前景的诊断工具，特别是在缺乏实验室基础设施的偏远农村地区。
