Prudhomme Jorian, Delabarre Aymeric, Alten Bulent, Berberoglu Umut, Berriatua Eduardo, Bongiorno Gioia, Cristovao José Manuel, Davidovich-Cohen Maya, Di Muccio Trentina, Erisoz Kasap Ozge, Fiorentino Eleonora, D Kirstein Oscar, Kniha Edwin, Maia Carla, Mungan Mesut, Muñoz-Hernández Clara, Nalçaci Muhammed, Oguz Kaskan Gizem, Ozbel Yusuf, Ozensoy Toz Seray, Parreira Ricardo, Platzgummer Katharina, Polat Ceylan, Risueño José, Studentsky Liora, Varol Gamze, Walochnik Julia, Yetişmiş Kardelen, Robert-Gangneux Florence
Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé Environnement Travail), UMR S 1085, Rennes, France.
Hacettepe University, Ankara, Turkey.
PLoS Negl Trop Dis. 2024 Dec 23;18(12):e0012543. doi: 10.1371/journal.pntd.0012543. eCollection 2024 Dec.
Leishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly.
METHODOLOGY/PRINCIPAL FINDINGS: EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 101 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load.
This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities.
利什曼病由白蛉传播的利什曼原虫寄生虫引起，是地中海盆地一个重大的公共卫生问题。有效监测感染利什曼原虫的白蛉需要标准化工具来比较其分布和感染率。一致的定量实时PCR（qPCR）参数和高效的DNA提取方案对于长期和跨地区获得可靠结果至关重要。然而，缺乏针对利什曼原虫DNA检测的标准化技术建议阻碍了有效监测。本研究旨在通过一项涉及九个参考实验室的多中心研究，比较不同的DNA提取方案，并进行基于qPCR的外部质量评估（EQA），重点是优化白蛉中利什曼原虫DNA的检测。
方法/主要发现：EQA样本包括婴儿利什曼原虫和硕大利什曼原虫，浓度范围为每毫升101至104个寄生虫。除一个中心外，所有中心都检测到了所有浓度，显示出很强的诊断能力。检测低浓度的能力突出了所用qPCR检测方法的稳健性，尽管Cq值的变化表明与技术能力或DNA提取试剂盒性能相关的灵敏度存在差异。对七种DNA提取方法的比较分析确定EZ1 DSP病毒试剂盒和QIAamp DNA微量试剂盒是最有效的，支持它们在标准化方案中的使用。该研究还评估了冻干和运输条件的影响，结果表明尽管Cq值略有变化，但在利什曼原虫检测方面没有显著影响。纳入实验感染的白蛉以模拟现场条件，所有中心都成功检测到了具有不同Cq值的阳性样本，这可能反映了感染负荷的差异。
本研究强调了标准化DNA提取方案和持续质量保证对于准确检测利什曼原虫DNA的重要性。结果突出了某些提取试剂盒的优越性能以及持续技术培训的必要性，这对于可靠的利什曼病监测至关重要，特别是在感染密度较低的现场环境中。
