BACKGROUND Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

中文翻译：

---

用于血液中基于RNA和DNA的利什曼原虫检测的核酸稳定剂作为内脏负担的代表的比较评估。

背景技术对于内脏利什曼病（VL）患者的诊断和治疗后随访，使用外周血的分子检测技术优于侵入性组织抽吸术。这项研究旨在确定合适的稳定剂，以防止DNA和RNA在储存和运输到进行分子诊断的专门实验室期间降解。方法学使用经过实验感染，治疗（米非福星或氨基吡唑DNDi-1044）和免疫抑制作用的叙利亚金仓鼠前鞭毛加标的人血和外周血，比较了不同市售试剂的稳定能力。结合建立的动塑料DNA（kDNA）qPCR和最近开发的用于检测利什曼原虫的剪接前导RNA（SL-RNA）分析，测试了各种存储温度条件的影响。主要发现无论使用哪种血型和稳定剂，通过SL-RNA qPCR获得的阈值（cT）值均系统地低于通过kDNA分析获得的阈值（cT），这证实了SL-RNA分析相对于广泛使用的kDNA分析具有优势低水平的利什曼原虫检测。外周血寄生虫水平与肝脏负担相对较好。RNA保护细胞试剂可在人和仓鼠血液中提供最佳的同时DNA和RNA稳定。然而，该稳定剂需要红细胞裂解步骤，这在田间条件下可能具有挑战性。DNA / RNA防护罩为下游kDNA和SL-RNA分析提供了很好的选择，特别是如果可以保证样品在4°C下的存储能力时。结论/意义推荐的稳定剂与VL仓鼠模型和人类血液加标中外周血中基于RNA和DNA的利什曼原虫检测兼容。由于使用外周血的分子检测技术的侵害性小于组织抽吸物的显微镜评估，因此本研究的结果可应用于人类VL临床研究。