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Use of Immunomagnetic Separation tool in Leishmania promastigotes capture
Acta Tropica ( IF 2.7 ) Pub Date : 2020-12-29 , DOI: 10.1016/j.actatropica.2020.105804
Imen Tayachi , Yousr Galai , Meriem Ben-abid , Nasreddine Saidi , Ines Ben-sghaier , Karim Aoun , Aïda Bouratbine

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media.

Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling.

Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture.

These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.



中文翻译:

免疫磁分离工具在利什曼原虫前鞭毛虫捕获中的使用

免疫磁分离(IMS)分析已用于分离可行的完整生物。我们工作的目的是生产抗利什曼原虫磁珠,并评估IMS技术在培养基中捕获利什曼原虫前鞭毛体的效率。

通过向兔静脉内注射大的利什曼原虫L.)的活的代谢环前鞭毛体来产生多克隆抗利什曼原虫抗体。评估纯化的抗利什曼原虫IgG对乳杆菌婴儿乳杆菌前鞭毛体的反应性,然后共价缀合至磁珠并用于IMS。后者可用于已知浓度或主要的前鞭毛体培养物或从皮肤利什曼病(CL)病变获得的组织液的Novy-MacNeal-Nicolle(NNN)培养的早期(24h,48h,72h)培养物中。煮沸后,通过显微镜或qPCR评估前鞭毛体的捕获。

间接免疫荧光分析表明,多克隆抗体同时针对乳酸杆菌和婴儿乳酸杆菌前鞭毛体反应。在50 µL溶液中,免疫磁珠能够捕获20个中的5个活前鞭毛体和2500个中的1050个,估计效率为25-42%。对于较少数量的寄生虫,IMS的效率较低,但仍可重复。在另一方面,IMS-qPCR的施加到确认的14个NNN培养利什曼病变表现出较高的灵敏度来检测比在72小时前鞭毛体生长(93%阳性的常规显微镜观察活寄生虫2-4周内阳性率为50%)。估计在72h捕获的寄生虫数量为1至100多个寄生虫/ 50 µL培养液相。

这些初步结果为将培养物用于利什曼病诊断和其他应用(例如从储水动物或沙蝇中提取的利什曼原虫进行检测)开辟了有趣的视角。

更新日期:2021-01-06
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