Laboratoire de recherche LR 16-IPT-06 "Parasitologie Médicale, Biotechnologies et Biomolécules"(1), Institut Pasteur de Tunis, Université Tunis El-Manar, 13 Place Pasteur, BP 74, 1002, Tunis, Tunisia; Faculté des Sciences de Bizerte, Université de Carthage, Bizerte, Tunisia.
Laboratoire de recherche LR 16-IPT-06 "Parasitologie Médicale, Biotechnologies et Biomolécules"(1), Institut Pasteur de Tunis, Université Tunis El-Manar, 13 Place Pasteur, BP 74, 1002, Tunis, Tunisia.
Acta Trop. 2021 Mar;215:105804. doi: 10.1016/j.actatropica.2020.105804. Epub 2020 Dec 29.
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.
免疫磁珠分离（IMS）法已被用于分离有活力的整个生物体。我们的工作目的是生产抗利什曼原虫的磁性珠，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。通过静脉注射活的循环前鞭毛体的方法在兔体内产生抗利什曼原虫的多克隆抗体。评估纯化的抗利什曼原虫 IgG 对大、小利什曼原虫前鞭毛体的反应性，然后将其共价偶联到磁性珠上，用于 IMS。将后者应用于已知浓度的大、小利什曼原虫前鞭毛体培养物，或来自皮肤利什曼病（CL）病变的早期（24 小时、48 小时、72 小时）Novy-MacNeal-Nicolle（NNN）组织液培养物。通过煮沸后显微镜检查或 qPCR 评估前鞭毛体的捕获。间接免疫荧光试验表明，多克隆抗体可与大、小利什曼原虫前鞭毛体反应。在 50µL 溶液中，免疫磁珠能够从 20 个中捕获 5 个活前鞭毛体，从 2500 个中捕获 1050 个，估计捕获效率为 25-42%。当寄生虫数量较少时，IMS 的效率较低，但仍可重复。另一方面，应用于已确认的利什曼病病变的 14 个 NNN 培养物的 IMS-qPCR 比常规显微镜观察前鞭毛体生长显示出更高的检测活寄生虫的敏感性（72 小时阳性率为 93%，而 2-4 周孵育期内阳性率为 50%）。72 小时内捕获的寄生虫估计数量从 1 个到 50µL 培养物液相中的 100 多个不等。这些初步结果为利用培养物进行利什曼病诊断开辟了新的途径，也为其他应用开辟了途径，如从储存动物或白蛉中采集的培养物中检测利什曼原虫。
