Bousslimi N, Ben Abda I, Ben Mously R, Siala E, Harrat Z, Zallagua N, Bouratbine A, Aoun K
Laboratoire de recherche LR 05-SP 03 « parasitoses émergentes », institut Pasteur de Tunis, 13, place Pasteur, BP74, 1002 Tunis, Tunisie.
Laboratoire de recherche LR 05-SP 03 « parasitoses émergentes », institut Pasteur de Tunis, 13, place Pasteur, BP74, 1002 Tunis, Tunisie; Laboratoire de parasitologie-mycologie, institut Pasteur de Tunis, 13, place Pasteur, BP74, 1002 Tunis, Tunisie.
Pathol Biol (Paris). 2014 Feb;62(1):30-3. doi: 10.1016/j.patbio.2013.10.004. Epub 2014 Feb 5.
Three forms of cutaneous leishmaniasis (CL) are endemic in Tunisia. The identification of the causative species is useful to complete epidemiological data and to manage the cases. The aim of this study is to assess PCR-RFLP technique in the identification of Leishmania species responsible of CL in Tunisia and to compare the results of this technique to those of isoenzyme analysis.
Sixty-one CL lesions were sampled. Dermal samples were tested by culture on NNN medium and analyzed by PCR-RFLP assay targeting the ITS1 region of ribosomal DNA. Species identification was performed by both iso-enzymatic typing for positive cultures and analysis of restriction profiles after enzymatic digestion by HaeIII of the obtained amplicons.
Thirty-eight (62%) samples were positive by culture. The iso-enzymatic typing of 32 isolates identified 3 L. infantum, 23 L. major MON-25 and 6 L. tropica MON-8. Sixty samples were positive by PCR. The PCR-RFLP digestion profiles of the 56 PCR products identified 12 L. infantum, 38 L. major and 6 L. tropica. The results of both techniques were concordant in the 32 strains identified by both techniques. Species identification correlated with the geographical distribution of CL forms endemic in Tunisia.
Results of PCR-RFLP revealed highly concordant with those of isoenzyme electrophoresis. Thanks to its simplicity, rapidity and ability to be performed directly on biological samples, this technique appears as an interesting alternative for the identification of Leishmania strains responsible of CL in Tunisia.
三种皮肤利什曼病（CL）在突尼斯呈地方流行。鉴定致病物种有助于完善流行病学数据并处理病例。本研究的目的是评估聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术在鉴定突尼斯CL致病利什曼原虫物种中的应用，并将该技术的结果与同工酶分析结果进行比较。
采集了61个CL病灶样本。皮肤样本在NNN培养基上进行培养检测，并通过针对核糖体DNA ITS1区域的PCR-RFLP分析进行分析。通过对阳性培养物进行同工酶分型以及对获得的扩增子用HaeIII酶切后的限制性图谱分析来进行物种鉴定。
38个（62%）样本培养呈阳性。对32株分离株进行同工酶分型鉴定出3株婴儿利什曼原虫、23株硕大利什曼原虫MON-25和6株热带利什曼原虫MON-8。60个样本PCR呈阳性。56个PCR产物的PCR-RFLP酶切图谱鉴定出12株婴儿利什曼原虫、38株硕大利什曼原虫和6株热带利什曼原虫。两种技术在通过两种技术鉴定的32株菌株中的结果一致。物种鉴定与突尼斯地方流行的CL形式的地理分布相关。
PCR-RFLP结果与同工酶电泳结果高度一致。由于其操作简单、快速且能够直接对生物样本进行检测，该技术似乎是鉴定突尼斯CL致病利什曼原虫菌株的一种有趣的替代方法。
