背景:利什曼病是地中海地区的一种人畜共患疾病,在该地区,婴儿利什曼原虫是人和犬感染的病原体。在亚种水平上对这种寄生虫的表征可用于流行病学研究,评估疾病的临床病程(例如耐药菌株,利什曼病的内脏和皮肤形式)以及确定感染库。多位点酶电泳(MLEE),一种目前公认的用于表征和鉴定利什曼原虫菌株的参考方法,既麻烦又耗时,需要培养寄生虫。这些缺点导致了其他方法的发展,如多位点微卫星分型(MLMT)和多位点序列分型(MLST),用于利什曼原虫寄生虫的分型;然而,这些方法尚未用于常规使用。在这项研究中,我们首先使用MLST来鉴定编码代谢酶的单拷贝基因的信息多态性,随后,我们开发了两种基于高分辨率熔解(HRM)分析的快速基因分型方法,以探索婴儿乳球菌寄生虫中的这些多态性。
方法:设计了针对14个管家基因的定制测序组,并对9个婴儿犬和人菌株/分离株进行了MLST分析。设计了两个定量实时PCR-HRM测定法来分析苹果酸酶(ME)和葡萄糖-6-磷酸异构酶(GPI)基因(390T/G和1831A/G,分别)。这两种方法被应用于来自意大利中部/南部和Pantelleria岛的73个临床样本/分离株,结果通过部分样本的DNA测序得到证实。
结果:MLST分析,连同Genbank数据库中的序列,能够鉴定编码ME和GPI的基因上的两个信息多态性。在73个临床样品/分离株中使用两种基于HRM的测定对这些多态性进行快速筛选,从而鉴定出7种基因型。总的来说,基因型1(序列类型390T/1831G)在整个样本中占比最高(45.2%),并且与最常见的婴儿乳球菌(MON-1,MON-72)相关.有趣的是,在Pantelleria岛,最普遍的基因型(70.6%)是基因型6(序列类型390T/1831A)。
结论:将我们的HRM分析应用于临床样本,使我们能够鉴定出七种不同的基因型,而无需寄生虫的分离和培养。我们已经证明,这些检测方法可以快速使用,常规和廉价的工具用于流行病学监测的婴儿乳杆菌或新的感染宿主的识别。
BACKGROUND: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites.
METHODS: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples.
RESULTS: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A).
CONCLUSIONS: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs.