High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping

Parasites & Vectors

Research

Marcello Ceccarelli¹  Mauro Magnani¹  Luca Galluzzi¹  Gloria Buffi¹  Michelalberto Abruzzese¹  Francesca Andreoni¹  Daniela Bencardino¹  Aurora Diotallevi¹  Fabrizio Vitale²  Germano Castelli²  Federica Bruno² 

[1] Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, PU, Italy;OIE Leishmania Reference Laboratory, Centro di Referenza Nazionale per le Leishmaniosi (C.Re.Na.L.), Istituto Zooprofilattico Sperimentale Della Sicilia, Palermo, PA, Italy;

关键词: Leishmania infantum;    Leishmaniasis;    MLST;    NGS;    qPCR;    HRM analysis;    Malic enzyme;    Glucose-6-phosphate isomerase;    Genotype;   

DOI  :  10.1186/s13071-023-05878-y

 received in 2023-02-24, accepted in 2023-07-11,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundLeishmaniasis 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.MethodsA 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.ResultsThe 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).ConclusionsApplying 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.Graphical abstract

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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