Genetic tools discriminate strains of Leishmania infantum isolated from  humans and dogs in Sicily, Italy

Abstract

Background Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection. Methodology/Principal Findings Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory. Conclusions/Significance Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The PLOS NEGLECTED TROPICAL DISEASES

Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity

“…In this study, we evaluated the diagnostic accuracy of the real-time PCR across multiple presentations associated with L. infantum infection. The high sensitivity and specificity of this method suggest the possibility of using it to diagnose CL, VL, CanL, and FeL, endemic diseases in Italy [ 4 , 9 , 56 , 57 ]. Leishmania infantum DNA was detected (%) in different clinical samples identifying the parasite load.…”

“…The study comprised 30 dogs, 20 cats, 30 humans living in Sicily, which is endemic for L. infantum [ 56 ]. Fifteen human patients showing forms of CL were examined at the Section of Dermatology of the University of Palermo; on the other hand, 15 patients affected by VL were examined at the Infectious Disease Department of the University of Palermo.…”

“…The cultivated strains were harvested, washed twice with NaCl 0.3%, and centrifuged. The total DNA was extracted from the promastigotes according to as described in Castelli et al (2020) [ 56 ] as follows: the pellet was lysed by heating at 96 °C for 20 min with 400 μL of a mixture containing 1% Tween 20 (Sigma, St. Louis, MO, USA), 1% Nonidet P-40 (Sigma, Tokyo, Japan) and 20% Chelex resin (Bio-Rad, Hercules, CA, USA) prepared in sterile distilled water. The mixture was centrifuged at 14,000× g for 10 min at 4 °C and the DNA-containing upper phase was then collected and stored at –20 °C until used.…”

Leishmania and the Model of Predominant Clonal Evolution

“…The subdivision of L. infantum into several NCs is confirmed by microsatellite analysis in Brazil [31], in Europe [28], in Northern Italy [36], in Sicilia [37], in Israel [38], in Tunisia [39], in Armenia [40] and in various other countries [30]. In all the cited studies, bifurcating trees cannot be explained by geographical separation alone, even if this last parameter plays a role (see population A in [37]; see their Figure 5). In [37,40], robustness of the branchings is corroborated by Bayesian, non-phylogenetic methods (STRUCTURE) [20].…”

“…In all the cited studies, bifurcating trees cannot be explained by geographical separation alone, even if this last parameter plays a role (see population A in [37]; see their Figure 5). In [37,40], robustness of the branchings is corroborated by Bayesian, non-phylogenetic methods (STRUCTURE) [20]. In Brazil, NCs are confirmed by phylogeny, STRUCTURE and factorial correspondence analysis [31].…”