This retrospective study aimed to genotype Leishmania parasites in samples from TL-affected patients diagnosed in two different southern European areas. The metropolitan city of Bologna is located within the Emilia-Romagna region in northern Italy and covers an area of 3,703 km2, with a population of approximately 1,020,000 inhabitants and a population density of 275.01 inhabitants/km2. The second area corresponds to the area served by the Hospital Universitario de Fuenlabrada, defined as Fuenlabrada, which includes approximately 222,500 inhabitants living in the cities of Fuenlabrada, Moraleja de Enmedio and Humanes in the south-west of the Madrid Autonomous Community, in central Spain.

The identification of a TL case was based on the World Health Organization (WHO) definition: a patient exhibiting suggestive cutaneous and/or mucosal lesion(s) with confirmation of Leishmania parasites or their genome by microscopy and/or molecular methods [3]. CL refers to lesion(s) of skin tissues, while ML refers to mucosal involvement regardless of the co-existence or prior presence of skin lesions. All diagnoses of TL cases from the metropolitan city of Bologna and Fuenlabrada were confirmed by real-time PCR as described by Gaspari et al. [18] and Chicharro et al. [19] for cases from Bologna and Fuenlabrada areas, respectively. The TL specimens included in the study were in total n = 87; of these n = 80 thin sections from formalin-fixed and paraffin embedded (FFPE) biopsies, n = 5 fresh biopsies, and two Leishmania strains (MHOM/IT/2023/IZSLER-Bo03 and MHOM/IT/2023/IZSLER-Bo04) obtained from skin/mucosal lesions.

Samples included in the study were collected from confirmed TL cases diagnosed in Bologna from January 1st 2016 to December 30th 2023 and in Fuenlabrada from January 1st 2013 to December 31st 2016. Data about the patients enrolled in the study were anonymized by using an alphanumerical code. The study was conducted in  accordance with the declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Area Vasta Emilia Centro (study number: n° EM414-2023_97/2017/O/Tess/AOUBo) for the Italian cases and by the Fuenlabrada University Hospital (APR12-66 and APR13-30) for the Spanish cases. All participants gave their written, informed consent.

For samples obtained from Italian TL cases, DNA extraction was performed as follows: the Maxwell® CSC DNA FFPE Kit (Promega, Madison, Wisconsin, United States) was employed in combination with the Maxwell CSC instrument (Promega, Madison, Wisconsin, United States) according to manufacturer instructions for FFPE biopsies, while the DNeasy® Blood&Tissue Kit (Qiagen, Hilden, Germany) was employed to extract DNA from fresh biopsies as well as clinical isolates. For samples obtained from Spanish TL cases, DNA was extracted as described by Chicharro et al. [19] using SpeedTools DNA/Tissue DNA extraction kit (Biotools B&M Labs, S.A., Madrid, Spain) after paraffin removal by treatment with 0.05% Tween 20 in phosphate-buffered saline. PCR reactions were performed using the Thermocycler T-Gradient ThermoBlock (Biometra, Göttingen, Germany) and the HotStarTaq plus kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Thermal profile, amplification and sequencing conditions of Hsp70 fragments were performed as described by Gritti et al. [11], with modifications aimed at improving amplification from FFPE samples, which often contain degraded or limited amounts of parasitic DNA. In brief, Hsp70 was amplified using a three-step nested PCR targeting the N- (593 bp), P- (295 bp), and Ps-fragment (262 bp) of the gene in the first, second, and third amplification rounds, respectively. For each subsequent step, the product from the previous amplification—purified using ExoSAP® (Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions—served as template. Thermal cycling conditions for N-fragment amplification were as follows: initial denaturation at 95 °C for 2 min; 35 cycles of 94 °C for 30 s (denaturation), 61 °C for 1 min (annealing), and 72 °C for 1 min (extension); followed by a final extension at 72 °C for 10 min. Amplification of the P- and Ps-fragments followed the same protocol, except for the annealing temperatures, which were set at 62 °C and 63.5 °C, respectively. The primers used for the smallest Ps-fragment amplification were also employed for sequencing all resulting Hsp70 amplicons. Amplification results were verified by electrophoretic run in a 2% agarose gel stained with GelRed® (Biotium, inc. Fremont, USA) and visualized by UV light. The Hsp70 consensus sequences were assembled by using the “Sequencing” and “Investigator” packages of GenomeLab™ System Software, version 11.0.24 (Beckman Coulter, Indianapolis, IN, United States). Sequences were identified as L. donovani complex spp. by a BLAST search in GenBank at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on October 14, 2024) and then aligned with the L. infantum reference strain JPCM5 Hsp70 sequence (XM_001470287) by using Clustal W, as implemented in BioEdit v.7.8.0 [20]. As previously described [11], we designated Hsp70(A) as the Hsp70 sequence corresponding to that of the reference L. infantum JPCM5 strain, while Hsp70(C), Hsp70(E), Hsp70(F), and Hsp70(G) were identified as variants diverging from the JPCM5 reference sequence.

Amplification of Cpb was performed by using primers described by Zackay et al. [13] with thermal conditions as follows: 95 °C for 2 min (activation), followed by 35 cycles at 94 °C for 30 s (denaturation), 65 °C for 30 s (annealing), and 72 °C for 30 s (extension), with a final extension at 72 °C for 10 min. In the case of negative results, a second round of amplification was performed using the same primer set and the previously amplified mix diluted 200 times as sample, and with thermal profile as follows: 95 °C for 2 min (activation), followed by 35 cycles at 94 °C for 30 s (denaturation), 63 °C for 30 s (annealing), and 72 °C for 30 s (extension), with a final extension at 72 °C for 10 min.

To confirm the amplification of the Cpb variants, two positive amplification controls were used for each reaction, including genomic DNA of 1) the L. infantum reference strain JPCM5 (MCAN/ES/98/LLM-724), generating 361 bp amplicons [defined as Cpb(E)], and 2) the L. donovani reference strain DD8 (MHOM/IN/80/DD8), whose amplification generates 400 bp long amplicons [defined as Cpb(F)] [13]. As positive control, DNA was extracted from cultured promastigotes at a concentration of 1 × 104 parasites/ml. Amplified products were verified by electrophoresis through a 4% agarose/TAE gel stained by soaking, for 30 min, in Ethidium Bromide bath (0.5 µg/ml in water) after the electrophoretic run.

The discriminatory power of the two typing methods was assessed by calculating the Simpson’s index of diversity using a free online tool (http://www.comparingpartitions.info/index.php?link=Tool, accessed on October 14, 2024). P values < 0.05 were considered significant.