Received: 26 February 2021 | Revised: 22 March 2021 | Accepted: 11 April 2021 DOI: 10.1111/tbed.14107 ORIGINAL ARTICLE Autochthonous and imported tegumentary leishmaniasis in Catalonia (Spain): Aetiological evolution in the last four decades and usefulness of different typing approaches based on biochemical, molecular and proteomic markers Anna Fernández-­Arévalo1,2,3 | Cristina Ballart1,4 | Jordana Muñoz-­Basagoiti1,2,3 | Leire Basarte1,2,3 | Gonzalo Lobato1,2,3 | Albert Arnau1,2,3 | Alba Abras5 | Silvia Tebar1 | Teresa Llovet3,6 | Patrick Lami7 | Francine Pratlong7 | Mercè Alsina8 | Esther Roe9 | Lluís Puig9 | Carme Muñoz2,3,6 | Montserrat Gállego1,4 1 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat Abstract de Farmàcia i Ciències de l'Alimentació, Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is en- Universitat de Barcelona, Barcelona, Spain 2 demic for both visceral and cutaneous leishmaniasis, the autochthonous aetiologi- Institut de Recerca Biomèdica Sant Pau, Barcelona, Spain cal agent being Leishmania infantum. Around the world, the L. donovani complex is 3 Servei de Microbiologia, Hospital de la associated with visceral symptoms, while any species of the Leishmania or Viannia Santa Creu i Sant Pau Barcelona, Barcelona, Spain subgenera affecting human can produce tegumentary forms. In a context of growing 4 ISGlobal, Hospital Clínic -­Universitat de numbers of imported cases, associated with globalisation, the aim of this study was Barcelona, Barcelona, Spain to analyse the aetiological evolution of human tegumentary leishmaniasis in a region 5 Laboratori d'Ictiologia Genètica, of Spain (Catalonia). Fifty-­six Leishmania strains, isolated from 1981 to 2018, were Departament de Biologia, Universitat de Girona, Girona, Spain analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI-­TOF. 6 Departament de Genètica i Microbiologia, The utility of these different analytical methods was compared. The results showed Universitat Autònoma de Barcelona, Bellaterra, Spain an increase in leishmaniasis over the two last decades, particularly imported cases, 7 National Reference Centre for which represented 39% of all cases studied. Leishmania infantum, L. major, L. tropica, Leishmaniasis, University Hospital Centre L. braziliensis, L. guyanensis and L. panamensis were identified. The combination of of Montpellier, University of Montpellier, Montpellier, France molecular and enzymatic methods allowed the identification of 29 different strain 8 Servei de Dermatologia, Hospital Clínic types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L. major de Barcelona & Departament de Medicina, Universitat de Barcelona, Barcelona, Spain types were relatable with geo-­temporal data. Among the autochthonous cases, type 9 Servei de Dermatologia, Hospital de la C prevailed throughout the studied period (39%). Minor types generally appeared Santa Creu i Sant Pau, Barcelona, Spain within a short time interval. While all the techniques provided identical identification Correspondence at the species complex level, MALDI-­TOF and rpoIILS or fh sequencing would be the Anna Fernández-­Arévalo, Secció de most suitable identification tools for clinical practice, and the tandem hsp70-­ITS2 Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia could substitute MLEE in the epidemiological field. i Ciències de l'Alimentació, Universitat de Carme Muñoz and Montserrat Gállego should be considered joint senior author. [Correction added on 25 June 2021, after first online publication: The copyright line was changed.] This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-­VCH GmbH. 1404 |  wileyonlinelibrary.com/journal/tbed Transbound Emerg Dis. 2022;69:1404–1418. | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FERNÁNDEZ-­ARÉVALO et al. 1405 Barcelona, Barcelona, Spain. KEYWORDS Email: annaferar@gmail.com aetiological evolution, characterisation methods, imported cases, Spain, tegumentary leishmaniasis 1 | I NTRO D U C TI O N are well established in laboratories worldwide (Da Silva et al., 2010; De Almeida et al., 2011; Mirahmadi et al., 2018; Van der Auwera Human leishmaniasis, present in 98 countries around the world, is et al., 2014) due to their relatively low cost, and because the results caused by Leishmania species (World Health Organization, 2020). are easy to interpret and compare between laboratories (Van der Cutaneous leishmaniasis (CL) has a wide variety of clinical manifes- Auwera & Dujardin, 2015). The targets of these methodologies vary tations, ranging from a small dermatosis on the inoculation point, considerably, the most common being ribosomal proteins, heat-­ usually with spontaneous recovery, to skin lesions of variable ap- shock proteins, kinetoplast regions, intergenic regions and other pearance that may lead to mucocutaneous and lymphatic dissemina- conserved genes (Akhoundi et al., 2017; Van der Auwera et al., 2016; tion (Burza et al., 2018; Reithinger et al., 2007). Other manifestations Van der Auwera & Dujardin, 2015). Lately, matrix-­assisted laser de- include diffuse CL, disseminated CL and leishmaniasis recidivans, as sorption/ionisation –­time of flight mass spectrometry (MALDI-­TOF well as numerous atypical forms (Meireles et al., 2017; Reithinger MS) has also been applied for species identification, due to its speed et al., 2007). While visceral leishmaniasis (VL) is mainly associated and simplicity (Lachaud et al., 2017; Mouri et al., 2014). with Leishmania donovani “senso lato”, human CL and its variant mu- The aim of this study was to evaluate the utility of different tech- cocutaneous leishmaniasis (MCL), both included in the term tegu- niques and markers for the identification of Leishmania strains stored mentary leishmaniasis (TL), can be caused by nearly 20 Leishmania in the Trypanosomatid Cryobank of the Universitat de Barcelona species of the subgenera Leishmania and Viannia (Van der Auwera & (UB) that have caused TL in Catalonia (NE Spain), and to analyse the Dujardin, 2015; World Health Organization, 2010). The main caus- aetiological evolution of TL in the last four decades. ative agents of TL in the Old World (Europe, Africa and Asia) belong to the species complexes L. donovani, L. tropica and L. major, and in Latin America to L. braziliensis, L. guyanensis and L. mexicana (Akhoundi 2 | M E TH O DS et al., 2017; World Health Organization, 2010). In Spain, the only au- tochthonous species involved in a zoonotic epidemiological cycle is 2.1 | Strains L. infantum, included in the L. donovani complex (Fernández-­Arévalo et al., 2020; Gállego et al., 2001; Jiménez et al., 1995; Martín-­Sánchez, Fifty-­six strains isolated from patients with CL or MCL in Catalan hos- Gramiccia, et al., 2004). As in other foci of the Mediterranean region, pitals from 1981 to 2018 were selected from the UB Trypanosomatid L. infantum causes both VL and CL, including non-­t ypical forms, and Cryobank. As all the strains had previously been identified by one of very rarely results in mucosal involvement (Aliaga et al., 2003; Alvar the techniques used in the current study, they were analysed with et al., 1997; Chicharro et al., 2003; Merino-­Espinosa et al., 2018; whichever methods not previously applied. All strains were thawed Pratlong et al., 2013; Cortés et al., 1997). The global incidence of the and cultured in Schneider's insect medium supplemented with 20% disease in Spain in 2018 was 0.64 cases/100,000 habitants (Centro foetal bovine serum and 1% sterile human urine until the exponential Nacional de Epidemiología, 2019). In the last decades, globalisation growth phase, when they were analysed. All the available epidemio- and migration have altered the classical geographical distribution of logical and clinical information was collected. Leishmania species and imported cases are becoming increasingly common (Giavedoni et al., 2015; Pérez-­Ayala et al., 2009). Lesion evolution and treatment effectiveness vary in each particular case, 2.2 | PCR-­sequencing being mainly influenced by the immunological response of the pa- tient as well as the species involved (Bañuls et al., 2011; World DNA from the cultured strains was obtained by the Qiamp DNA Health Organization, 2010). As microscopical examination does Mini Kit (Qiagen) following the manufacturer's instructions. Four not allow discrimination between Leishmania species, various tech- different regions were amplified and sequenced: heat-­s hock pro- niques based on biochemical, molecular or proteomic markers have tein 70 (hsp70), RNA polymerase II largest subunit (rpoIILS), inter- been developed as identification tools (Akhoundi et al., 2017; Van nal transcribed spacer 2 (ITS2) and fumarate hydratase (fh). For der Auwera & Dujardin, 2015). Multilocus enzyme electrophoresis hsp70, rpoIILS and ITS2, primers and cycling conditions previously (MLEE), a classic technique based on the evaluation of metabolic en- described by Van der Auwera et al., (2013), Ravel et al., (2006) and El zyme phenotypes (Rioux et al., 1990), has been considered the gold Tai et al., (2001), respectively, were used. For fh, the forward primer standard for typing purposes (World Health Organization, 2010), al- described in Zemanová et al., (2007) was used for the Leishmania though it is currently available only in a few laboratories. In contrast, subgenus. An alternative forward primer was designed for the molecular-­based techniques, mainly PCR-­RFLP and PCR-­sequencing, Viannia subgenus (fhnm_F, 5′-­TCGTCTTCGTCTTCTGTGC-­3′), | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1406 FERNÁNDEZ-­ARÉVALO et al. F I G U R E 1 Leishmania complex identification by MLEE, PCR-­sequencing, and MALDI-­TOF. Different tonalities indicate different types within each complex. The number of types per complex are bracketed. Cpx, complex as well as a new reverse primer for both subgenera (fhint_R, (MPI) EC 5.3.1.8 and glucose phosphate isomerase (GPI) EC 5.3.1.9], 5′-­G GCAATGAAGAGGAACTCGTA-­3′). Amplification conditions following a previously described procedure (Rioux et al., 1990). For were an initial denaturalisation step of 5 min at 95°C followed New World strains, MDH, ICD and GLUD enzymes were excluded by 35 cycles of 1 min at 95°C, 1 min at 57°C and 1 min at 72°C (Chouicha et al., 1997). Protein extracts were run on starch gels and a final elongation step of 10 min at 72°C. All the amplicons (7 cm, 90 V) at different pH's (7.4, 8.6, 9.4) with or without coen- were enzymatically purified using EXOSAP-­I T (Affymetrix USB) zymes (NAD, NADP) and then revealed, using the conditions suitable and double-­s trand sequenced at the Scientific and Technologic for each enzyme. Migration distances were measured and compared Centres of the UB by the Sanger method. The sequences ob- with those from marker strains to assign the corresponding electro- tained were edited using Bionumerics software (Applied Maths, morph and zymodeme. The isoelectric focusing technique was oc- version 7.6.3) and uploaded to GenBank under the accession casionally used to confirm the NP1 mobility (Piarroux et al., 1994). numbers MT497923-­M T497978 and MT498854-­M T499021. For the analysis, sequences were aligned and compared in MEGA X software (Kumar et al., 2018). For the ITS2, each complex was 2.4 | MALDI-­TOF analysed separately. A neighbour joining (NJ) tree was con- structed from the concatenation of hsp70, rpoIILS and fh genes Six replicate samples of each strain were prepared as previously using the p-­d istance substitution method and a bootstrap of described (Lachaud et al., 2017). Mass spectra were obtained with 1,000 replicates. the AutoFlex II MALDI TOF/TOF instrument and the Flex Control version 3.4 software (Bruker Daltonics) on default parameters and submitted to the mass-­spectral identification (MSI) application avail- 2.3 | MLEE able on https://biolo​gical​-­mass-­spect​romet​r y-­ident​ifica​tion.com/ msi/. The species result that predominated among the replicates was Protein extracts were obtained from mass cultures as previously de- considered as the strain identification, and in case of a tie, the spe- scribed under the aforementioned conditions (Piarroux et al., 1994). cies with the best score was chosen. MLEE was performed on 12 or 15 enzymes, depending on the previ- ous identification result or the possible origin of the strains. For Old World strains, MLEE was performed on a total of 15 enzymes [malate 3 | R E S U LT S dehydrogenase (MDH) E.C 1.1.1.37, malic enzyme (ME) EC 1.1.1.40, isocitrate dehydrogenase (ICD) EC 1.1.1.42, phosphogluconate dehy- 3.1 | Strain identification drogenase (PGD) EC 1.1.1.44, glucose-­6-­phosphate dehydrogenase (G6PD) EC 1.1.1.49, glutamate dehydrogenase (GLUD) EC 1.4.1.3, The four genomic regions were sequenced in all the strains, NADH diaphorase (DIA) EC 1.6.2.2, nucleoside phosphorylase pu- whereas MLEE and MALDI-­TOF analyses were only performed rine (NP) −1 and 2-­EC 2.4.2.1, glutamate oxaloacetate transaminase in 53 of the 56 selected strains due to culture issues. For three (GOT) −1 and 2-­ EC 2.6.1.1, phosphoglucomutase (PGM) EC 2.7.5.1, L. major strains, the ITS2 sequences had zones of poor qual- fumarate hydratase (FH) 4.2.1.2, mannose phosphate isomerase ity; however, discriminative regions were sufficiently clear to be | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FERNÁNDEZ-­ARÉVALO et al. 1407 included in the analysis. All the methodologies coincided 100% MHOM/EC/2002/BCN-­545 MHOM/EC/2016/BCN-­885 MHOM/PA/2014/BCN-­859 MHOM/GT/2005/BCN-­717 in the identification at the species complex level (Figure 1), but below that, there were variations depending on the discrimination power of each technique. Thus, by consensus identification, the Strain WHO code studied strains belonged to six different taxa: L. infantum (n = 36), L. major (n = 10), L. tropica (n = 1), L. braziliensis (n = 6), L. guyanensis (n = 2), and L. panamensis (n = 1) (Figure 1). All the complexes were represented by one species except L. guyanensis (L. guyanensis and L. panamensis). Multilocus enzyme electrophoresis grouped strains in a total of GPI 17 zymodemes, 11 of which belonged to the Leishmania subgenus 87 87 87 87 and 6 to the Viannia subgenus (Figure 1). Intra-­complex variability was due to different mobilities of three and up to five enzymes. Four MPI a 60 60 70 70 L. (Viannia) strains (one L. braziliensis, two L. guyanensis and L. pan- amensis) presented new zymodemes. The new isoenzyme profiles were different combinations of known electrophoretic mobilities or FH 82 87 87 87 new electromorphs for the taxa. The new zymodemes, coded from MON-­327 to MON-­330, are given in Table 1. Molecular techniques showed nineteen, fifteen, eleven and 108.5 108.5 108.5 109.6 PGM seven different sequence types for ITS2, hsp70, rpoIILS and fh, re- spectively (Figure 1). Although nucleotide transitions and transver- sions between different complexes were observed, interspecific GOT2 90a 120 125 118 variation was mainly due to the presence of heterozygous alleles or, in the case of ITS2, diverse repetition of the microsatellite re- gions (Table 2). The latter resulted in different lengths for the ITS2 GOT1 amplicons among the different complexes, but also among strains 148 132 145 145 of the same taxa. Complexes of the Leishmania subgenus (L. dono- vani, L. major and L. tropica complexes) showed more intra-­complex NP2 nucleotide differences compared to those of the Viannia subgenus 100 100 100 112 (L. braziliensis and L. guyanensis complexes). Below the complex level, species of the subgenus Viannia had more inter-­strain variability in NP1 nucleotide positions or regions than the species of the Leishmania 800 380 0 800 subgenus. Consequently, in the NJ tree produced from the concate- nation of the hsp70, rpoIILS and fh gene, greater distances were ob- DIA served between the complexes of the Leishmania subgenus, whereas 50 50 50 50 the L. (Viannia) complexes appeared more diversified (Figure 2). Based on MALDI-­TOF, the strains were confidently associated G6PD with five complexes (Figure 1). At the species level, MALDI-­TOF 90 106 103 103 Enzymatic profile of the new zymodemes agreed with the consensus identification obtained by the other techniques in 87% of the strains. L. braziliensis and L. guyanensis complexes were the most controversial (Table 3, Figure 1). The MSI PGD 60 b 70 45 45 application did not allow any further distinction below the species level. ME The discrimination power of the techniques varied depending 87 87 87 87 on the complex analysed: the highest diversity in L. donovani was New mobilities at taxon level. revealed by MLEE, in L. major by ITS2, in L. braziliensis by both ITS2 New mobility at genus level. MON 330 329 328 327 and hsp70, and in L. guyanensis by all three markers. When the results from molecular and enzymatic data were combined, the strains were classified into 29 types (A to AC) (Figure 2). The greatest diversity L. panamensis was observed in the species of the Viannia subgenus. Only between L. guyanensis L. guyanensis L. braziliensis TA B L E 1 L. infantum and L. major strains it was possible to observe clusters Taxon sharing identical features with all the techniques used. For strains without MLEE results, the most prevalent MLEE type was assumed. b a | 1408 TA B L E 2 Detailed intra-­complex variability for hsp70, rpoIILS, fh and ITS2 sequences hsp70 rpoIILS fh ITS2 Variable Variable Variable No. No. intra-­ No. intra-­ No. intra-­ Intracomplex variable sequence Variable intra-­ sequence complex sequence complex sequence complex repeated and deleted Complex Length types complex positions Length types positions Length types positions Length types positions regions L. donovani (n = 36) 1,245 2 504G>R 529 1 –­ 615 1 –­ 610–­614 4 –­ 36(AT)5–­6, 62(TA)4–­5, 320(G)5–­9 L. major (n = 10) 1,245 3 102C>Y, 954C>Y 529 2 9T>A 615 1 –­ 669–­683 5 490A>G, 106(AT)8–­9, 478(TA)6–­ 515T>G 11, 605(AT)4–­5 L. tropica (n = 1) 1,245 1 NA 529 1 NA 615 1 NA 540 1 NA NA L. braziliensis (n = 6) 1,245 6 51G>R, 264G>A, 529 5 57G>K, 615 2 89G>R, 529–­537 6 46A>T, 45(TA)4–­5, 94(TA)6–­ 561G>A/R, 129G>K, 114C>Y, 204G>C, 11, 413(CT)4–­5, 1213T>A, 175G>K, 414G>R 280G>A, 452(TA)4–­8, 1237G>A/R 204A>R, 527G>A 460_463dupCATA, 213A>R, 503(G)4–­6, 519(G)7–­8 324G>R, 333C>Y, 375G>R, 387G>S L. guyanensis (n = 3) 1,245 3 13C>Y, 564G>R, 529 2 396C>A 615 2 9A>G 524–­536 3 449T>C 95(TA)4–­9, 1078T>G, 1017C>T, 221(TCCTCTC)2–­3, 1102A>G, 247_253delTCTCTTC, 1181A>R, 254_257delTCC, 1213A>G, 270(G)5–­6, 309(G)7–­ 1236C>G 9, 516(G)7–­8 Note: Sequence variants were noted according to den Dunnen et al., (2016) and the International Union of Pure and Applied Chemistry (IUPAC) nomenclature. n: number of strains, NA: not applicable. FERNÁNDEZ-­ARÉVALO et al. 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FERNÁNDEZ-­ARÉVALO et al. 1409 F I G U R E 2 Characterisation of all the strains and association with epidemiological and clinical data. From left to right: NJ tree constructed from concatenated sequences of hsp70, rpoIILS and fh; typing results (i.e., sequence types for the different loci and zymodemes −each one of the same species complex colour-­coded in different tonalities of the same colour−and global strain type) and epidemiological and clinical data. Strains from the same type are boxed. Strains from the same patient are indicated with the same sub-­index number next to the clinical presentation. NR, no results; ND, no data; NA, not applicable; CL, cutaneous leishmaniasis and MCL, mucocutaneous leishmaniasis 3.2 | Analysis of typing results considering 25% female (gender was unknown for the remaining 2%) (Figure 3), epidemiological and clinical data of the sample and 24% were immunocompromised. Skin involvement was more abundant than mucocutaneous forms (45 versus. 5 patients and The 56 strains analysed were isolated in a 37-­year period (from one patient having both clinical forms) (Figure 2). Although there 1981 to 2018), 46% of them in the decade from 2001 to 2010 was a high percentage of undescribed cutaneous lesions (20%, (Figure 2). They belonged to 51 patients aged between 8 and 9/46), those reported were mainly located on extremities (25 pa- 76 years (average age of 43 years). Patients were 73% male and tients, 13 with lesions on upper limbs and 13 on lower limbs) or the | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1410 FERNÁNDEZ-­ARÉVALO et al. TA B L E 3 MALDI-­TOF results according to the species identified as either. Between 2011 and 2018, imported cases were consensus identification more numerous than autochthonous ones (nine versus seven, re- Consensus ID MALDI-­TOF ID spectively, and one undetermined). The greatest taxonomic diversity was found in strains isolated in the last two decades (2001–­2018) L. donovani complex (L. infantum, n = 33) L. infantum (n = 33) (Figure 4). L. major complex (L. major, n = 10) L. major (n = 10) All cases considered as undoubtedly autochthonous (28 pa- L. tropica complex (L. tropica, n = 1) L. killicki (n = 1) tients and 33 strains) were caused by L. infantum strains. Among L. braziliensis complex (L. braziliensis, n = 6) L. braziliensis (n = 2) these, the predominant patient profile was composed by males L. peruviana (n = 4) aged between 30 and 40 years, although the overall average age L. guyanensis complex (L. guyanensis, n = 2; L. panamensis (n = 3) in the study was 48 years. Cutaneous lesions, frequently single (in L. panamensis, n = 1) at least 17 patients), were usually found on the face or extremities Correct species ID 46 strains (87%) (9 and 10 patients, respectively). Five patients had MCL (Figure 2), Correct complex ID 6 strains (11%) and two of them had Human Immunodeficiency Virus infection Note: Consensus ID refers to the identification obtained with MLEE and (HIV+). molecular techniques. In five patients, four documented as immunocompromised Abbreviation: ID, identification and two previously diagnosed with VL, two different isolates of L. infantum were obtained (Table 4). In two cases (patients 2 and 3), the two strains were identified as identical by all the analytical techniques used. In two other cases (patients 1 and 5), the two strains were different according to MLEE, the results being MON-­1 and MON-­3 4 in both patients. In patient 4, the strains differed in at least the hsp70 type (MLEE was not performed in one of the strains). After combining molecular and enzymatic data, the 33 autoch- thonous strains were divided into 11 different types (from A to K) (Figure 5). Type C strains were the most prevalent (n = 13, 39%) and appeared throughout the studied period. They belonged to zymo- deme MON-­1 and presented the L. infantum consensus sequence for all the genes. Four strain types (E, F, G and I) differed from type C F I G U R E 3 Age and sex distribution of the patients. For patients only in the zymodeme, MON-­29 being the second most frequent. with more than one isolate, age data refer to the first isolate. ND, Type H differed only in the hsp70 sequence, while five types (A, B, D, no data J and K) diverged in both zymodeme and sequence type. The other groups had short temporary distributions, especially when a molecu- lar change was detected, except for types F and G that were isolated over 12 and 13 years, respectively. Cases classified as undoubtedly imported (20 patients and strains) were caused by five different taxa (L. major, L. tropica, L. bra- ziliensis, L. guyanensis and L. panamensis). Most of these patients were aged between 21 and 30 years, but the mean age was 32. Fifty percent (7 males, 3 females) were Spanish nationals who had trav- elled to other endemic areas, 40% were migrants (7 males and 1 female) and 10% were of unknown nationality. The only immuno- compromised patient was a migrant with CL. CL lesions were single or multiple (8 and 10 cases, respectively) and affected mainly the FIGURE 4 Species identification variability for each studied extremities (14 cases). There was one case of MCL caused by L. bra- decade ziliensis. As shown in Figure 2, the L. major strains came from North Africa and the Middle East and were usually associated with mul- face (11 patients); other locations were neck, lumbar area, back, tiple lesions. The seven different profiles found among the strains buttocks, each in one patient as either single or multiple lesions (in differed in the geographical origin or year of isolation and were as- 27 and 16 patients, respectively). Mucocutaneous lesions affected sociated with MLEE and ITS2 types and to a lesser extent with the the nose (2 patients), palate, tongue and rectum (1 patient each). hsp70 types. In three clusters (types N, O and Q), all the techniques Among the patients, 28 (55%) represented autochthonous cases, gave identical characterisation results, and each was composed of whereas 20 (39%) were imported, and three cases could not be two strains, which temporarily coincided in Jordan, Mauritania and | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FERNÁNDEZ-­ARÉVALO et al. 1411 TA B L E 4 Detailed information and identification results of autochthonous patients with more than one isolate (L. infantum) MLEE Lesion Patient OMS code hsp70 rpoIILS fh ITS2 (MON) Type Interval localisation VIH 1 MHOM/ES/94/BCN-­113 ST1 ST1 ST1 ST1 1 C 17 months Palate Sublingual pos MHOM/ES/95/BCN-­137 ST1 ST1 ST1 ST1 34 F 2 MHOM/ES/96/BCN-­148 ST1 ST1 ST1 ST1 1 C 2 months Rectum pos MHOM/ES/96/BCN-­168 ST1 ST1 ST1 ST1 1 C 3a MHOM/ES/2002/BCN-­466 ST1 ST1 ST1 ST1 1 C simultaneously Healthy skin pos MHOM/ES/2002/BCN-­467 ST1 ST1 ST1 ST1 1 C Lesion 4a MHOM/ES/2005/BCN-­657 ST1 ST1 ST1 ST1 NR (C) 17 months ND pos MHOM/ES/2006/BCN-­732 ST2 ST1 ST1 ST1 1 H ND 5 MHOM/ES/2007/BCN-­763 ST1 ST1 ST1 ST1 34 F 31 months Face ND MHOM/ES/2010/BCN-­816 ST1 ST1 ST1 ST1 1 C Nose Note: Bracketed type indicates that the predominant zymodeme was assumed. Abbreviations: ND, no data; NR, not carried out; pos, positive; ST, sequence type. a Patients with diagnosed VL. F I G U R E 5 Temporal distribution of the L. infantum types from confirmed autochthonous cases. The number of strains is indicated when there were multiple isolates. Stripes denote the types deduced as probable due to the lack of MLEE results Morocco, respectively. As only one L. tropica strain was found, ac- (García-­Almagro, 2005; Gil-­Prieto et al., 2011; Portús et al., 2007; quired in Israel, a more in-­depth analysis was not possible. L. bra- Suárez Rodríguez et al., 2012). Even so, in the last two decades, TL ziliensis strains belonged to different countries of Latin America, strains deposited or obtained by sample cultivation have quadrupled L. guyanensis strains were isolated from patients originating from and tripled, respectively. This increase could be a consequence of or visiting Ecuador, and the L. panamensis strain was from Panama. imported cases involving other Leishmania species, the occurrence Each L. (Viannia) strain constituted a unique type, although some of rare or atypical lesions requiring differential diagnosis of CL, and strains shared geographical origins. the implementation of identification protocols derived from treat- ment differences according to the species and lesion characteristics. Although strain isolation is less sensitive than other diagnostic meth- 4 | D I S CU S S I O N ods (Aronson & Joya, 2019), a similar increase in TL cases has been observed in other studies conducted in Spain and Italy (Garrido-­ Since the Trypasomatid Cryobank of the UB (Spain) was created in Jareño et al., 2020; Giavedoni et al., 2015; Riera et al., 2016). 1983 (Gállego et al., 2003), more than 1,000 strains, belonging prin- Regarding the patient profile in autochthonous cases, the aver- cipally to Leishmania genera, have been deposited. The Leishmania age age was 48 years, which coincides with the average observed strains were acquired mainly from patients affected by VL or are for leishmaniasis patients in other studies in Spain (Fernández collection strains. At the end of 2018, strains isolated in hospitals Martínez et al., 2019). However, the infant population, one of the in the Catalonia region from patients with TL represented only 5% most commonly affected by leishmaniasis, was not represented. of the total deposited. Despite fluctuating collaboration between One explanation is that the hospital providing 33% of the samples the University and the hospitals over the years, the low number of does not offer paediatric health care, but another factor is that TL strains is most likely to be due to under-­diagnosis. As TL does reported cases among children correspond predominantly to VL not often require hospitalisation, it is frequently diagnosed and (Fernández Martínez et al., 2019; Garrido-­Jareño et al., 2020). The treated in primary healthcare or private dermatological centres majority of patients were male (71%), as in numerous other reports | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1412 FERNÁNDEZ-­ARÉVALO et al. (Fernández Martínez et al., 2019; Garrido-­Jareño et al., 2020; Gil-­ cases (Bart et al., 2013; Di Muccio et al., 2015; Giavedoni et al., 2015; Prieto et al., 2011; Herrador et al., 2015; Portús et al., 2007; Riera Pérez-­Ayala et al., 2009; Vandeputte et al., 2020). Patient character- et al., 2016). Among the hypotheses proposed by researchers for istics in terms of age, origin or destination matched those reported this gender bias, those more applicable in our context could be for the immigrant population in Spain and the profile of the Spanish the dimorphic immunological response driven by sexual hormones traveller in several studies on imported diseases (Instituto Nacional (Lockard et al., 2019) or the higher prevalence of Leishmania/HIV de Estadística, 2020; Jaén-­Sánchez et al., 2016; Valerio et al., 2005; coinfection in males (Desjeux & Alvar, 2003; Herrador et al., 2015). Zamarrón Fuertes et al., 2010). It is worth mentioning that most of the patients in the study were In three cases (1 MCL and 2 Cl) with documented travel his- immunocompetent, or at least no immunosuppression had been tory it was not possible to determine if the infection caused by reported. In the case of immunocompromised patients (39%), who L. infantum had occurred in Spain or during the stay in a foreign were mainly coinfected with HIV, all the strains were isolated be- country (Mexico, Italy and Portugal). In South America, L. infan- tween 1994 and 2010, and only two patients were female. tum predominantly causes VL and some cases of CL have been Mucocutaneous lesions are principally associated with South reported in Costa Rica, Honduras, Brazil and Venezuela (Castro American Leishmania species, although they can also be caused by et al., 2016; De Lima et al., 2009; Noyes et al., 1997; Zeledón Old World species (Strazzulla et al., 2013), primarily in immunosup- et al., 1989). In Mexico, L. infantum has been reported to cause pressed patients (Mejia, 2018; World Health Organization, 2010). In VL, but it is not associated with skin lesions (Pan American Health this study, five autochthonous patients presented mucocutaneous Organization, 2019). Without species characterisation, the case lesions and only two of them were immunocompromised. Although would have likely been classified as imported, based on the history mucosal involvement is rarely associated with L. infantum, other of travel to Mexico and the clinical form (MCL), but the identifica- cases of MCL in immunocompetent patients have been described tion of L. infantum as the aetiological agent opens up the possibil- in Spain and elsewhere (Aliaga et al., 2003; Casolari et al., 2005; ity of infection in Spain. In the other two cases, since Portugal and Freites-­Martinez et al., 2015; Garrido-­Jareño et al., 2020). In the cur- Italy are included in the large Mediterranean area where L. infan- rent work, only one out of six MCL cases was imported, and was tum is endemic, together with Spain (Alvar et al., 2012; Campino caused by L. braziliensis, the species most frequently responsible for et al., 2006; Gramiccia et al., 2013; Pratlong et al., 2013), infection this clinical form (Reithinger et al., 2007). was equally possible in any of the three countries. Confirmed imported cases represented 39% of the anal- Similarly, in three other imported cases it was not possible to ysed strains from the last decades, which constitutes an import- identify the country of infection, since multiple destinations with ant increase, in agreement with other studies in Spain (Giavedoni circulating species in common were reported. One patient had et al., 2015; Riera et al., 2016). An increasing tendency of imported travelled to Jordan and Syria, both endemic for L. major, the spe- leishmaniasis has been observed around the world as a result of glo- cies identified. However, its typing profile was identical to that balisation, even affecting non-­endemic countries (Bart et al., 2013; of another temporally close Jordanian strain. It should be men- Di Muccio et al., 2015; Söbirk et al., 2018; Stark et al., 2008; tioned that L. tropica also causes CL in that area (Alvar et al., 2012; Vandeputte et al., 2020). According to data from the World Tourism Postigo, 2010), although usually resulting in a single lesion, whereas Organization, the number of international tourist arrivals worldwide L. major is more associated with multiple lesions, as suffered by this in the studied period rose from 277 million in 1981 to 1,407 million patient (Aoun & Bouratbine, 2014). Another patient, also with a L. in 2018 (World Turism Organization, 2020). Additionally, the immi- major isolate but producing a single lesion, had travelled to differ- grant population in Spain increased from nearly 20,000 people in ent countries of West Africa, where this species is responsible for the mid-­1980s to over 600,000 people in 2018 (Instituto Nacional human CL (Kone et al., 2018). The third case was a patient who had de Estadística, 2020; Romero Valiente, 2003). It is noteworthy that visited Peru, Bolivia and Colombia and presented multiple CL lesions among samples deposited in 2011–­2018, imported cases outnum- caused by L. braziliensis. This species is present in all three coun- bered autochthonous cases. A certain bias towards imported cases tries, where it cohabits with other species (Pan American Health could be related to their association with more complex lesions, in Organization, 2019). contrast with autochthonous cases, with predominantly single le- The aforementioned examples, as well as the overall taxonomic sions (Giavedoni et al., 2015; Merino-­Espinosa et al., 2018). However, diversity found (six taxa from five complexes), indicate the growing growing awareness among clinicians of the need for species identi- risks of deducing the aetiological agent based only on epidemiolog- fication in patients reporting travel history may also be occurring. ical data, which may only be conclusive for autochthonous cases In imported cases, patients were younger than in autochthonous without any history of travel abroad, and support the use of spe- cases, with similar numbers of Spanish travellers and immigrants (10 cies identification tools. Today, the treatment of CL varies in each and 8, respectively, and two patients of unknown nationality). The patient, depending on the Leishmania species as well as the num- immigrants were often established in Spain but had recently trav- ber, appearance and duration of the lesions, which could determine elled to their countries of origin. Infections were acquired to a similar the self-­healing nature of the disease, the response to certain drugs extent in the Old and New World (11 and 9, respectively), unlike in and risk of metastatic complications (Aronson & Joya, 2019; Mensa other studies, which usually report a predominance of Old World et al., 2020). From an epidemiological point of view, the imported | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FERNÁNDEZ-­ARÉVALO et al. 1413 species identified are unlikely to represent a risk in Spain, due to a mixed infection (Cortés et al., 1997). An alternative hypothesis the absence of reservoirs or sand fly vectors. The exception is the is that as long as MON-­34 differs from MON-­1 exclusively in the anthroponotic species L. tropica, whose vector P. sergenti is pres- mobility of the MDH enzyme, a sequence mutation could have oc- ent elsewhere in Spain (Ballart et al., 2014; Barón et al., 2008; Gil curred specifically affecting enzyme mobility, as reported for other Collado et al., 1989). genes (Mauricio et al., 2006; Zemanová et al., 2007). Similarly, in pa- The use of high resolutive approaches or the combination of dif- tient 4, the presence of a heterozygous allele of the hsp70 gene in ferent methods can contribute other valuable information besides the second isolate, together with a HIV and VL background, again species identification. For example, the combination of MLEE with suggests a relapse. As the hsp70 sequence type was also observed ITS2 allowed us to evaluate the variation of autochthonous strains in two other strains isolated within a short period of time, a mixed through four decades. There was a prevailing strain type (type C) infection would be a reasonable explanation. A different situation throughout the period (39%), which was probably equivalent to was observed for patient 5. Here, the change in zymodeme, together the predominant group found in other studies (Kuhls et al., 2005). with a higher time interval between isolates, a slight difference in the Variations of type C, most of them due to different zymodemes, ap- localisation of the lesion and no records of immunosuppression leads peared only in narrow intervals of time, except types F and G, which us to think of a possible reinfection. had longer distribution periods. Types F and G differed from type An additional use of strain characterisation is for identification in C in being zymodemes MON-­34 and MON-­29, respectively, which outbreaks of disease. As we are all aware, a consequence of globali- are frequently found in CL cases in other parts of Spain (Chicharro sation is that health issues are increasingly crossing borders. Among et al., 2003; Jiménez et al., 1995; Pratlong et al., 2013). It should be our data, we observed three L. major strains isolated between 2017 noted that in the later years of the study, four strains (types J and K) and 2018 that were acquired in Morocco, and with similar results for showed a particular ITS2 sequence type, which differed from the con- the different typing methods (two were identical and the third dif- sensus sequence in the number of repetitions of three microsatellite fered in one repetition of a microsatellite in the ITS2). In this period, regions and which probably corresponds to the Lombardi type found an outbreak of leishmaniasis was active in Morocco (El Hamouchi by Chicharro et al., (2013) in Madrid among strains with zymodemes et al., 2019), as also occurred in other countries at some point of MON-­1 and MON-­24. The zymodeme for type J was MON-­27, which the studied period (Mosleh et al., 2018; Oliveira-­Neto et al., 1988; to the best of our knowledge has only been recorded in Italy and in a Postigo, 2010). It, therefore, seems plausible that some of the identi- Spanish man living in Argentina with an uncertain origin of infection fied strains could have originated in an outbreak abroad. (Martín-­Sánchez, Navarro-­Mari, et al., 2004; Pratlong et al., 2013). In Many markers and techniques have been used for Leishmania our study, the affected patient was Spanish and without a history of identification, as reviewed (Akhoundi et al., 2017; Kuhls & travel. The zymodeme of type K strains was MON-­24, which is abun- Mauricio, 2019; Van der Auwera & Dujardin, 2015). Nevertheless, dant in Spain but did not appear in the current study among the au- in most studies, they have been applied to a low number of strains, tochthonous strains before 2016. Another interesting finding was a to strains isolated only from the Old or New World, without cover- strain of MON-­284 (type I). Rarely reported, this zymodeme is typi- ing both Leishmania and Viannia subgenera, or are limited to a sin- cally associated with HIV+ patients (Chicharro et al., 2003), as in this gle technique. In this study, we used biochemical (MLEE), molecular case. Regarding the L. infantum strain whose geographical origin was (sequencing) and proteomic (MALDI-­TOF MS) techniques to identify either Spain or Mexico, MLEE typing results (MON-­34) point towards 56 strains from different complexes isolated from autochthonous an autochthonous case. As far as we are aware, this zymodeme has and imported Leishmania species in Catalonia. Although not always not been found in America (Ribeiro Coutinho et al., 2011) and only in a straightforward manner, all the techniques and markers used one L. infantum strain from Panama has shown a profile other than allowed the correct identification at the complex level, unlike the MON-­1 in the New World (Franssen et al., 2020). In this case, se- experience of another study carried out in different European labo- quencing methods alone would not allow to discriminate between an ratories (Van der Auwera et al., 2016). autochthonous or an imported case since the predominant sequence Since not all identification techniques provide the same degree types showed by the strain (Figure 2, BCN 698) have also been found of discrimination, choosing the most suitable methodology depends in the New World (Kuhls et al., 2005; Van der Auwera et al., 2013). on the specific purpose of the research. Grosso modo, MLEE discrim- More in-­depth characterisation could also be useful to evalu- inates below the species level with a high level of correspondence ate possible relapses or reinfections. In our case, five patients had among zymodemes and geographical origin. Used to type nearly all two isolates. For patient 3, and probably also patient 2, both iso- the described species, MLEE constitutes the basis of current taxon- lates were taken during the same episode and the strain type did omy and is considered by many as the gold standard typing method not change. In the other three cases, 17 to 31 months elapsed be- (Van der Auwera & Dujardin, 2015). However, it requires large culture tween the two isolates, and in all of them there was a change in the volumes, is time-­consuming, expensive and cumbersome, and inter- strain type. Strains of patient 1 differed in the zymodeme, which pretation of results is not straightforward, which has limited its use changed from MON-­1 to MON-­34. However, based on the locali- to specialized laboratories. Moreover, the phenotype observed does sation of the lesion, relapse seems the most plausible scenario, and not always correspond to the real genotype (Mauricio et al., 2006; the MLEE variation could be explained by culture selection from Zemanová et al., 2007). In contrast, MALDI-­TOF is faster, easier to | 18651682, 2022, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tbed.14107 by Readcube (Labtiva Inc.), Wiley Online Library on [26/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1414 FERNÁNDEZ-­ARÉVALO et al. use and cost-­effective. However, certain difficulties could arouse in also increased, are not caused by a single strain type, which could the identification below the complex level of taxonomically contro- affect the severity of the disease or the healing process. Therefore, versial groups and different interpretation criteria of MSI data can the identification and further characterisation of the circulating produce slight differences in the results. Regardless, identification at strains affords valuable information for both the clinical and epi- the species complex level is sufficient to manage Leishmania. demiological management of the disease. While MLEE, sequencing A third approach is sequencing, which generally takes up an in- and MALDI-­TOF are all useful tools, they are not equally suitable termediate position regarding discriminative power, workability and for all contexts. rpoIILS and fh sequencing or MALDI-­TOF (despite time consumption, but its pros and cons are hardly influenced by its culture dependence) could be key identification tools in the clini- the target used (Kuhls & Mauricio, 2019). In the current study, the cal area, where speed and simplicity are the most valuable features. four analysed genetic regions permitted a confident identification of Whereas hsp70 and ITS2, due to their respective confidence and res- the Leishmania subgenus at the species level. However, if punctual olution, could constitute an interesting tandem to substitute MLEE sequence-­t ype mutations were not considered, far fewer consti- in the epidemiological field. tutive differences were observed among complexes of the Viannia subgenus, up to the point that the L. braziliensis and L. guyanensis AC K N OW L E D G E M E N T S complexes differed by only a single position in the fh fragment. The authors would like to thank Professor M. Portús for starting Differences among taxa within the L. guyanensis complex were espe- the Trypanosomatid Cryobank of the Universitat de Barcelona, cially scarce and indistinguishable by the fh marker. However, since which has not only served as the basis of much research work in only one L. panamensis strain was found, it is uncertain if the ob- our group but has also contributed to expanding our knowledge of served variations in the other genes are characteristic of the taxon leishmaniasis in our area. ISGlobal members participate in the group or polymorphisms of the strain. Below the species level, hsp70 and GREPIMER (Grup de Recerca en Patologia Importada i Malaties ITS2 were the markers that provided additional inter-­strain diver- Emergents i Re-­emergents) which is supported by AGAUR (2017 gence for the entire genus. The high variability obtained among the SGR 00924), the Tropical Disease Cooperative Research Network ITS2 sequences, together with the degree of geographical and/or (RICET) (RD12/0018/0010), and the Spanish Ministry of Science, temporal correspondence found among the sequence types, render Innovation and Universities through the “Centro de Excelencia this molecular target suitable for epidemiological purposes beyond Severo Ochoa 2019-­2023” Program (CEX2018-­0 00806-­S). ISGlobal species identification. Nevertheless, the differences blurred the lim- is member of the CERCA Program, Generalitat de Catalunya. its between the L. braziliensis and L. guyanensis complexes. The low variability observed with the fh fragment could be a consequence of C O N FL I C T O F I N T E R E S T the reduction in the fragment length made to amplify all the clinically Authors declare no competing interests. important complexes. At least for the L. donovani complex, studies using the full-­length gene have found greater diversity; the strains E T H I C A L A P P R OVA L were also geographically diverse, unlike in our study (Zemanová The authors confirm that the ethical policies of the journal, as noted et al., 2007). In terms of workability and time efficiency, rpoIILS and on the journal's author guidelines page, have been adhered to. No fh were the simplest targets, due to their constant and relatively ethical approval was required for this original research. short length. For the whole hsp70 sequencing, the concatenation of two shorter fragments rendered better results than a full-­length DATA AVA I L A B I L I T Y S TAT E M E N T amplification, making it less straightforward. In ITS2, the abundant Sequence data are available in GenBank under the accession num- microsatellites hampered sequencing reads and their interpretation. bers MT497923-­MT497978 and MT498854-­MT499021. In addition, the final length variability among strains did not permit the alignment of all the sequences and their addition to the NJ tree. ORCID Nevertheless, the amplicon length could preliminarily orientate to- Anna Fernández-­Arévalo https://orcid.org/0000-0003-4925-7184 wards certain complexes. Finally, even if not applied here, another factor worth considering is that some of the used regions work well REFERENCES with small amounts of DNA, being directly available from a clinical Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., sample (Kuhls & Mauricio, 2019; Montalvo et al., 2017; Schönian Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets et al., 2003); a trait inapplicable for MLEE or MALDI-­TOF. This re- and diagnosis. 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