PLOS NEGLECTED TROPICAL DISEASES RESEARCH ARTICLE Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona Aroa Silgado1, Mayuli Armas1, Adrián Sánchez-Montalvá2, Lidia Goterris ID1, Maria Ubals ID3, Jordi Temprana-Salvador ID4, Gloria Aparicio3, Carmen Chicharro ID5, Núria Serre-Delcor2, Berta Ferrer4, Israel Molina ID2, Vicenç Garcı́a-Patos ID3, Tomas Pumarola1, Elena Sulleiro ID1* a1111111111 1 Department of Microbiology, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, a1111111111 PROSICS Barcelona, Barcelona, Spain, 2 Department of Infectious Diseases-Drassanes, Vall d’Hebron a1111111111 University Hospital, Universitat Autònoma de Barcelona, PROSICS Barcelona, Barcelona, Spain, a1111111111 3 Department of Dermatology, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Department of Pathology, Vall d’Hebron University Hospital, Universitat Autònoma de a1111111111 Barcelona, Barcelona, Spain, 5 National Center of Microbiology, Majadahonda, Spain * esulleir@vhebron.net OPEN ACCESS Abstract Citation: Silgado A, Armas M, Sánchez-Montalvá A, Goterris L, Ubals M, Temprana-Salvador J, et al. (2021) Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a Background referral center in Barcelona. PLoS Negl Trop Dis Leishmaniasis is a neglected disease caused by different species of the protozoa Leish- 15(11): e0009884. https://doi.org/10.1371/journal. mania spp. Cutaneous lesions are the most common clinical manifestation. This disease is pntd.0009884 prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Editor: Alvaro Acosta-Serrano, Liverpool School of Leishmania (L.) infantum is the only endemic species, but imported cases are often diag- Tropical Medicine, UNITED KINGDOM nosed. Different classical parasitological methods can be performed for cutaneous leish- Received: May 18, 2021 maniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the Accepted: October 7, 2021 detection and characterization of Leishmania parasites. We aimed to evaluate clinical and Published: November 10, 2021 epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hos- Copyright: © 2021 Silgado et al. This is an open pital over a six-year period. access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Methodology/Principal findings reproduction in any medium, provided the original author and source are credited. Clinical, epidemiological and microbiological data were retrospectively collected and ana- lyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, Data Availability Statement: All data generated or analysed during this study are within the showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 manuscript. between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies Funding: The author(s) received no specific samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant funding for this work. when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most Competing interests: The authors have declared frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus that no competing interests exist. (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 1 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). Conclusions/Significance An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases. Author summary Leishmaniasis is a neglected disease caused by different species of the genus Leishmania. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, the only endemic species is L. (L) infantum, although there are cases of imported leishmaniasis due to travel and migration of people from other endemic areas. Cutaneous lesions are the most common clinical form, but L. (L) infantum can also cause mucosal or visceral involvement. Although microscopic diagnosis and skin biopsy speci- mens are still used in routine diagnosis for cutaneous leishmaniasis, they are time-con- suming and invasive, respectively. Currently, molecular techniques, such as real-time PCR, are very sensitive tools for the detection and identification of Leishmania parasites. In addition, the skin swab sample seems to be a non-invasive alternative for the diagnosis of cutaneous leishmaniasis. In the present work, we describe the clinical characteristics of 59 patients diagnosed by real-time PCR of cutaneous leishmaniasis, as well as the molecu- lar epidemiology of the causative species. We show that there has been an increase in cuta- neous leishmaniasis cases, both autochthonous and imported cases, over the study period. The presence of Leishmania species other than L. (L) infantum is also highlighted. Introduction Leishmaniases are neglected infectious diseases caused by at least 20 different species of the protozoan Leishmania spp. transmitted by the bite of a phlebotomine sandfly [1]. Leishmania- sis is endemic in more than 98 countries, including tropical and subtropical areas and the Mediterranean basin. More than one billion people are at risk of acquiring the infection and approximately 0.7–1 million cutaneous leishmaniasis (CL) cases are reported annually [2,3]. Leishmania species have a different geographic distribution around the world. Old World leishmaniasis, usually manifested as mild cutaneous disease, occurs in Africa, Asia and Europe and the main causative species are L. (L.) tropica, L. (L.) major, and L. (L.) infantum. New World leishmaniasis, with lesions range from mild cutaneous disease to severe mucosal lesions, occurs in America and is mostly caused by L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, L. (L.) mexicana, and L. (L.) amazonensis [4]. Depending on the characteristics of the parasite and the host response, various clinical forms of leishmaniases are present, such as CL, mucocutaneous leishmaniasis, visceral leish- maniasis, and post-kala-azar dermal leishmaniasis. CL is the most frequent clinical form worldwide [5]. Because of the Leishmania species involved, this clinical form presents a variety of clinical features, treatment options, and differ- ent prognosis [4]. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 2 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona In the Mediterranean region, including Spain, L. (L.) infantum is the endemic species, which causes mainly CL and, less frequently, visceral or mucosal leishmaniasis (ML) [6,7]. In this coun- try, the two species of sand flies involved in the transmission of L. (L.) infantum are Phlebotomus perniciosus and P. ariasi [8] and the main reservoir is the dog [7]. Although autochthonous trans- mission occurs in Spain, imported cases caused by other Leishmania species are often diagnosed in migrants or travelers returning from other endemic areas [4,9]. Imported cases may present atypical clinical forms that make clinical diagnosis difficult and, consequently, have different ther- apeutic approaches [4]. In addition, the presence of imported species and of the sand flies involved in their transmission could represent a risk of spread of this species within Spain. CL diagnosis depends on clinical examination of skin lesions and a compatible epidemiol- ogy. The definitive diagnosis is based on direct demonstration the parasite or its genetic mate- rial [10,11] in clinical specimens, using microscopic examination (Giemsa staining, histopathology, immunochemistry) or molecular analysis [12]. Currently, molecular techniques mainly based in PCR protocols, serve as a relevant tool for the diagnosis of leishmaniasis due to their high sensitivity, specificity and performance feasibil- ity in a wide variety of clinical samples, including non-invasive procedures [13]. Moreover, as allow the identification of Leishmania species [14,15], such techniques prove useful in epide- miological surveillance, outbreak characterization and optimal treatment care [16,17]. In addi- tion, real-time PCR allows for the use of an internal control of DNA integrity and/or PCR inhibition in multiplexed reactions [18]. The objective of this study was to describe the epidemiological and clinical features of patients with autochthonous and imported CL diagnosed by real-time PCR in skin swab and/ or biopsy sample in a tertiary hospital of the Mediterranean basin over a six-year period. Methods Ethics statement Epidemiological, clinical and laboratory data were obtained from patients’ medical records in cases diagnosed with CL. Review Board approvals were obtained from the Ethics Committee of the Vall d’Hebron Research Institute, according to the principles expressed in the Declara- tion of Helsinki. All data were anonymized before being analyzed. Study population, samples and microbiological procedures A retrospective study of patients who presented with clinical suspicion of CL between January 2014 and December 2019 at Vall d’Hebron University Hospital (Barcelona, Spain) was per- formed. The clinical charts of the patients were reviewed to obtain additional information including patient age and gender, presence of immunosuppression, travels, type of lesions, and microbiological results. For CL diagnosis in typical single ulcerative skin lesions, first skin swab sample was col- lected and processed for real-time PCR. If the skin swab sample obtained a negative result by real-time PCR or the clinical diagnosis was unclear, a biopsy sample was collected and ana- lyzed for real-time PCR and, when possible, for immunohistochemistry. Definitive CL diagnosis was considered by detecting Leishmania DNA by real-time PCR in skin swab and/or biopsy sample (Fig 1). Molecular techniques DNA extraction from skin swab was carried out via silica–membrane technology using the NucliSens easyMAG system (bioMérieux Diagnostics, Marcy-l’Etoile, France). DNA PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 3 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona Fig 1. Algorithm of the samples used in this study and which assays were performed. https://doi.org/10.1371/journal.pntd.0009884.g001 extraction from skin biopsy was performed after digestion of proteinase K, using the Qiagen Biorobot EZ1 (Qiagen, Hilden, Germany) or the MagCore Plus HF16 (RBC Bioscience, New Taipei City, Taiwan) without distinction. All protocols were performed in accordance with manufacturers’ instructions. A duplex real-time PCR targeted to a specific region of Leishmania spp. kinetoplast DNA and human RNase P gene (Taq Man Human RNase P detection reagent; Applied Biosystems, Foster City, CA) was performed. Real-time PCRs were performed following the protocol described by Mary et al. [19] yet with some modifications: the final conditions in the PCR mix- ture were 1x Quantitec Muliplex PCR kit (Qiagen), 400 nM of both primers for Leishmania target, 100 nM of the TaqMan Leishmania probe, and 0.8x RNase P detection reagent. Reac- tions were performed using 5 μL of eluted DNA in a final volume of 25 μL. Amplifications were carried out in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The cycling conditions were as follows: an initial step of 15 minutes at 95˚C, followed by 40 cycles at 95˚C for 15 seconds and 55˚C for one minute. In all cases, a sample was considered positive for Leishmania DNA when the threshold cycle (Ct) for the Leishmania target was <40. A sample was considered as invalid when the internal control (RNase P human gene) was inefficiently amplified. Positive and negative sam- ples were included as external controls in each real-time PCR run. Molecular characterization of Leishmania species was performed in the National Center of Microbiology laboratory (Madrid, Spain) on samples with a positive result by real-time PCR from patients who came from other countries where CL is endemic or had atypical forms of CL. Briefly, ribosomal internal transcribed spacer 1 (ITS1) was amplified and then PCR prod- ucts were sequenced, following the protocol described by Kuhls et al. [20]. A dendogram was PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 4 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona constructed in order to determine the relationship of the obtained sequence with the reference set [21]. Immunohistochemistry Biopsy tissues were fixed in 5% buffered formalin, embedded in paraffin blocks and cut into 4μm tissue sections. For routine diagnosis purposes, samples were stained with hematoxylin and eosin. Immunohistochemical detection of Leishmania amastigotes was performed on 4μm sections of formalin-fixed, paraffin-embedded tissues. The polyclonal antibody was obtained by immunizing rabbits with a Leishmania antigen. Briefly, sections were deparaffinized and rehydrated through graded alcohol and water. Antigen retrieval with CC1 solution (pH 8.4) for 20 minutes was performed. The sections were incubated in a humidified chamber with the antibody anti-Leishmania diluted 1/1000 for 32 minutes. Staining was performed with Bench- Mark Ultra (Ventana/Roche; Roche Diagnostics, Manheim, Germany) using the ultraVIEW Universal DAB Detection Kit (Ventana/Roche). Statistical analysis Qualitative variables were expressed as absolute frequencies and percentages. Categorical vari- ables were described as mean or median and standard deviation or interquartile range accord- ing to normal distribution. Statistical analyses were done using SPSS Statistics v22.0 (SPSS Inc, Chicago, IL). Results From 2014 to 2019, a total of 210 samples from 188 patients with cutaneous lesions clinically suspected as CL were included in the study. Leishmaniasis was confirmed in 59 of 188 (31.4%) patients by real-time PCR. Thirty-two of 59 (54%) patients were men, and the median age was 34 years (IQR 8–52.5): 20 of 59 (34%) patients were aged under 15 years; 29 (49%) patients were aged between 16 and 65 years; and the remaining 10 (17%) were aged over 65 years. Immunosuppressive conditions were detected in 11 of 59 (18.6%) patients: 10 of 11 (91%) for anti-TNF drugs and 1 patient (9%) referred alcoholism. When comparing the total number of patients diagnosed in a 3-year period, 11 of 59 patients received a CL diagnosis between 2014 and 2016, and 48 of 59 patients between 2017 and 2019 (Fig 2). In the entire study period, 27 (45.8%) patients had previous travel history to a country where CL is endemic, in whom the skin lesion appears after the trip, and had a diag- nosis of imported leishmaniasis. These patients reported having traveled to Morocco (20 of 27; 74%), other countries in the Mediterranean basin (4; 14.8%, including Italia, France, and Israel), Senegal (1; 3.7%), Peru (1; 3.7%) and Costa Rica (1; 3.7%). Localized cutaneous leishmaniasis (LCL) was diagnosed in 26 of 59 (44.1%) patients, being papule the most common skin manifestation (11 of 26 patients; 42.3%). Nineteen of 59 (32.2%) patients presented with more than two lesions (multifocal leishmaniasis), of which 5 patients (26.3%) presented two different types of skin lesions: nodules and plaques (2 of 5; 40%), papules and nodules (2; 40%) or plaques and papules (1; 20%). Three of 59 (5%) patients had oral involvement (none of them with immunosuppressive status) and were considered to have ML. Clinical presentations of skin lesions, differentiating between autochthonous and imported cases, are shown in Table 1. Skin samples from patients with CL suspicion were distributed as follows: 113 of 210 (53.8%) were skin biopsies; 96 (45.7%) were ulcer swabs; and 1 (0.5%) was a laryngeal biopsy. Leishmania DNA was detected by real-time PCR in 30 of 113 (26.5%) skin biopsies and 37 of PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 5 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona Fig 2. Distribution of CL cases from 2014 to 2019. https://doi.org/10.1371/journal.pntd.0009884.g002 96 (38.5%) swab samples. In 13 patients, both swab and biopsy samples were performed simul- taneously, obtaining concordant results; seven (53.8%) of these patients had a positive real- time PCR result. The laryngeal biopsy also tested positive for Leishmania DNA. Retrospectively, in 39 skin biopsies immunohistochemistry and real-time PCR have been performed in parallel. A total of 17 of 39 (43.6%) samples were positive with both techniques, 18 of 39 (46.1%) samples were negative by both techniques, and 4 of 39 (10.3%) samples were negative by immunohistochemistry but positive by real-time PCR. Species identification could not be performed in 8 of 59 (13.6%) cases and in 11 of 59 (18.6%) patients it was not performed because they were autochthonous cases with typical lesion. Molecular characterization of Leishmania species was determined in 40 of 59 (67.8%) confirmed patients. L. (L.) infantum and L. (L.) major were found to be the most frequent spe- cies in 50% (20/40) and 45% (18/40) of cases, respectively. Almost all the samples typed as L. (L.) major (17 of 18; 94.4%) were from patients in Morocco, except one sample from a patient who traveled to Israel (1 of 18; 5.6%). The vast majority of samples typed as L. (L.) infantum (17 of 20; 85%) were from autochthonous cases and the remaining three (3 of 20; 15%) were Table 1. Different clinical presentation of CL per the source of infection. Autochthonous (N = 32) Imported (N = 27) Total (N = 59) Local cutaneous lesion (LCL) 18 8 26 Nodule 5 1 6 Papule 9 2 11 Plaque 3 4 7 a Non-defined 1 1 2 Multifocal leishmaniasis 7 12 19 Nodule 1 2 3 Papule 1 1 2 Plaque 1 4 5 Combinationb 2 3 5 Non-defined 2 2 4 Mucosal leishmaniasis 1 2 3 Non-defineda 6 5 11 a no data available in the patient’s medical record b combination of two different types of skin lesions https://doi.org/10.1371/journal.pntd.0009884.t001 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 6 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona Fig 3. Geographical origins of different Leishmania species. https://doi.org/10.1371/journal.pntd.0009884.g003 from patients who traveled to other Mediterranean countries. Two (5%) samples were classi- fied under the Viannia subgenus, of which one corresponded with a traveller returning from Costa Rica and the other to a Peruvian migrant. The first case could be typed as L. (V.) brazi- lensis but in the second case the specific species could not be determined. The Leishmania spe- cies and their geographical origins are shown in Fig 3. Clinical infections caused by L. (L.) infantum were mostly (10 of 20; 50%) classified as LCL (Table 2). This species was also the causative agent of multifocal CL in 4 of 20 (20%) cases and ML in 3 of 20 (15%) cases. L. (L.) major caused a LCL in 6 of 18 (33%) cases and multifocal CL in 7 of 18 (38.9%) cases. The two (100%) patients infected by Leishmania species from the Viannia subgenus were clas- sified as multifocal CL. Discussion This study shows the results of 59 patients diagnosed with CL by real-time PCR in a Barce- lona-based tertiary hospital over a 6-year period. Clinical and epidemiological presentation observed in this work were similar to that in other studies conducted in Spain, in which patients with CL were mainly adult males [22–25]. During the study period, an increase in the number of both autochthonous and imported CL cases in the last, few years was observed; this rise was similar to that in other cohorts [23,26,27]. According to data published in our country, an elevated number of immunosup- pressed patients [28] and migrants/travelers from highly endemic CL settings [8], could Table 2. Clinical presentation of CL according to the species identified. Localized CL Multifocal CL Mucosal CL Non-definedb L. (L.) infantum 10 4 3 3 L. (L.) major 6 7 5 Viannia subgenus 2 Undetermineda 10 6 3 a molecular typing could not be performed b no data available https://doi.org/10.1371/journal.pntd.0009884.t002 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 7 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona explain this rise in leishmaniasis cases. In addition, the use of sensitive molecular techniques could have contributed to confirming presumptive clinical diagnoses and, thereby, more cases of infection [25]. In this study, leishmaniasis diagnosis was performed by real-time PCR on skin biopsies and/or swab samples. Despite the high sensitivity of molecular techniques, the performance of real-time PCR obtained in this work is quite low in general (38.5% and 26.5% for skin swab and biopsy samples, respectively). This low percentage of positivity may be due to the fact that leishmaniasis presents a great diversity of clinical manifestations [29,30], which makes its diag- nostic orientation difficult if not a typical cutaneous lesion is observed with a compatible epidemiology. Skin biopsy is the most robust sample to diagnose CL. However, swab scraping confers sub- stantial benefits regarding biopsy (less invasive and painful, easier to perform and does not make permanent scar) and should be recommended as the first approach [31,32]. Our data show a higher percentage of positivity for Leishmania DNA detection in swab samples than the biopsy sample (38.5% vs. 26.5%, respectively). The lower positivity of real-time PCR on skin biopsy samples may be due to the fact that this type of sample is obtained to diagnose atypical lesions that may correspond to clinical entities other than leishmaniasis. In our study, few patients had both skin biopsy and swab sample, with 100% concordance between the real- time PCR results of both samples. Yet, few attempts have been made to simultaneously com- pare the utility of different lesion sampling procedures for PCR-based CL diagnosis; sensitivity reached approximately 93–98% when a swab sample was used [31,33]. In 39 samples, both real-time PCR and immunohistochemistry could be performed in par- allel; however, both techniques attained only 89% agreement, with 4 negatives for immunohis- tochemistry. This finding supports the use of real-time PCR as a diagnostic method for CL, with particular utility in disease types with low parasitic loads [34]. In a large number of stud- ies, sensitivity results obtained from PCR protocols is better when compared with classical parasitological methods, including immunohistochemistry, with values reaching higher than 90% [15,25,35,36]. Due to high sensitivity, accessibility and simplicity, molecular assays have been included in diagnostic strategies within many settings. In our cohort, at least four Leishmania species belonging to both Leishmania and Viannia subgenus caused infections in patients with CL. However, the vast majority of infections were due to species from the Leishmania subgenus; L. (L.) infantum was the most frequent causative agent (50% of cases). As expected, in all autochthonous cases whose samples could be typed, L. (L.) infantum was identified as the causative agent. Species identification of imported cases, including L. (L.) infantum, L. (L.) major and Vian- nia subgenus, was fully consistent with the travel history of all patients. In our clinical practice, molecular typing was not performed in autochthonous patients with a typical single ulcerative lesion, and these infections were interpreted as L. (L.) infantum. If we considered these autochthonous patients and molecular typed-confirmed cases, the per- centage of L. (L.) infantum would rise from 50% to 60.8%, and L. (L.) major and Viannia would drop from 45% to 35.3% and from 5% to 3.9%, respectively. Similar detection rates of Leishmania species, in both autochthonous and imported cases, were reported in a study done by Merino-Espinosa et al. from another Spanish hospital (70% L. (L.) infantum vs. 30% L. (L.) major) [37]. To know the circulating Leishmania species will contribute to a better under- standing of the epidemiology of leishmaniasis [21]. Most of our patients infected with L. (L.) major came from Morocco. In the last, few decades, an important epidemic focus of CL has occurred in the arid pre-Saharan regions. L. (L.) major is the most important causative agent of these outbreaks in the southern of Morocco (79% between 2015 and 2016) resulting primarily in multifocal skin lesions [38,39]. In focus, PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 8 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona P. papatasi sand fly, the main vector of L. (L.) major [40], is distributed in large regions of Spain [41], making possible that this Leishmania specie could become established as an autochthonous specie in our country. Thus, infected travelers/migrants could also represent potential reservoir hosts for anthroponotic Leishmania parasite acquired abroad [42]. In this sense, appropriate surveillance network is essential in order to monitoring the potentially introduction of non-endemic Leishmania species in endemic areas with local autochthonous transmission [43]. Characterization of causative species of Leishmania is relevant when deciding adequate treatment and managing the disease [4,9,14]. CL caused by Old World Leishmania species tends to resolve spontaneously, but CL caused by New World species causes serious infections and only a few percentage of cases resolve spontaneously [12]. Clinical infections caused by L. (L.) infantum were mostly (51.4%) classified as LCL. How- ever, there were three cases of ML, with L. (L.) infantum being the etiological agent. Although mucosal involvement is not common [24], cases of ML caused by L. (L.) infantum have been reported in Spain and other European regions [44]. Our study does, however, have limitations due to its retrospective nature. For example, the study lacks information regarding some patients because we had no clinical and/or epidemio- logical information. Additionally, in those patients with skin biopsy sample, not in all cases we were able to obtain information regarding the immunohistochemistry technique. Due to the low number of skin swabs and biopsies samples collected in parallel, a significant difference in real-time PCR positivity could not be determined. Conclusions Autochthonous and imported cases of CL diagnosed by real-time PCR have been increasing over the last, few years. Due to the high number of imported leishmaniasis cases detected, other Leishmania species than L. (L.) infantum was also increased, especially L. (L.) major in travelers and/or migrants from Morocco. Molecular characterization provides important epidemiological information about patients from other geographical regions or with atypical forms of leishmaniasis, as well as what approaches to undertake for optimal treatment care. Acknowledgments The authors thank Anthony Armenta for his assistance with the English. Author Contributions Conceptualization: Aroa Silgado, Mayuli Armas, Elena Sulleiro. Data curation: Aroa Silgado, Mayuli Armas, Adrián Sánchez-Montalvá, Lidia Goterris, Maria Ubals, Jordi Temprana-Salvador, Gloria Aparicio, Carmen Chicharro, Núria Serre-Delcor, Berta Ferrer, Israel Molina, Vicenç Garcı́a-Patos, Tomas Pumarola, Elena Sulleiro. Formal analysis: Aroa Silgado. Investigation: Aroa Silgado, Mayuli Armas, Adrián Sánchez-Montalvá, Lidia Goterris, Maria Ubals, Jordi Temprana-Salvador, Gloria Aparicio, Carmen Chicharro, Núria Serre-Delcor, Berta Ferrer, Israel Molina, Vicenç Garcı́a-Patos, Tomas Pumarola, Elena Sulleiro. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009884 November 10, 2021 9 / 12 PLOS NEGLECTED TROPICAL DISEASES Diagnosis and epidemiology of cutaneous leishmaniasis in Barcelona Methodology: Aroa Silgado, Mayuli Armas, Adrián Sánchez-Montalvá, Lidia Goterris, Maria Ubals, Jordi Temprana-Salvador, Gloria Aparicio, Carmen Chicharro, Núria Serre-Delcor, Berta Ferrer, Israel Molina, Vicenç Garcı́a-Patos, Tomas Pumarola, Elena Sulleiro. Validation: Elena Sulleiro. Writing – original draft: Aroa Silgado. Writing – review & editing: Aroa Silgado, Mayuli Armas, Adrián Sánchez-Montalvá, Lidia Goterris, Maria Ubals, Jordi Temprana-Salvador, Gloria Aparicio, Carmen Chicharro, Núria Serre-Delcor, Berta Ferrer, Israel Molina, Vicenç Garcı́a-Patos, Tomas Pumarola, Elena Sulleiro. References 1. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018; 392: 951–970. https://doi.org/10.1016/ S0140-6736(18)31204-2 PMID: 30126638 2. Gradoni L, López-Vélez R, Mokni M. 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