Clinical diversity and treatment results in Tegumentary Leishmaniasis: A European clinical report in 459 patients.

BACKGROUND: Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management.
METHODOLOGY: Demographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients. PRINCIPAL
FINDINGS: Compared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%). CONCLUSION/SIGNIFICANCE: CL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates.

Introduction

Ninety-eight countries and 3 territories are considered endemic for leishmaniasis, a vector-born parasitic disease caused by a protozoan from the Leishmania genus [1]. While visceral leishmaniasis is a severe disease, cutaneous leishmaniasis (CL), that affects the skin and can involve the mucosa of nose and mouth, is not life-threatening. It causes however disfiguring lesions, scarring and stigma [2]. CL affects 0.7 to 1 million individuals each year and its global burden is increasing [3,4].

According to existing surveillance networks, CL is not infrequent in travellers visiting endemic areas [5]. The absence of mandatory notification in most countries and the self-curing course of a proportion of lesions hamper robust estimation of the burden of CL in travellers. Recent conflicts in the Middle East have been linked to a rise in the incidence of CL in migrants and refugees [6].

Textbook and reference reviews have long proposed a dichotomic view, with Old World CL often described as a benign cutaneous disease in contrast to New World CL strongly associated with destructive mucosal lesions. Thus, assessment of the mucosal involvement in travellers is discussed only for New World CL and almost never for Old World CL in clinical practice. To provide a detailed description of the clinical presentation and risk of mucosal involvement of CL, both directly impacting treatment decisions, we analysed its presentation in travellers from data deposited in a large international database. The harmonized data collection by clinicians attending patients infected in many transmission foci worldwide enables direct comparisons of clinical patterns induced by different Leishmania species, and the outcome following treatment with either local or systemic regimens.

Methods

Ethics statement

This observational study (DR-2013-041; N°912650) was approved by the French National Agency regulating data protection (Commission Nationale de l’Informatique et des Libertés). Patients (or their legal representative) provided verbal or written consent according to national regulations for use of anonymized data on clinical findings, treatment received, clinical outcome and laboratory results. No genetic analyses of human DNA were performed.

Data collection

From 2006 through 2012 data collection was initiated by a French referral network as reported elsewhere [7,8]. A standardized case report form was used for baseline demographical, clinical and biological data and a second case report form was used to collect outcomes, identification of Leishmania species and adverse events, at least 42 days after the expert had provided treatment advice to the attending physician. From 2012 through 2019, the analysis was extended to several European countries by teams belonging to the LeishMan network.[9] LeishMan is a multicentre international medical project aiming to improve the management of leishmaniasis through harmonization of medical practices and collection of data in a common system. The variables of the LeishMan database were translated from those of the initial French database. Currently, the consortium gathers 50 experts affiliated to 30 institutions in 11 European countries (Belgium, France, Germany, Italy, Norway, Portugal, Spain, Sweden, Switzerland, the United Kingdom, The Netherlands). Data were pseudonymously and prospectively collected by experts from each institution. The database is hosted by Epiconcept since 2012 and has been certified as “Health Data Host” (ISO standards ISO 27001 and elements of ISO 2000–1 and ISO 27018). Demographic, clinical and biological data were filled in an electronic case report form.

Definitions

A patient was considered to have cutaneous/mucocutaneous leishmaniasis if she/he had: (1) cutaneous and/or mucosal lesions; (2) laboratory confirmation of the presence of Leishmania as follows: presence of amastigotes in smears or tissue sections and/or promastigotes in culture and/or positive molecular testing by Polymerase Chain Reaction (PCR) on a skin sample.

Mucosal leishmaniasis (ML) refers to the presence of mucosal lesion(s) without skin involvement. Muco-cutaneous leishmaniasis (MCL) refers to the simultaneous presence of both mucosal and skin lesions. Mucosal involvement refers to both ML and MCL. Post-Kala Azar Disease (PKDL) was defined as new skin lesions in a patient who had recovered from visceral leishmaniasis. Disseminated cutaneous leishmaniasis was defined as CL with more than 10 lesions in 2 non-contiguous anatomical sites. Immunocompromised patients include patients with at least one of the following treatments or conditions: immunosuppressive therapy (e.g >5mg/day equivalent prednisone during more than 3 months, chemotherapy, methotrexate, monoclonal antibodies or small molecules targeting immune cells or their products [e.g. anti-TNF agents], HIV infection, primary immunodeficiencies). HIV testing was not systematically done and/or collected as variable.

Healing in CL was defined as complete re-epithelialization for an ulcer or disappearance of induration for a papular lesion at least 42 days after treatment start [10]. Treatment regimen and dosage mostly followed national or international guidelines [11-13].

Information regarding the infecting Leishmania species was captured in the case report form by the expert based on the molecular identification performed by local laboratories in each centre or by national reference laboratories. Clustering of (sub)species in complexes followed a recent classification of Leishmania species [14]. Briefly, L. infantum was included in the L. donovani Complex, L. major was included in the L. major Complex. L. braziliensis and L. peruviana were included in the L. braziliensis Complex, and L. panamensis and L. guyanensis were merged into L. guyanensis Complex.

Patients eligible for local therapy based on clinical parameters

We determined the proportion of patients with CL potentially eligible for a local therapy (eg, paromomycin cream or intralesional antimony + cryotherapy). Criteria for eligibility were as per the LeishMan consensus for Treatment of Cutaneous and Mucosal Leishmaniasis in Travellers and WHO recommendations [12,15]. Criteria were: localized CL, ≤ 4 lesions, ≤ 4cm in largest induration diameter, no immunosuppression, lesion type (papulo-nodular or dry crust or wet crust), lesion site compatible with treatment method (eyelid and peribuccal lesions excluded).

Statistics

Continuous variables are presented as median [interquartile range] and categorical variables as numbers (frequencies). Categorical variables were compared using chi-square test or Fisher’s exact test as appropriate. Continuous variables were compared using Student’s t-test or Wilcoxon-Mann-Whitney test as appropriate. A two-tailed p-value < .05 was considered statistically significant. Statistical analyses were performed using R software 3.1 version (R Development Core Team, 2008) using GMRC Shiny Stat application developed by CHU de Strasbourg (2017).

Results

Geography

A total of 459 patients with CL were recruited into the study cohort between 2006 and 2019, corresponding to 464 infections, i.e., 5 patients had a second episode (. Patients were included by 10 centres from 7 countries in Europe (Tables A and B in . There were 279 cases (60%) and 185 cases (40%) acquired in Old World and New World, respectively. The infection had been acquired in 47 different countries (. Top 3 countries of acquisition were French Guiana (n = 41), Peru (n = 37) and Costa Rica (n = 28) for the New World and Spain (n = 48), Syria (n = 36) and Morocco (n = 34) for the Old World (Tables C and D in . Countries of acquisition could not be determined in 10 cases (2%) because patients had travelled to multiple endemic countries in a short period of time.

Flow chart of entries in the LeishMan database and selected analyses.

Infection, Relapse or Reinfection correspond to a documented episode of visceral or cutaneous leishmaniasis, with or without mucosal involvement. * patients with multiple follow-up visits or samples. Notes: A “Patient” file corresponds to demographic information. A “Sample” file corresponds to one sample collected in one site with one technique at one date (eg. PCR on skin biopsy of right hand collected the 25th of May 2017). Abbreviations: CL, cutaneous leishmaniasis; VL, visceral leishmaniasis

Number of cases reported from each country of acquisition (464 infections in 459 patients).

Created with mapchart.net.

Demography

The median age of patients in the study cohort was 30 years; 99 infections (21%) occurred in children <16 years. There were marked differences in age distributions between travellers Visiting Friends or Relatives and tourists (. The main reasons for travel in children were Visiting Friends or Relatives (57%) or migration (25%) while travelling reasons in adults were more heterogeneous and influenced by travel-destination. For example, tourists returning from the New World (often referred to as “backpackers”) were younger than tourists from the Old World (median age 30 years vs 48 years; p = 0.03). Migration-related infections were only observed in the Old World while military personal or expatriates acquired the infection almost exclusively in the New World. Patients were immunocompromised in 5% of cases, and a previous history of leishmaniasis was reported by 8% of patients in the cohort. Compared to those infected in the Old World, patients infected in the New World were more frequently males (75% versus 54.5%), and more rarely immunocompromised (0.6% versus 8%) (.

Clinical features of Cutaneous Disease

Most patients had lesions limited to the skin (n = 440; 95%), with typically one or two ulcerated lesions of the limbs that had been present for 3 months prior to diagnosis and were 10 to 30 mm-wide (Table E in . Compared to lesions acquired in the Old World, lesions acquired in the New World were more frequently ulcerative (75% versus 47%), larger (median diameter 30 mm versus 20 mm), more frequently localized on the limbs (61% versus 47%) and more frequently associated with nodular lymphangitis (30% versus 6%) (.

Identification of the infecting Leishmania complex in 198 patients (Table F in showed species-associated demographic and clinical patterns. For patients with no species identification, diagnosis was mostly confirmed by histology or smear, i.e., samples on which identification cannot be performed. Excluding rare species and incomplete species (Table F in ), we focused on the 5 most frequent infecting complex species (affecting a sub-cohort of 166 patients (). This analysis showed that typically, children infected with L. major in Africa had rapidly evolving multiple lesions of the limbs; children and young adults infected with L. tropica had a lesion of the face without mucosal involvement; 40–70 year-old tourists infected with L. infantum in Southern Europe had a slowly evolving papulo-nodular lesion in the face and mucosal involvement in 22% of cases; young adults infected in Latin America with L. braziliensis or L. guyanensis had a rapidly evolving ulcer of the lower limbs with lymphangitis and mucosal involvement in 35% and 6% of cases, respectively (. Immunosuppression was reported in 22 patients and was related to ongoing therapy with anti-cancer chemotherapy (n = 1), ustekinumab (n = 1), methotrexate (N = 5), TNFα antagonists (n = 5), prolonged corticosteroids with or without other immunosuppressive drugs (azathioprine (n = 1), mycophenolate mofetil (n = 1) or corticosteroids alone (n = 4)), or HIV infection (n = 3), or Good syndrome (n = 1).

Mucosal involvement

Mucosal involvement was observed in 20 patients (4.3%), 15 were infected in the Old World and 5 in the New World (. In the 5 patients from the New World 2 had ML and 3 had MCL, while in the 15 patients from the Old World 7 had ML and 8 had MCL. The risk of having mucosal involvement at presentation was 5.3% in patients with lesions acquired in the Old World and 2.7% in patients infected in the New World. Countries of acquisition of New World mucosal or muco-cutaneous leishmaniasis were Bolivia (2 cases), Costa Rica (1 case), French Guiana (1 case), Nicaragua (1 case). For Old World leishmaniasis with mucosal involvement, countries of acquisition were Spain (7 cases), France (2 cases), Italy (2 cases), Greece (2 cases), Oman (1 case), Turkey (1 case). We observed 10 MCL, 9 ML, and one MCL with visceral involvement and inaugural skin lesions in a patient with AIDS (CD4 count 67/mm3). Mouth and laryngeal lesions were observed in 6/13 cases from Old World (missing data in 2 patients) and 1/5 case from New World, while lesions of nasal cavity were observed in 7/13 cases from Old World and 5/5 cases from New World. Seven of 15 infections (47%) with mucosal involvement in the Old World were observed in immunocompromised patients while no patient was immunocompromised in the New World subgroup with mucosal involvement. A previous history of leishmaniasis was reported in 5 of 20 patients with mucosal involvement. CL, ML or VL had been diagnosed in 1, 2 and 2 patients respectively. In all patients except one (a previous VL episode 20 years before), the mucosal involvement occurred less than 5 years (median = 4 years) after the first episode. Analysis in the subgroup of patients with an identified Leishmania species found a prevalence of mucosal involvement of 22%, 5.8%, 0% and 0% for L. (infantum)/donovani Complex, L. braziliensis/L. guyanensis Complex, L. tropica Complex and L. major respectively (.

Local or systemic therapy

More than 10 different treatment regimens were used by physicians in the network (). Compared to patients who received systemic therapy, patients treated with local therapy were more frequently infected in the Old World (88% vs 40%, p< 0.0001), had smaller lesions (median 19mm vs 31 mm, p < 0.0001), less frequently associated with nodular lymphangitis 7.5% vs 28.3%, p < 0.0001), and similar number of lesions (median 2 vs 1.5, p = 0.51) (. These different approaches applied in these different patient populations resulted in similar cure rates (89% vs 86%) at first evaluation with a median full duration of follow-up of 80 days [IQR 59–111]. As previously observed “No specific treatment”, which corresponds to washing lesions with soap and water followed by semi-occlusive dressing, was associated with high (81%) cure rate [7]. Patients treated with this approach were mainly infected in OW (90%; 19/21). Due to small numbers within each subgroup, analysis of treatment outcome has not been made in respect to infecting species within the groups Old World CL and New world CL.

Patients treated with non-systemic therapy

In patients with CL acquired in the New World, healing after day 42 occurred in 12 of 13 (92%) managed with local therapy, and in 55 of 68 (81%) managed with systemic therapy (p = 0.32). In patients with Old World CL, healing after day 42 occurred in 83 of 94 (88%) managed with local therapy and in 42 of 45 (93%) managed with systemic therapy.

Patients with CL eligible for local therapy

Among 348/440 patients with localized CL and all available clinical criteria (see Methods), we found that at least 208/348 patients (60%) were eligible for local therapy. Among them, 90 and 118 patients had been infected in the New World and Old World respectively.

Discussion

Cutaneous Leishmaniasis (CL) displays a diversity of lesion aspect, number, size, and location on the body surface, including the potential extension to mucosae of the nose, mouth, pharynx and larynx. These features determine the selection of the most appropriate treatment regimens, from simple wound dressing to long courses of potentially toxic parenteral drugs. In this cohort of 459 European patients with CL infected in 47 countries from 4 continents, we show that mucosal involvement (that includes both purely mucosal or mixed cutaneous and mucosal infections) was present in less than 5% of patients and was similarly infrequent in subjects infected either in Latin America (“New World”, 2.7%) or in Europe, Africa or Asia (“Old World”, 5.3%). Forms caused by the 5 most frequent infecting complex species (L. major, L. tropica, L. infantum, L. braziliensis, and L. guyanensis), identified in 166 patients, were each associated with distinct features. In particular, no mucosal involvement was observed in patients infected with either L. major or L. tropica, whereas it affected 6% of patients infected with L. braziliensis or L. guyanensis complex (2 MCL, 1 ML) and 22% of patients infected with L. infantum (4 ML, 4 MCL), in whom this complication was strongly (though not exclusively) related to pre-existing immunosuppression. This new set of data on lesions characteristics and on the risk of mucosal involvement will form a solid basis to refine treatment recommendations, based on an optimized benefit-to-risk analysis.

We observed 4 typical patterns of CL presentation. Young adults infected in the New World with L. braziliensis or L. guyanensis complex typically had a single, large ulcerative lesion on the lower limb. L. major- and L. tropica-infected subjects were predominantly children and young adults, who typically had either multiple lesions located on the upper limbs or a single lesion on the face. L. infantum-infected subjects were predominantly older than 40 years, and typically had a single small lesion in the face. Interestingly, travelers returning from South and Central America are predominantly young backpackers visiting deep forests. In terms of age and lesion characteristics, this population resembles that of young infected native patients living in areas where leishmaniasis is endemic. Taken together, these clinical patterns should help attending physicians recognize typical forms of CL, hopefully reducing the diagnostic delay that currently averages 3 months. Discriminating the respective contributions of parasite-, vector- and host-related factors in these new phenotypes is beyond the scope of our current approach but the sustained, multisite, multiparametric surveys performed by the LeishMan network and other groups worldwide will provide information to more precisely revisit the pathogenesis of human leishmaniasis [9].

It was surprising that infections acquired in the Old World (and more specifically in Europe or some Mediterranean countries) had higher rates of mucosal involvement than infections with L. braziliensis or L. guyanensis complex acquired in Latin America. The prevalence of mucosal involvement at baseline was 4.3% in the cohort which is consistent with the rate (usually <7%) observed in studies including travellers [5,16]. In our study, the unexpected, low rate of mucosal involvement in infections acquired in the New World contrasts with the unexpected, relatively high rate in infections acquired in the Old World. High rates of mucosal involvement patients with Old World leishmaniasis, especially in L. infantum infection, has been recently observed in some but not all studies in travellers [17-19]. The increasing number of immunocompromised patients, probably more keen to travel to European countries than to Latin America, likely contributes to the rising incidence of this complication [20]. A previous study from France also suggested a risk of visceral dissemination that we did not observe in our large cohort [21]. Immunosuppression seems to play an important role in the pathogenesis of ML in the Old World, while its role in ML in New World is more difficult to determine, as many apparently immunocompetent patients do develop ML.

Systemic treatment is warranted in patients with L. infantum mucosal infection, in whom miltefosine and liposomal amphotericin B seem effective [8,22]. Therefore, in immunosuppressed patients with cutaneous lesions due to L. infantum, miltefosine and liposomal amphotericin B should probably be used, in the hope of preventing the subsequent occurrence of mucosal involvement that is frequently multifocal with this species (Fig 3). Of interest however, we confirm here recent observations showing that liposomal amphotericin B is not highly effective in CL caused by Leishmania species other than L. infantum [8,23].

Fig 3

Comparative features of 166 cases of cutaneous leishmaniasis by main infecting species.

Abbreviations: IQR, interquartile range; VFR, visiting friends and relatives.

In the subpopulation of patients in whom it was used, local therapy was effective. Compared to patients who received systemic therapy, patients treated with local therapy were more frequently infected in the Old World, had smaller lesions, and less frequent nodular lymphangitis. Arguably, these differences reflect predominantly treatment choices by physicians, based on the continent of infection and, to a lesser extent, on the applicability of local treatment. Local and systemic therapy were thus used in different patient populations but resulted in similarly high cure rates (89% vs 86%). This tells little on the intrinsic power of these different approaches but suggests that local treatment was appropriate in most patients in whom it was used, i.e., that criteria defined in the LeishMan consensus were accurate [11,12,16]. Local therapy is now proposed by national and international recommendations not only for Old World but also for a proportion of patients with New World CL [24]. In the small number of patients with New World CL treated locally, healing was almost the rule in this cohort, suggesting that more patients may benefit from this approach in the future. These results match those of prospective studies in endemic countries but larger prospective studies are needed before a robust conclusion can be reached in the specific context of CL in travellers [16,25-27]. Not all patients may be eligible to local therapy. In particular, whether infection acquired in Bolivia is compatible with local therapy is still a matter of controversy. A higher risk of mucosal involvement in travellers returning from Andean countries especially Bolivia has indeed been reported [16].

Solomon and colleagues found that 17 of 145 (11.7%) Israeli travellers with CL acquired in Amazon Basin in Bolivia received a diagnosis of ML [28]. We observed a prevalence of 9.5% (2/21) of mucosal involvement in travellers from Bolivia in our study, which matches the 5–15% rate indicated in a quasi-exhaustive review of the literature and the 11.5% rate reported in a recent large cohort of travellers by Boggild and al. [5,16]. Detecting subsequent mucosal relapse through an extended follow-up was beyond the immediate scope of our study. However, as most teams involved in the LeishMan consortium are reference centres for leishmaniasis in their respective country, they are expected to attend most patients with secondary mucosal involvement, and capture corresponding information in the common database. The similar risks of mucosal involvement across these different reports suggest that very few if any cases of were missed by our consortium during the study period. In our cohort, a previous episode of leishmaniasis was reported in 20% of patients with mucosal involvement. Several teams in our consortium give to CL patients a note mentioning the diagnosis of leishmaniasis and the significant (though relatively low) risk of oro-nasal complications that may occur years to decades after the initial episode. Our results suggest that a similar information should also be provided after a VL episode.

Approximately two thirds of patients of our cohort were eligible for local therapy when following guidelines in travellers based on lesions number, size, location and preexisting conditions [7,12,15]. Even though there may have been some heterogeneity in therapeutic management during the study period, the general algorithm we described in 2013 has remained our consortium’s therapeutic management backbone. Cryotherapy followed by intralesional injections of pentavalent antimony is difficult to perform on several skin locations (lips, eyelids, genitalia, hands and feet), and in remote endemic areas where liquid nitrogen is not available and where injections are potentially harmful. In young patients with many and/or large lesions this effective yet painful approach is vastly suboptimal [11]. By contrast, application of paromomycin-based ointments is limited only by the site of application (ie. peribuccal and eyelids), and is well accepted by children and patients with many lesions. It has proved effective in several randomized trials in the Old and New World [25,26,29,30]. Gaps and issues in its development have been thwarted by some teams who developed local yet effective paromomycin formulations, including in Bolivia (Guéry & Buffet, personal data) [27].

This study has limitations. The number of mucosal or mucocutaneous episodes was too small to enable a robust multivariate analysis of the risk factors for mucosal involvement. Because this is a common surveillance program and not a prospective, comparative clinical trial, we could not capture follow-up data in all patients, hence the need for a careful interpretation of our observations regarding therapy. Not least, HIV testing was not systematically done which may limit our conclusions regarding the impact of immunosuppression on the clinical spectrum of cutaneous leishmaniasis. Nevertheless, this robust description of clinical, parasitological and therapeutic features of cutaneous leishmaniasis in almost half a thousand of travelers, reveals a moving landscape, where the risk of mucosal involvement is not limited to travels in the New World and effective treatments of CL are not limited to systemic therapy.

(A). Number of patients attended at each centre of the LeishMan consortium. (B). Number of cases according each country of LeishMan consortium. Abbreviations: UK, United Kingdom. (C). Suspected countries of acquisition in Old World. (D). Suspected countries of acquisition in New World. (E). Demographic and clinical characteristics of total cohort. Note. Results are expressed as number (%), unless otherwise stated. There are some missing data for each variable (<10%) explaining incomplete count proportion for categorical variables. Abbreviations: DissCL, disseminated cutaneous leishmaniasis; IQR, interquartile range; MCL, muco-cutaneous leishmaniasis, PKDL, post Kala-azar dermal leishmaniasis; VL, visceral leishmaniasis. (F). Subgenus, complex species, species identified in 198 cutaneous leishmaniasis infections. Note. *Viannia subgenus not further identified to species level.

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Age distribution of the main categories of travellers.

Panel A: age of distribution in total cohort; Panel B: age distribution in travellers visiting friends and relatives; Panel C: age distribution in tourists. Abbreviations: VFR visiting friends and relatives.

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Dataset 1.

(CSV)

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Dataset 2.

(CSV)

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22 Jan 2021

Dear Mr. Guery,

Thank you very much for submitting your manuscript "Clinical diversity, mucosal involvement and treatment strategy in cutaneous leishmaniasis: a European clinical report in 459 patients." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated [YES] with a clear testable hypothesis stated[NOT APPLICABLE]?

-Is the study design appropriate to address the stated objectives [YES]?

-Is the population clearly described and appropriate for the hypothesis being tested[YES]?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested [NOT APPLICABLE?

-Were correct statistical analysis used to support conclusions[YES]?

-Are there concerns about ethical or regulatory requirements being met[NO]?

Reviewer #2: This is an elegant, well-conceived, well-planned, and well-executed work. Provided information is very useful in the understanding of relationships and differences between the Old and New World integumentary forms of leishmaniasis and includes a very good number of patients with complete information, which is usually a major deficiency in case series reports. No observation or concerns regarding ethics and statistic analysis. Processes and steps are clearly mentioned, definitions and outcomes are precise.

Reviewer #3: The objectives are clear and the design study is appropriated for the hypotesis. It is a convenience cohort study, with 459 patients included, however the number of patients approached is not described in the methodoly, only in teh supplement file. Please make this information in teh text.

Reviewer #4: This is an interesting report on clinical and therapeutic aspects of travelers acquiring cutaneous leishmaniasis in both New and Old World countries. Data were systematically collected by members of a consortium following a harmonized protocol during two decades. The objectives are clear and the study was mainly descriptive with some comparisons between Old and New World acquired cases and also between cases due to different parasite species.

In spite of the clear usefulness of the presented data for improving clinical care of such group of patients, some issues should be properly addressed in order to make clear for the potential readers the limitations of the exploratory analysis performed for possible determinants of the mucosal involvement and the lack of data on parasite species and treatment approach of a relevant number of the included patients.

Then, I strongly suggest the following:

1. The inclusion of the details of the multivariate approach for exploring the possible ML determinants in the method section, e.g. the criteria for inclusion of the variables in the final model (it is unusual just include de variables with p<0.05 significance level in the univariate analysis without other considerations related to the biological plausibility), the exploration of confusion and potential interactions between variables and the final evaluation of the model fitness.

2. A description of the loss of data for at least the therapeutic approach and parasite identification in the flow chart (figure 1), i.e., how many patients were analyzed in accordance of parasite species? and how many patients were included for the therapeutic approach comparison?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Does the analysis presented match the analysis plan?[THESE STANDARD PLOS QUERIES ABOUT STATISTICS ARE NOT APPLICABLE FOR THIS DESCRIPTIVE STUDY]

-Are the results clearly and completely presented[YES]?

-Are the figures (Tables, Images) of sufficient quality for clarity [YES]?

Reviewer #2: 111: "...a previous history of leishmaniasis was reported by 8% of patients in the cohort". These were patients of CL with a previous episode of CL or ML patients with a previous episode of CL? Or ML with a previous episode of ML?

114, Table 1: Demographic and clinical characteristic in this population is strikingly similar to that of infected native patients probably because travellers coming to latinamerican countries are young people looking for extreme experiences and, for such reason, are going to deep forests where this zoonotic disease is endemic and where most of native patients are infected.

114, Table 1: The low number of patients who traveled to the New World and developed mucosal lesions could be explained by the fact that several years are usually necessary between skin lesions and mucosal compromise (range 1 to 50 or more but usually 2 to 5). Do you agree or do you have another explanation? Many of your patients went to countries like Peru and Bolivia with high prevalence of mucosal disease and your results might suggest that mucous extension is not so frequent.

114, Table 1: "Delay from first symptoms to parasitological diagnosis (months)". Why this 2 or 4 months delay? Aren't patients looking for diagnosis? Or tests to confirm diagnosis are not easily available? It could be interesting to know which other diagnostics received before confirmation of leishmaniasis, specially because such info could be helpful for doctor with not wide experience with leishmaniasis, to keep in mind all possible differential diagnosis.

114, Table 1: Includes SCAR as a type of lesion could be confussing because scar is usually the final result of leish when cured. Please explain me why was included and what happen with those 4 out of 271 OW subjects reported.

135: "lymphangitis 35%" is really high. Is this "pure" leish lymphangitis or bacterial co-infections that are quite common and also produce lymphangitis?

137, Table 2: "Duration of disease". Is this from first symptom to the consultation? Do you have info about time from the trip and first symptom? It could be useful for other doctors to know this "incubation" time to make the differential diagnosis exercise.

161: In native patients age is also a very correlated factor in mucosal disease, although most of them had initial infection (with or without cutaneous lesion) when were younger. In your over 50 patients ML was close in time to CL? In natives ML usually need a long time to appaer, what in yours?

183, Table 4: 107 patients treated with local therapies and 113 with systemic. What happen with the other 239 patients?

183, Table 4: "No specific treatment". Is this spontaneous cure? Because soap, water and dressing are good for any ulcer but 81% of cures deserve an explanation.

Reviewer #3: The analyzes presented are in accordance with the proposed methodology and the results are clearly presented.

Regarding the results some points need to be clear.

1) In the table 1, the authors shown the following results: Delay from first symptoms to parasitological diagnosis (months), median [IQR] [1-3] 4 [2-6] <0·01. Duration of disease (in months), median [IQR] 3 [2-4] 4 [3-7] <0·01. How to explain that the delay in diagnosis is longer than the time of illness?

2) The authors mention that ulcerated injuries were more frequent in travelers traveling from the new world (75.5%) than from the old world (47.5%), but there was no statistical difference. How to affirm or explain this finding?

3) Likewise, they state that the lesions are more frequently localized on the limbs (61% versus 47%) and

more frequently associated with nodular lymphangitis (30% versus 6%), comparing New and Old world. there is no statistical difference presented.Please, clarify these point.

4) Another important point is relaed to treatment. The most interesting data would be to evaluate the therapeutic response in relation to the species of Leishmania. I believe that you cannot compare systemic treatment with local, if there is no well-defined parameter. For example: compare response to different treatments in patients with similar lesions.

Reviewer #4: 1. The most curious result was related to the mucosal involvement rate detected in patients infected with L. infantum. Also, the evidence of immunocompromise as a factor associated with such an involvement. However, the merit of the comparison of the L. infantum group against the New World acquired cases infected with parasites belonging to L. braziliensis complex should be revised. In fact, epidemiology and pathogenesis of ML associated to L. braziliensis complex is pretty different of the pattern described for the L. infantum mucosal disease. The relevant comment on that is slightly mentioned in the discussion when authors refer to the long-term follow-up needed for concluding on ML rates for patients who received local treatment. It could be misleading if readers were induced to consider that ML is more relevant for L. infantum infected patients. Instead of that, the results of this study confirm that for immunocompetent individuals ML continues to be more relevant in cases infected with parasites belonging to L. braziliensis complex. Then, an additional analysis should be done excluding the immunocompromised patients.

2. The results of the multivariate analysis for ML determinants should be summarized in a table with the unadjusted and adjusted ORs and their respective 95%CI.

3. Therapeutic data were available from 220 patients. This represents a large data loss of ~50%. An explanation should be offered and this issue should be highlighted as a relevant study limitation.

4. Were all the patients tested for HIV infection? If not all the patients had an HIV test done, it would be mention also among the limitations of the study.

5. What type of immunocompromise were registered? This is a relevant information that could appear at least as a supplementary file.

6. Finally, the standard of care could be changed during the study period and this aspect deserves at least a comment in the discussion section.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented [YES]?

-Are the limitations of analysis clearly described [YES]?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study [YES]?

-Is public health relevance addressed [YES]?

Reviewer #2: 213: I agree with you that clinical characteristics are changing and that therapeutic recomendations must be reviewed in the near future. However a new group of patients from L.b regions, mainly Brazil, Perú and Bolivia, with severe, extensive, chronic and recurrent disease are emerging. According with your data (and I agree) around 75% of CL could be treated with local, simple, safe and cheap therapies. And systemic drugs have to be reserved to treat other forms of CL, ML, recurrent or relapsing diseases, leish in immunocompromised hosts, etc.

223: Yes, this comment goes in the same line with my previous (line 114) and that's why a list of initial diagnosis done before to confirm leish could be very useful for doctors with less experience in this disease.

271: "Detecting subsequent mucosal relapse through an extended follow-up was beyond the immediate scope of our study". OK, but at least you could tell us if any of the patients attended during the initial years developed ML during the time when the study was still active. My concern is because people with no big experience in CL and ML can read the paper and understand that ML appears soon after CL and forget long-term surveillance.

Reviewer #3: The main conclusion of this manuscritp is be immunocompromised and be over fifty years old are risk factors to develop mucosal leishmaniasis.

The authors not mentioned the limitations of manuscript.

The topic addressed in this manuscript is very important for public health, mainly to alert health professionals from non-endemic areas to leishmaniasis.

Reviewer #4: Most of the conclusions are supported by the data. The study limitations should be clearly stated as requested in above the comments . After considering the study limitations, the abstract section deserves some attention to be less emphatic for conclusions based on limited data.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The Title “Clinical diversity, mucosal involvement and treatment strategy in cutaneous leishmaniasis: a European clinical report in 459 patients” is a little off. I think the authors mean “Clinical diversity, mucosal involvement and treatment RESULTS in cutaneous leishmaniasis: a European clinical report OF 459 patients.” Note that the title is not really accurate with respect to mucosal involvement, because it implies that all cases of mucosal involvement were seen in concomitant cutaneous disease, but I am not sure how to fix this. “Clinical diversity, mucosal involvement and treatment RESULTS in cutaneous and mucosal leishmaniasis” is redundant (of course there is mucosal involvement in mucosal leishmaniasis). “Clinical diversity and treatment RESULTS in cutaneous, mucocutaneous, and mucosal leishmaniasis:” is probably best.

The heading “Clinical Features” at line 122 should be “Clinical Features of Cutaneous Disease.

“ Mucosal Involvement”, the second focus of the report, is tricky. Mucocutaneous disease (mucosal disease with cutaneous disease) and mucosal disease per se (only mucosal disease) are correctly defined in Definitions, but it might be there added that mucosal involvement includes both presentations. I found it hard to track mucocutaneous disease separately from mucosal disease. In Table 2, “mucosal disease” might be replaced by “mucocutaneous disease” to make it clear that for Table 2 (cutaneous disease), all “mucosal disease” was mucocutaneous. In the paragraph beginning on line 145, the sentence “We observed 10 MCL, 9 ML, and one MCL with visceral involvement and inaugural skin lesions in a patient with AIDS (CD4 count 67/mm3)” is welcome because it separates the several presentations. It would be helpful if in the following sentences “Mouth and laryngeal lesions were observed in 6/13 cases from Old World and 1/5 case from New World, while lesions of nasal cavity were observed in 7/13 cases from Old World and 5/5 cases from New World. Seven of 15 infections (47%) with mucosal involvement in the Old World were observed in immunocompromised patients while no patient was immunocompromised in the New World subgroup with mucosal involvement”, the authors could give an idea of the breakout of mucocutaneous vs mucosal disease. In Discussion, it is said that “mucosal involvement was observed in…22% of patients infected with L. infantum, in whom this complication was strongly related to pre-existing immunosuppression.” At least this reviewer could not figure out the L infantum cases. Were these mucosally-involved cases mucosal spread from a cutaneous focus (mucocutaneous disease) or frank mucosal disease without other organs (skin, viscera) being involved? In spite of these issues, the sentence in Discussion “In our study, the unexpected, low rate of mucosal involvement in infections acquired in the New World contrasts with the unexpected, relatively high rate in infections acquired in the Old World” is both cleverly worded and accurate.

Reviewer #2: Not needed

Reviewer #3: The authors mentioned that 198 species of Leishmania were identified, but in the results and discussion, there are references to only 166 isolates. Please correct or explain this difference.

One conclusion was missing from this manuscript, electing the main findings that contribute to current literature.

Reviewer #4: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: PNTD-D-20-01699

Guery et al report on “Demographic and clinical data from 459 travellers infected in 47 different

countries were collected by members of the European LeishMan consortium.” [Abstract].

The authors are right that “[This] harmonized data collection by clinicians attending patients infected in many transmission foci worldwide enables direct comparisons of clinical patterns induced by different Leishmania species, and the outcome following treatment with either local or systemic regimens”[Intro] and the implication, that this comparison within one report will be frequently quoted in preference to separate reports on each Leishmania species, is also right. A second focus is on mucosal disease.

This complex report is extremely well written, especially since the first and senior authors are not native-English speakers. Lines 129-136 summarize the massive amount of demographic and clinical presentation data for cutaneous disease (tables 1-2 and figures 1-3) nicely. The presentation of mucosal disease is in Table 3 and the results of therapy for all presentations are in Table 4.

Discussion is thoughtful.

Reviewer #2: Congratulations for an excellent work; I enjoy and learn reading it.

Reviewer #3: I added my comments and sugestion for the authors, mainly in the section of results.

Reviewer #4: The study is relevant, the information is important for clinicians dealing with travelers with cutaneous lesions suspected of having leishmaniasis. It seems to me that this is the report of the largest clinical cohort of travelers with leishmaniasis ever reported in the literature.

--------------------

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Reviewer #1: Yes: Dr Jonathan D Berman

Reviewer #2: Yes: Jaime Soto, MD. FUNDERMA - Fundación Nacional de Dermatología, Santa Cruz de la Sierra, Bolivia. jaime.soto@infoleis,com

Reviewer #3: No

Reviewer #4: Yes: Gustavo Romero

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25 May 2021

Submitted filename: 2020-05-21-response-to-reviewers.docx

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10 Aug 2021

Dear Mr. Guery,

Thank you very much for submitting your manuscript "“Clinical Diversity and Treatment Results in Cutaneous, Mucocutaneous, and Mucosal Leishmaniasis: a European clinical report in 459 patients." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Methods:good

Reviewer #2: Due to the wide diversity of species and clinical presentations but with very few cases in each specific situation, I consider that the most appropriate title should be Clinical diversity and treatment of tegumentary leishmaniasis (there is no enough information on ML to be specifically mentioned in the title).

47 to 53: definition of CL overlaps with that of MCL: "...A patient was considered to have CL if she/he had: (1) cutaneous and/or mucosal lesions..." and "...Muco-cutaneous leishmaniasis (MCL) refers to the simultaneous presence of both mucosal and skin lesions...". I think CL is only cutaneous lesion (not "and/or mucosal").

77: "..., lesion type (papulo-nodular or dry crust or wet crust)..." Not ulcers? Ulcers are the type of lesions usually treated with local therapies.

Reviewer #3: The objectives are intrinscially related with hypothesis. The study design is appropriate to descritive of cohort study proposed. The population is clearly described, however the sample size is not sufficient to power of the hyphotesis. For example, to estimate the prevalence or frequence of mucosal leishmaniasis, or evaluate the response to local therapy the numeber need to be higher.

Statistical analysis support the conclusions.

There was compliance with ethical precepts. All regulations were followed.

Reviewer #4: Authors have made all the modifications requested and they also have offered the proper clarifications. Methods are adequate.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: REsults: good

Reviewer #2: 111: "... and a previous history of leishmaniasis was reported by 8% of patients in the cohort". I insist that it would be important to know if these patients had had skin or mucosal lesions as a previous episode.

131: "...Excluding rare species and incomplete species (supplementary table 6), we focused on the 5 most frequent infecting complex species...". Good decision.

197, Table 4: "Wash lesion and wound dressing" it could be apparently enough to cure OW CL at similar rates to systemic or topical therapies; so this local care could be recommended as treatment at least to be tested in a clinical trial? It could be THE treatment of many patients in the old world and some in the new world? Or, in the opinion of the authors, are these local measures an adjunctive management of a local or systemic treatment with drugs?

Reviewer #3: The results are clearly presented, however some points in the table 1 (for example) is not complete.

Reviewer #4: Authors have made all the modifications requested an.d they also have offered the proper clarifications. The results section is adequate.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: conclusions: Good

Reviewer #2: If immunocompromised patients make mucosal lesions in the old world is understandable but that new world patients with all their immune system functioning make recent or late mucosal lesions is more worrying. In fact, what we observe is that mucosal patients in the New World improve with treatment but have frequent recurrences.

Infiltration and ulceration in tegumentary leishmaniasis is the consequence of an active host immune system fighting against parasites. Accordingly, how can mucosal presentation be explained in the immunosuppressed from the old world compared to the immunocompetent from the new world? Would the authors write a paragraph considering this aspect?

"...the risk of mucosal involvement is not limited to travels in the New World and effective treatments of CL are not limited to systemic therapy". I fully agree that these are the main findings of this paper, as the authors say in the added paragraph in which they mention the limitations of the study.

Reviewer #3: The conclusions are supported by the results and the limitations are described.

The manuscript present relevant data for public health

Reviewer #4: Authors have made all the modifications requested and they also offered the proper clarifications. The discussion and conclusions sections are adequate.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: With the modifications and adjustments in tables and texts it becomes clearer

Reviewer #3: It is necessary rewrite the table 1, containing information about nodular lymphangitis

Reviewer #4: None

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Well revised

Reviewer #2: Paper improved with changes in text and tables. The limitations are now more evident to the reader and allow him/her to read with a better perspective.

Reviewer #3: The authors present comparing tegumentary leishmaniasis from the Old and New World. The topic is quite interesting and current and really requires more information in the literature to support decision-making in public health. However some point need to be improved. Bellow I send my considerations and suggestions.

1. Abstract

# In table 1, the authors present data on the size of the lesion in mm. I suggest keeping the same unit as shown in table 1 (mm) or leaving cm in the table.

# The authors refer that in the old world there was 5% of mucosal involvement, however, in table 1, the prevalence of mucosal damage was between 2 and 2.5%. Please review this information. It is unclear.

2. Mehtodology

#It is not clear whether all patients with skin lesions were evaluated for the presence of mucosal lesions, as there may be concomitant lesions. If they were evaluated, how was this evaluation done. This information can vary the frequency of mucous involvement, described in this manuscript.

#The authors report a low association with immunodeficiency, however not all patients were tested for HIV. How to state this?

It would be interesting to describe how many were tested for HIV, to try to make a prevalence of co-infection in those who were tested.

#Regarding the methodology for identifying Leishmania species, was there a standard methodology used in all countries or did each center have its own methodology?

3. Results

# In table 1 there is an asterisk referring to lymphangitis, however there is no lymphangitis data in the table. In the text, the authors described nodular lymphangitis (30% and 6%). Please review this data.

#Out of 459 patients, only 198 had identification of the Leishmania species. In the others it was not tried to identify or was it not possible to identify? Please make this information as clear as possible.

#In table 2, the authors list the Leishmania species identified, with different variables. Regarding the New World, they describe 12 from Peru, as L. braziliensis, and 5 from French Guiana. Regarding L. guyanensis, the authors describe 8 samples from Costa Rica and 7 from French Guiana. Were the other 25 samples from Peru, 20 from Costa Rica and 29 from French Guiana not identified? This information is essential, as in French Guiana there is a prevalence of L. guyanensis and in Costa Rica there is L. infantum causing atypical cutaneous leishmaniasis.

#In table 3, the authors compare the number of lesions between mucous involvement and without mucosal involvement. Was this analysis performed only on patients who had cutaneous and mucosal lesions? Please make this information clearer.

#In table 4, the authors compare local and systemic treatment in old world patients. However, I believe that there may be a bias, since the indication for treatment is based exclusively on the characteristics of the lesion and the number of lesions.

#Regarding treatment, it would also be interesting to analyze the therapeutic response to the species, as some species are known to respond better to certain drugs than others.

Discussion

#From 216 to 220 pages the author mentioned "In particular, no mucosal involvement was observed in patients infected with either L. major or L. tropica, whereas it affected 6% of patients infected with L. braziliensis or L. guyanensis complex (2 MCL, 1 ML) and 22% of patients infected with L. infantum (4 ML, 4 MCL), in whom this complication was strongly (though not exclusively) related to pre-existing immunosuppression."

I suggest review this affirmation. I believe there is a bias here, as patients who came from the New World had skin lesions. There is no reference for evaluating mucosal lesions in these patients. Furthermore, the mucosal form in the new world usually occurs after years of primary infection, mainly caused by L. braziliensis.

Reviewer #4: None

--------------------

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Reviewer #1: No

Reviewer #2: Yes: jaime soto

Reviewer #3: No

Reviewer #4: Yes: Gustavo Romero

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References

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9 Sep 2021

Submitted filename: 2021-09-07-Rebuttal-2.docx

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28 Sep 2021

Dear Mr. Guery,

We are pleased to inform you that your manuscript 'Clinical Diversity and Treatment Results in Tegumentary Leishmaniasis: a European clinical report in 459 patients.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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8 Oct 2021

Dear Mr. Guery,

We are delighted to inform you that your manuscript, "Clinical Diversity and Treatment Results in Tegumentary Leishmaniasis: a European clinical report in 459 patients.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Table 1

Comparative features of cutaneous leishmaniasis by continent(s) of acquisition.

		New World	Old World	p-value
		185 infections	279 infections
Age, median [IQR]		30 [24–38]	32 [10–58]	0·81
Male		75% (138)	54% (152)	<0·01
Immunocompromised		0·6% (1/177)	8% (21/264)	<0·01
Type of traveller				NA
Tourist		61% (109/180)	36% (96/269)
Visiting Friends and Relatives		7% (12/180)	43% (117/269)
Migrant		0% (0/180)	14% (39/269)
Expatriate (worker, missionary)		12% (21/180)	3% (8/269)
Soldier		13% (24/180)	0% (0/269)
Others		8% (14/180)	3% (9/269)
Type of cutaneous leishmaniasis				NA
Localized Cutaneous		96% (178)	94% (262)
Muco-cutaneous		2% (3)	2·5% (7)
Mucosal		1% (2)	2·5% (7)
PKDL		0·5% (1)	0·3% (1)
Disseminated cutaneous leishmaniasis		0·5% (1)	0·3% (1)
Muco-cutaneous and visceral leishmaniasis		0% (0)	0·3% (1)
Delay from first symptoms to the first consultation (in months), median [IQR]		3 [2–4]	4 [3–7]	<0·01
Number of lesions, median [IQR]		1 [1–2·2]	2 [1–4]	<0·01
Type of lesions				< 0.01
Ulcer (wet crust)		75·5% (139/184)	47·5% (128/271)
Papulo-nodular		5·5% (10/184)	25·5% (69/271)
Dry crust		14% (26/184)	16% (44/271)
Squamous plaque		3% (5/184)	8·5% (23/271)
Others		2% (4/184)	1% (3/271)
Scar or new papule on a previous scar		0% (0/184)	1·5% (4/271)
Lesion localization				<0.01
Upper limb		32% (58/183)	31% (84/274)
Face, neck and scalp		17% (31/183)	38% (104/274)
Lower limb		29% (53/183)	16% (45/274)
Hand		8% (15/183)	8% (21/274)
Trunk		7% (13/183)	2% (6/274)
Feet		4% (7/183)	4% (10/274)
Neck and Scalp		3% (6/183)	1% (4/274)
Diameter of largest lesion (millimeter), median [IQR]		30 [20–43·5]	20 [10–35]	<0·01
Nodular lymphangitis*		30% (56)	6% (16)	<0·01

Notes.

*Nodular lymphangitis was defined as subcutaneous nodules in proximity to the primary lesion and/or dilated palpable lymphatic vessels in the form of a “beaded cord,” and/or regional lymphadenitis

Univariate analysis. Categorical variables not included in the univariate analysis (indicated p-value as “NA”). Data are % (n) unless indicated.

Abbreviations: IQR, interquartile range; PKDL, Post Kala-azar Dermal Leishmaniasis.

Table 2

Comparative features of 166 cases of cutaneous, mucocutaneous and mucosal leishmaniasis by main infecting complex species.

		L. braziliensis complexn = 34	L. guyanensis complexn = 18	L. major complexn = 52	L. tropica complexn = 26	L. donovani complexn = 36
Age, median [IQR]		27 [23–33]	32 [27–37]	18.5 [6–46]	26 [12–50]	56 [47–66]
Male		76% (26)	61% (11)	54% (28)	50% (13)	50% (18)
Immunocompromised		0% (0)	0% (0)	6% (3)	0% (0)	18% (6)
Type of traveller
	Tourist	73% (24)	50% (9)	24% (12)	8% (2)	70% (25)
	Visiting Friends & Relatives	3% (1)	17% (3)	69% (35)	44% (11)	25% (9)
	Migrants, Expatriate, Soldiers and Others	24 % (8)	33% (6)	7.8 % (4)	48% (12)	5.5 % (2)
First 3 countries of acquisition
		Peru (12)	Costa Rica (8)	Tunisia (10)	Syria (10)	Spain (11)
		Bolivia (9)	French Guiana (7)	Algeria (10)	Tunisia (4)	France (4)
		French Guiana (5)	Brazil, Suriname, Peru (1+1+1)	Senegal (8)	Morocco (3)	Italy + Malta (4)
Delay from first symptoms to the first consultation, median [IQR]		2 [1,5-3]	3 [2-3]	3 [2-3]	6 [5-12]	7 [3-12]
Number of lesions, median [IQR]		1 [1-2]	2 [1-3]	4 [2-8]	1 [1-2]	1 [1-2]
Type of lesions
	Ulcer (wet crust)	85% (28)	88% (16)	63% (32)	35% (9)	47% (15)
	Papulo-nodular	0% (0)	6% (1)	23% (12)	31% (8)	25% (8)
	Dry crust	9% (3)	6% (1)	12% (6)	11% (3)	19% (6)
	Squamous plaque	0% (0)	0% (0)	2% (1)	19% (5)	9% (3)
	Others	6% (2)	0% (0)	0% (0)	4% (1)	0% (0)
Lesion localization
	Upper limb	18% (6)	28% (5)	44% (23)	34% (9)	24% (8)
	Face, neck and scalp	15% (5)	16% (3)	19% (10)	46% (12)	40% (13)
	Lower limb	43% (14)	28% (5)	29% (15)	8% (2)	18% (6)
	Hand	6% (2)	22% (4)	6% (3)	12% (3)	12% (4)
	Trunk	6% (2)	6% (1)	0% (0)	0% (0)	6% (2)
	Feet	12% (4)	0% (0)	2% (1)	0% (0)	0% (0)
Diameter of largest lesion (mm) , median [IQR]		30 [20-39]	40 [25-50]	30 [15-43]	20 [10-30]	7 [3-12]
Nodular lymphangitis		35% (12)	44% (9)	12% (6)	12% (3)	6% (2)
Mucosal involvement		6% (2)ML (1)MCL(1)	5% (1)MCL (1)	0% (0)	0% (0)	22% (8)ML(4)MCL(4)

Note. Data are % (n) unless indicated. Abbreviations: IQR, interquartile range

Table 3

Comparative features in patients with or without mucosal involvement at presentation.

	No mucosal involvement	With mucosal involvement
	444 infections	20 infections	p value
Age, median [IQR]	30 [18–51]	58 [33–65]	<0·001
Age > 50 years	27% (119)	65% (13)	<0·001
Male	62% (274)	80% (16)	0·160
Immunocompromised	3% (15)	37% (7)	<0·001
Region of acquisition			0·250
Old World	59·5% (264)	75% (15)
New World	40·5% (180)	25% (5)
Delay from first symptoms to the first consultation, median [IQR]	3 [2–6]	5 [3–12]	0·057
Number of lesions, median [IQR]	2 [1–3]	1.5 [1–3]	0·287
Face, neck or scalp involvement (excluding pure mucosal forms)	29% (123/428)	64% (7/11)	0.020
Large lesion *	50% (196)	64% (7)	0.560
Nodular lymphangitis	15·5% (69)	15% (3)	1

Data are % (n) unless indicated.

*: > 4 cm2 (corresponding to a diameter of 23mm)

Abbreviations: IQR, interquartile range.

Table 4

Clinical characteristics of patients and healing rates according to treatment administration modality (systemic versus local).

	Local treatment		Systemic treatment
	n = 107		n = 113		p value
Patients infected in Old World	94 (88)		45 (40)		<0,0001
Age, median [IQR]	30 [15–57]		30 [21–51]		0·71
Number of lesions, median [IQR]	2 [1–4]		1·5 [1–3]		0·51
Diameter of largest lesion (mm), median [IQR]	19 [10–30]		31 [20–50]		<0·0001
Delay from first symptoms to the first consultation, median [IQR]	4 [3–6]		3 [2–4]		0·008
Nodular lymphangitis	8 (4,5)		32 (28,5)		<0·0001
Immunosuppression or diabete mellitus	12 (11)		5 (4,5)		0·05
Healing rate	95/107 (89)		97/113 (86)		0·05
	Treatment received	Healing rate	Treatment received	Healing rate
	No specific treatment *	17/21 (81)	Antimonial therapy (MA or SSG)	30/34 (88)
	Intralesional MA or SSG +/- Cryotherapy **	70/75 (93)	Amphotericin B (liposomal or deoxycholate***)	7/12 (58)
	Topical Paromomycin	7/9 (78)	Fluconazole	11/12 (92)
	Others: surgery (n = 1), imiquimod (n = 1)	1/2 (50)	Miltefosine	38/43 (88)
			Pentamidine Isethionate	10/11 (91)
			Other: SSG + pentoxifylline (n = 1)	1/1 (100)

Data are n (%) unless indicated. Healing rate = first evaluation after at least 42 days of treatment initiation (median follow-up of 80 days [IQR 59–111]).

Note

*Wash lesion and wound dressing

**two patients received cryotherapy alone

*** all patients except one received liposomal Amphotericin B

Abbreviations: MA, meglumine antimoniate; SSG, sodium stibogluconate

First Name	First Initial	Surname
Isabelle	I	ALCARAZ
Florence	F	AMELOT
Fanny	F	ANDRY
Adela	A	ANGOULVANT
Julie	J	ARATA BARDET
Yercanik	Y	ARMAGAN
Jean Philippe	JP	ARNAULT
Selma	S	AZIB-MEFTAH
Dominique	D	BARTHELME
Bernhard	B	BECK
Sorya	S	BELAZ
Muriel	M	BELLIAH-NAPPEZ
Nathalia	N	BELLON
Nathalie	N	BENETON
Astrid	A	BLOM
Olivia	O	BOCCARA
Nathalie	N	BODAK
Mathilde	M	BON MARDION
Daphné	D	BOSSET
Emmanuel	E	BOTTIEAU
François	F	BOUQUEAU
Emmanuelle	E	BOURRAT
Romain	R	BRICCA
Olivier	O	CARPENTIER
Eline	E	CASASSA
Eric	E	CAUMES
Sophie	S	CHARLES
Angèle	A	CLABAUT
Heloise	H	CLERC
Hélène	H	COIGNARD
Domenica	D	CUSTER
Hervé	H	DARIE
Madeleine	M	DE DARUVAR
Henry JC	HJC	DE VRIES
Sebastien	S	DEBARBIEUX
Marine	M	DELOBEAU
Charlotte	C	DENTAN
Julie	J	DI LUCCA
François	F	DURUPT
Olivier	O	EPAULARD
Catherine	C	ESCHARD
Jean-Louis	JL	ESTIVAL
William	W	ETIENNE
Marie	M	FERNEINY
Fanny	F	FICHEL
Françoise	F	FOULET
Alexandre	A	GALLOULA
Florence	F	ROBERT-GANGNEUX
Amélie	A	GANTZER
Gilles	G	GARGALA
Pauline	P	GELOT
Céline	C	GIRARD
Hedvig	H	GLANS
Jeremy	J	GOTTLIEB
Emmanuel	E	HEAU
Michel	M	JANIER
Kaoutar	K	JIDAR
Judith	J	KARSENTY
Christine	C	KATLAMA
Natalia	N	KIRSTEN
Diane	D	KOTTLER
Nora	N	KRAMKIMEL
Barthélémy	B	LAFON-DESMURS
Brigitte	B	LAGRANGE
Pauline	P	LANSALOT-MATRAS
Noël	N	LE CAM
Laurence	L	LE CLEACH
David	D	LEBEAUX
Delphine	D	LEBRUN
Maeva	M	LEFEBVRE
Regine	R	LEVET
Audrey	A	LORRIAUX
Devy	D	LU
Hervé	H	MAILLARD
Mylene	M	MAILLET
Fredrik	F	MANSSON
Guillaume	G	MARTIN-BLONDEL
Valérie	V	MARTINEZ-POURCHER
Pierre	P	MARTY
Guillaume	G	MELLON
Oussama	O	MOURI
Justine	J	MUNOZ
Kristina	K	OPLETALOVA
André	A	PAUGAM
Alice	A	PERIGNON
Jean-Luc	JL	PERROT
Eskild	E	PETERSEN
Antoine	A	PETIT
Catherine	C	PICARD-DAHAN
Emily	E	POLLOCK
Christelle	C	POMARES
Charlotte	C	POUPLARD
Olivier	O	ROGEAUX
Mahtab	M	SAMIMI
Mathilda	M	SANDSTROM
Anne	A	SAUSSINE
Pierre	P	SCHNEIDER
Robert	R	SEBBAG
Patricia	P	SENET
Amandine	A	SERVY
Patrick	P	SOENTJENS
Cornelis	C	STIJNIS
Marc	M	THELLIER
Feyrouz	F	TOUKAL
Mathis	M	TREPP
Raoul	R	TRILLER
François	F	TRUCHETET
Aude	A	VALOIS
Steven	S	VAN DEN BROUCKE
Jef	J	VAN DEN ENDE
Erwin	E	VAN DEN ENDEN
Alfons	A	VAN GOMPEL
Pieter-Paul	PP	VAN THIEL
Michèle	M	VAN VUGT
Francisco	F	VEGA-LOPEZ
Filip	F	VERHAEGHE
Mireille	M	VERNIER
Michele	M	WOLTER-DESFOSSES
Daniela	D	ZAHARIA
Stéphane	S	ZEMIRO
Elina	E	ZUELGARAY