Dermatology: Practical and Conceptual Research Letter | Dermatol Pract Concept. 2024;14(4):e2024271 1 Non-Invasive Imaging Techniques (Dermoscopy, Reflectance Confocal Microscopy, and Line-Field Confocal Optical Coherence Tomography) for the Diagnosis of Cutaneous Leishmaniasis Giulio Cortonesi1, Vittoria Cioppa1, Carolina Donelli1, Flavio Giulio Liso1, Pietro Rubegni1, Elisa Cinotti1 1 Dermatology Section, Department of Medical, Surgical and Neurological Sciences, Santa Maria Alle Scotte Hospital, Siena, Italy Key words: Dermoscopy, Confocal microscopy, Cutaneous leishmaniasis Citation: Cioppa V, Cortonesi G, Donelli C, Liso FG, Rubegni P, Cinotti E. Non-invasive imaging techniques (dermoscopy, reflectance confocal microscopy and line-field confocal optical coherence tomography) for the diagnosis of cutaneous leishmaniasis. Dermatol Pract Concept. 2024;14(4):e2024271. DOI: https://doi.org/10.5826/dpc.1404a271 Accepted: June 5, 2024; Published: October 2024 Copyright: ©2024 Cioppa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding Author: Vittoria Cioppa, MD, Dermatology Section, Santa Maria alle Scotte Hospital, Viale Mario Bracci, 53100, Siena – Italy. E-mail: v.cioppa@student.unisi.it Introduction Cutaneous leishmaniasis (CL) is an infectious disease caused by the Leishmania genus of flagellate protozoan. The disease is widespread in most countries in the Mediterranean basin, including Italy. Dermoscopy is a noninvasive diagnostic tool that has been used to describe different skin lesions: neoplas- tic, inflammatory, infectious, hair disorders [1,2]. Other non- invasive diagnostic technique, such as reflectance confocal microscopy (RCM) and line-field confocal optical coherence tomography (LC-OCT), are nowadays used to help the clini- cian to make the correct diagnosis. Case Presentations We describe a series of two patients with CL studied with dermoscopy, RCM, and LC-OCT in the Dermatology Sec- tion of University of Siena, Italy. The clinical presentation was similar: a single well-circumscribed nodular lesion on head and neck, with an orangish erythematous color with a tendency to enlarge (Figure 1A and B). The only relevant anamnestic datapoint was that both patients used to spend time in the countryside. Dermoscopy showed generalized erythema, with streaks and enlarged vascular polymorphous structures were present 2 Research Letter | Dermatol Pract Concept. 2024;14(4):e2024271 Figure 1. (A, B) Clinical images of cutaneous leishmaniasis. (C) Dermoscopy, ×10 magni- fication and (D) ×40. Figure 2. (A-D) Reflectance confocal microscopy of inflammatory cells (yellow circles [A, C]), dendritic cells (white circles [B, D]), and giant cells (white arrows [A, D]). (E-H) Line-field confocal optical coherence tomography of vacuolized areas in the epidermis and upper dermis (blue circles, [F, G]) and polymorphous infiltrate in shape and size (red circles [E, H]). (I, J) Histological exam- ination of cutaneous leishmaniasis. Research Letter | Dermatol Pract Concept. 2024;14(4):e2024271 3 in all CL lesions (Figure 1C and D). RCM (Vivascope 1500, Mavig) revealed a typical honeycomb epidermal pattern, small bright cells, a dense infiltrate of dendritic cells within the basal layer, and a well-represented inflammatory infil- trate in the upper dermis (Figure 2A-D). In the dermis there were hyper-reflecting interwoven fibers forming roundish structures. Multinucleated giant cells were also observed in the upper dermis. LC-OCT (DeepLive®, DAMAE Médi- cal) showed mild hyperkeratosis, a dense inflammatory infiltrate with some vacuolized areas in the epidermis and upper dermis. The inflammatory infiltrate was extremely polymorphous in shape and size, and it can be well cor- related with the classical inflammatory infiltrate present in CL (Figure 2E-H). Histopathology exams (Figure 2, I and J) in a skin sample obtained by punch biopsy revealed a dense mixed infiltrate (lymphocytes, plasma cells, and histiocytes) with small epithelioid granulomas and multinucleated giant cells. In some histiocytes, Donovan bodies were observed. Diagnosis of CL was extremely evocative. The patients were then sent to the regional referral Infectious Diseases Depart- ment, which confirmed the diagnosis. Discussion Even if the diagnosis remains clinical and requires validation with laboratory tests and histopathological examination, LC-OCT, like other noninvasive diagnostic techniques, can represent a further tool for noninvasive assessment of cuta- neous infection and infestation [2,3]. Our preliminary results found that most of the histological features of CL can be visualized on LC-OCT. Our findings suggest that LC-OCT may be useful for the noninvasive diagnosis of CL, allowing it to be distinguished from other neoplastic or infectious dis- eases for the specific skin patterns and alterations that can be found [4]. LC-OCT may help decrease the need for surgical excision for confirmation of the diagnosis of benign tumors, which can be removed with less invasive surgical techniques. Conclusion Although Italy is not a country where CL has a prevalent epidemiology, more and more cases are being recorded in suburban areas [5]. The increase in the incidence of cases of zoonotic diseases can be traced back to a higher prev- alence of immigrant and traveler carriers, the application of the newest and integrated diagnostic techniques (such as noninvasive diagnostics), and the greater clinical skill in rec- ognizing pathologies that were once of tropical relevance. However, further studies with a larger number of cases are necessary to demonstrate the diagnostic utility of LC-OCT in the clinical setting. References 1. Buljan M, Zalaudek I, Massone C, Hofmann-Wellenhof R, Fink-Puches R, Arzberger E. Dermoscopy and reflectance confo- cal microscopy in cutaneous leishmaniasis on the face. Australas J Dermatol. 2016; 57(4): 316-8. DOI:10.1111/ajd.12404. 2. Orsini C, Trovato E, Cortonesi G, et al. Line-field confocal optical coherence tomography: New insights for psoria- sis treatment monitoring. J Eur Acad Dermatol Venereol. 2024;38(2):325-331. DOI: 10.1111/jdv.19568. 3. Campoli M, Cortonesi G, Tognetti L, Rubegni P, Cinotti E. Noninvasive imaging techniques for the diagnosis of cutaneous larva migrans. Skin Res Technol. 2022; 28(2): 374-6. DOI: 10 .1111/srt.13126. Epub 2021 Dec 19. PMID: 34923675; PMCID: PMC9907695. 4. Cortonesi G, Donelli C, Rubegni P, Miracco C, Cinotti E. Non- invasive imaging techniques for the diagnosis of clear cell ac- anthomas: dermoscopy, reflectance confocal microscopy and line-field confocal optical coherence tomography. Dermatol Pract Concept. 2023; 13(4):e2023231. DOI: 10.5826/dpc.1304a231. PMID: 37992357; PMCID: PMC10656184. 5. Van der Auwera G, Davidsson L, Buffet P, et al; LeishMan Surveillance network. 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