Leishmania spp. amastigotes surrounding sensory nerve fibers in human painless skin ulcers: Evidence of pathogen-neuron proximity and absence of neuronal apoptosis.

In this present study, carried out between November 2020 and July 2023 at Londrina's University Hospital, patients with active lesions of cutaneous leishmaniasis (CL) were analyzed regarding pain perception and anatomopathological aspects of the ulcers. Pain was assessed using a numerical rating scale (NRS) to compare five patients diagnosed with CL with four control patients diagnosed with vascular skin ulcers. Histopathological evaluations were used to investigate the nociceptor neuron-Leishmania interface. Patients with CL ulcers reported less pain compared to patients with vascular ulcers (2.60 ± 2.30 and 7.25 ± 0.95, respectively, p = 0.0072). Histopathology evidenced Leishmania spp. amastigote forms nearby sensory nerve fibers in profound dermis. Schwann cells marker (S100 protein) was detected, and caspase-3 activation was not evidenced in the in the nerve fibers of CL patients' samples, suggesting absence of apoptotic activity in nerve endings. Additionally, samples taken from the active edge of the lesion were negative for bacilli acid-alcohol resistant (BAAR), which excludes concomitant leprosy, in which painless lesions are also observed. Thus, the present data unveil for the first time anatomopathological and microbiological details of painless ulcers in CL patients, which has important clinical implications for a better understanding on the intriguing painless clinical characteristic of CL.

Temporal Evaluation of the Surface Area of Treated Skin Ulcers Caused by Cutaneous Leishmaniasis and Relation with Optical Parameters in an Animal Model: A Proof of Concept.

Cutaneous leishmaniasis (CL) is a neglected disease caused by an intracellular parasite of the Leishmania genus. CL lacks tools that allow its understanding and treatment follow-up. This article presents the use of metrical and optical tools for the analysis of the temporal evolution of treated skin ulcers caused by CL in an animal model. Leishmania braziliensis and L. panamensis were experimentally inoculated in golden hamsters, which were treated with experimental and commercial drugs. The temporal evolution was monitored by means of ulcers' surface areas, as well as absorption and scattering optical parameters. Ulcers' surface areas were obtained via photogrammetry, which is a procedure that allowed for 3D modeling of the ulcer using specialized software. Optical parameters were obtained from a spectroscopy study, representing the cutaneous tissue's biological components. A one-way ANOVA analysis was conducted to identify relationships between both the ulcers' areas and optical parameters. As a result, ulcers' surface areas were found to be related to the following optical parameters: epidermis thickness, collagen, keratinocytes, volume-fraction of blood, and oxygen saturation. This study is a proof of concept that shows that optical parameters could be associated with metrical ones, giving a more reliable concept during the assessment of a skin ulcer's healing.

Detection of cutaneous leishmaniasis in three communities of Oti Region, Ghana.

BACKGROUND: Cutaneous leishmaniasis (CL) is the most common type of leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania. In Ghana, some studies in the Volta region have detected Leishmania parasites among persons with skin ulcers. METHODOLOGY/PRINCIPAL FINDINGS: Using a cross-sectional study design, the prevalence of CL in three communities of the Oti Region of Ghana was investigated. Demographic and epidemiological data were obtained by a structured interviewer administered questionnaire. A total of 426 (12.4%) out of 3,440 participants screened had at least one skin ulcer. Of 595 skin ulcers sampled and tested by PCR for Leishmania infection, 150 (25.2%) ulcers from 136 individuals tested positive, accounting for an overall CL prevalence of 31.9% among persons with skin ulcers. Individual community CL prevalence of 23.2%, 29.8%, and 36.8% was observed in Ashiabre, Keri, and Sibi Hilltop respectively among persons with skin ulcers. CONCLUSIONS/SIGNIFICANCE: Confirmation of CL in the study area suggests an active cycle of transmission of Leishmania infection. The observation of skin ulcers which tested negative to Leishmania infection suggests a need to test for additional causes of skin ulcers such as Treponema pallidum pertenue and Mycobacterium ulcerans in the study area.

PCR performance for the diagnosis of cutaneous leishmaniasis caused by Leishmania viannia complex using biopsy samples, compared with exudate samples from skin lesions on filter paper.

BACKGROUND: Cutaneous leishmaniasis (CL) is generally diagnosed by molecular methods, including PCR, using biopsy samples, skin scrapings and clinical exudates. In this study, we assessed the PCR performance for diagnosis of CL using skin of biopsy samples vs PCR of skin lesion exudate samples on filter paper and compared the diagnostic concordance of PCR using both sampling methods. METHODS: We assessed the PCR performance using 80 skin biopsy samples and 80 filter paper samples containing exudates from skin lesions obtained from 74 patients with clinical suspicion of CL in Cusco, Peru. RESULTS: : PCR using skin biopsy samples had superior diagnostic accuracy compared with filter paper PCR (62.5% [50/80] vs 38.7% [31/80], respectively; p˂0.005) and the diagnostic concordance between both sampling methods was 'moderate' (kappa coefficient=0.50, 95% CI 0.98 to 1.0). CONCLUSIONS: PCR using biopsy samples remains the standard for diagnosis of CL.

Estimation of Biological Parameters of Cutaneous Ulcers Caused by Leishmaniasis in an Animal Model Using Diffuse Reflectance Spectroscopy.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that requires novel tools for its understanding, diagnosis, and treatment follow-up. In the cases of other cutaneous pathologies, such as cancer or cutaneous ulcers due to diabetes, optical diffuse reflectance-based tools and methods are widely used for the investigation of those illnesses. These types of tools and methods offer the possibility to develop portable diagnosis and treatment follow-up systems. In this article, we propose the use of a three-layer diffuse reflectance model for the study of the formation of cutaneous ulcers caused by CL. The proposed model together with an inverse-modeling procedure were used in the evaluation of diffuse-reflectance spectral signatures acquired from cutaneous ulcers formed in the dorsal area of 21 golden hamsters inoculated with Leishmanisis braziliensis. As result, the quantification of the model's variables related to the main biological parameters of skin were obtained, such as: diameter and volumetric fraction of keratinocytes, collagen; volumetric fraction of hemoglobin, and oxygen saturation. Those parameters show statistically significant differences among the different stages of the CL ulcer formation. We found that these differences are coherent with histopathological manifestations reported in the literature for the main phases of CL formation.

Role of Histopathology in the Diagnosis of Cutaneous Leishmaniasis: A Case-Control Study in Sri Lanka.

Cutaneous leishmaniasis (CL) displays a spectrum of manifestations clinically and histologically. Then, it becomes a diagnostic challenge and must discern from the other clinical and histological mimics, especially when the Leishman-Donovan bodies are inattentive. In this study, we compared the distinguishing histomorphological characteristics of CL against the other skin diseases with similar clinical and histological features. Skin biopsies of 181 patients, which suspect CL clinically, are evaluated histologically. Pertaining to the first case-control comparison, which performed between skin lesions of CL with or without discernible organisms and the other granulomatous dermatitis, highlighted that the ill-formed coalescent granulomata (OR = 14.83) and diffuse dense dermal plasma cell infiltrate (OR = 74.25) are significantly associated with the skin lesions of CL. The second case-control analysis was between CL without discernible organisms and the other granulomatous dermatitis, and identified a significant association in the presence of ill-formed coalescent granulomata (OR = 16.94) and diffuse dense (>50/HPF) dermal plasma cell infiltrate (OR = 74.5) in the skin lesions of CL. Pertaining to epidermal changes, acanthosis (OR = 2.38), spongiosis (OR = 9.13), and the presence of ulceration (OR = 20.26) are among the major concerns in CL. In conclusion, in the presence of clinical suspicion, dermal granulomata in ill-formed coalescent morphology with high plasma cell density in a diffuse arrangement are positive factors for the diagnosis of CL, especially when the discernible Leishmania amastigotes are absent. Resource utilization such as polymerase chain reaction and other ancillary techniques during the diagnosis of CL can be minimized by using a range of histopathological features and special attention should be focused on this in the future.

Ambiguous skin ulcer on the ear pinna.

Cutaneous leishmaniasis (CL) is a parasitic disease which has a biphasic life cycle; infection by promastigotes from the sandfly reaches a wound where it is phagocytosed by macrophages, producing the amastigote (the Leishmania donovani body) in the host. A protozoan parasite transmitted by the phlebotomous sandfly causes human leishmaniasis. Cutaneous forms include classical cutaneous, mucocutaneous and post-kala-azar dermal leishmaniasis. It affects c. 300 million individuals in more than 90 nations around the globe. The cutaneous form in the Old World is caused at low altitudes mainly by L. major (which has an animal reservoir, rodents such as mouse) and in swampy regions and high altitudes by L. tropica (which has no animal reservoir). L. aethiopica and L. major lead to disseminated ulcers in Saudi Arabia, Yemen, Iraq, Iran, Pakistan, India, Tunisia, Sudan and Ethiopia, whose main electrophoretic isozyme pattern Zymodeme in Saudi Arabia is LON-4.

Characterization of the Histopathologic Features in Patients in the Early and Late Phases of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL), characterized by an ulcerated lesion, is the most common clinical form of human leishmaniasis. Before the ulcer develops, patients infected with Leishmania (Viannia) braziliensis present a small papule at the site of the sandfly bite, referred to as early cutaneous leishmaniasis (E-CL). Two to four weeks later the typical ulcer develops, which is considered here as late CL (L-CL). Although there is a great deal known about T-cell responses in patients with L-CL, there is little information about the in situ inflammatory response in E-CL. Histological sections of skin biopsies from 15 E-CL and 28 L-CL patients were stained by hematoxilin and eosin to measure the area infiltrated by cells, as well as tissue necrosis. Leishmania braziliensis amastigotes, CD4+, CD8+, CD20+, and CD68+ cells were identified and quantified by immunohistochemistry. The number of amastigotes in E-CL was higher than in L-CL, and the inflammation area was larger in classical ulcers than in E-CL. There was no relationship between the number of parasites and magnitude of the inflammation area, or with the lesion size. However, there was a direct correlation between the number of macrophages and the lesion size in E-CL, and between the number of macrophages and necrotic area throughout the course of the disease. These positive correlations suggest that macrophages are directly involved in the pathology of L. braziliensis-induced lesions.

A Potential Role for Mononuclear Phagocytes in Cutaneous Ulcer Development in Human Immunodeficiency Virus-Leishmania braziliensis Coinfection.

Skin ulcer development in cutaneous leishmaniasis due to Leishmania braziliensis infection is associated with a mononuclear cell infiltrate and high levels of tumor necrosis factor (TNF). Herein, we show that despite the absence of Leishmania-driven TNF, a cutaneous leishmaniasis patient with acquired immunodeficiency syndrome developed a skin ulcer. The presence of mononuclear phagocytes and high levels of TNF, chemokine (C-C motif) ligand 2 (CCL2), and metalloproteinase-9 in tissue are identified as potential contributors to immunopathology observed in L. braziliensis-infected patients.

Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load.

BACKGROUND: Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion. METHODOLOGY/PRINCIPAL FINDINGS: We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4). CONCLUSION/SIGNIFICANCE: Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.

Lesson of the month 2: Chronic erythematous painless plaque on the eyelid co-presenting with multiple ulcerated nodules on the extremities.

Cutaneous leishmaniasis is a parasitic disease caused by the Leishmania species, transmitted by the bite of an infected sandfly. The typical cutaneous lesion is a painless ulcer with a raised, indurated margin and often covered with an adherent crust. The lesions are mostly located on exposed sites such as the face and the extremities. Eyelid involvement is rare, making up only 2-5% of cases with facial cutaneous leishmaniasis. Herein, we report a 50-year-old male who presented with an erythematous plaque on the upper eyelid and multiple ulcerated nodules located on the extremities. Following microscopic examination of the lesional smear, a diagnosis of cutaneous leishmaniasis was made, and the patient was successfully treated with intramuscular meglumine antimonate therapy.

Imported new world cutaneous leishmaniasis in a traveller from Slovakia.

We present a case of imported leishmaniasis in a 31-year-old woman from Slovakia, who visited the countries of South America for three months in 2011. On 29 and 31 August 2011, she was probably infected with Leishmania parasites in the jungles of Ecuador. Approximately one week after returning to Slovakia, a small papules appeared on patient's left leg. Another wound was found after two weeks. Both ulcers were enlarging. We proved amastigote forms of Leishmania spp. only in repeated dermal scrapings from the edge of the ulcer by Giemsa staining after negative results from examination of a wound scrape and biopsy specimen. We identified the species Leishmania (Viannia) panamensis as a causative agent by using the polymerase chain reaction (PCR) method and subsequent sequencing of the ITS region. Closure of wounds and scab formation were observed after 20 days of treatment with sodium stibogluconate. In the control microscopic examination after the end of the treatment, parasites were not present, and the PCR confirmed the negative result (Fig. 2, Ref. 31).

Experimental Infection of the Mayan Cichlid Cichlasoma urophthalmus with the Oomycete Aphanomyces invadans.

This study aimed to determine the susceptibility of the Mayan cichlid Cichlasoma urophthalmus to infection with the fungus Aphanomyces invadans (also known as epizootic ulcerative syndrome [EUS]). A total of 27 C. urophthalmus were exposed to the original A. Invadans 2006/86/EC strain by intramuscularly injecting the fish with 25,000 zoospores/ml or exposing the fish to a suspension of 25,000 zoospores/ml in 6-L aquaria for 30 days. To assess the infectious capacity of A. invadans, 3 golden barbs (Puntius semifasciolatus) were infected intramuscularly with 200,000 zoospores/ml. A second experiment using 100 C. urophthalmus was performed for 60 days with 50 fish in each treatment group. A polymerase chain reaction (PCR)-based diagnostic method was used; muscle and gills were the target tissues. In the first experiment, none of the exposed C. urophthalmus developed skin lesions related to A. invadans infection. However, PCR analysis revealed that infection had occurred. For the intramuscular treatment, there were significant differences between the controls and the muscle samples (Fisher's exact test; P < 0.05) but not between the controls and the gill samples (P > 0.05). All golden barbs became infected, as indicated by PCR, and developed skin lesions typical of A. invadans infection. We concluded that C. urophthalmus was infected with A. invadans but was an asymptomatic carrier because skin lesions did not develop. In the second experiment, all fish were negative, suggesting that the fish had cleared the infection by the end of the experiment.