Proteome profiling of cutaneous leishmaniasis lesions due to dermotropic Leishmania donovani in Sri Lanka.

BACKGROUND: Characterization of the host response in cutaneous leishmaniasis (CL) through proteome profiling has gained limited insights into leishmaniasis research compared to that of the parasite. The primary objective of this study was to comprehensively analyze the proteomic profile of the skin lesions tissues in patients with CL, by mass spectrometry, and subsequent validation of these findings through immunohistochemical methods. METHODS: Eight lesion specimens from leishmaniasis-confirmed patients and eight control skin biopsies were processed for proteomic profiling by mass spectrometry. Formalin-fixed paraffin-embedded lesion specimens from thirty patients and six control skin specimens were used for Immunohistochemistry (IHC) staining. Statistical analyses were carried out using SPSS software. The chi-square test was used to assess the association between the degree of staining for each marker and the clinical and pathological features. RESULTS: Sixty-seven proteins exhibited significant differential expression between tissues of CL lesions and healthy controls (p < 0.01), representing numerous enriched biological processes within the lesion tissue, as evident by both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Among these, the integrated endoplasmic reticulum stress response (IERSR) emerges as a pathway characterized by the up-regulated proteins in CL tissues compared to healthy skin. Expression of endoplasmic reticulum (ER) stress sensors, inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6) in lesion tissue was validated by immunohistochemistry. CONCLUSIONS: In conclusion, proteomic profiling of skin lesions carried out as a discovery phase study revealed a multitude of probable immunological and pathological mechanisms operating in patients with CL in Sri Lanka, which needs to be further elaborated using more in-depth and targeted investigations. Further research exploring the intricate interplay between ER stress and CL pathophysiology may offer promising avenues for the development of novel diagnostic tools and therapeutic strategies in combating this disease.

Proteome profiling of cutaneous leishmaniasis lesions due to dermotropic Leishmania donovani in Sri Lanka.

Characterization of the host response in cutaneous leishmaniasis (CL) through proteome profiling has gained limited insights in leishmaniasis research, in comparison to that of the parasite. The primary objective of this study was to comprehensively analyze the proteomic profile of the skin lesions tissues in patients with CL, by mass spectrometry, and subsequent validation of these findings through immunohistochemical methods. Sixty-seven proteins exhibited significant differential expression between tissues of CL lesions and healthy controls (p<0.01), representing numerous enriched biological processes within the lesion tissue, as evident by both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Among these, the integrated endoplasmic reticulum stress response (IERSR) emerges as a pathway characterized by the up-regulated proteins in CL tissues compared to healthy skin. Expression of endoplasmic reticulum (ER) stress sensors, inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6) in lesion tissue was validated by immunohistochemistry. In conclusion, proteomic profiling of skin lesions carried out as a discovery phase study revealed a multitude of probable immunological and pathological mechanisms operating in patients with CL in Sri Lanka, which needs to be further elaborated using more in-depth and targeted investigations.

Prevalence of cutaneous leishmaniasis infection and clinico-epidemiological patterns among military personnel in Mullaitivu and Kilinochchi districts of the Northern Province, early war-torn areas in Sri Lanka.

BACKGROUND: The 30-year-old armed conflict in Sri Lanka resulted in a general breakdown of civil administration in the Northern and Eastern provinces, leading to mobilisation of many armed forces personnel to assist with reconstruction and resettlement. This occupational group has been identified as a priority risk group for leishmaniasis. METHODS: Individuals enlisted at all military establishments in Mullaitivu and Kilinochchi districts, Northern Province of Sri Lanka were included. Five thousand individuals were screened for skin lesions between September 2018 and August 2019. Persons with lesions suspected as cutaneous leishmaniasis (CL) were further investigated. Information on sociodemographic/other potential risk factors was obtained through an interviewer-administered structured questionnaire. The diagnosis was confirmed by microscopic visualization of parasitic stages from different samples obtained (skin scraping, lesion aspirate and tissue impression smears), histopathology and polymerase chain reaction DNA amplification. RESULTS: Among 5000 individuals screened, 74 individuals were suspected of having CL. Of these, 67.6% (n = 50) patients were confirmed for CL by microscopy. Around two third of both males (67.6%; n = 48) and females (66.6%; n = 2) were positive for Leishmania. The soldiers belonging to 26-35-year age group reported the highest susceptibility (83.3%; OR: 4.83, 95% CI: 3.49-6.20%). Of the sociodemographic factors, age, wearing short-sleeved upper body clothing as the uniform and non-use of insect repellents were identified as significant risk factors. Most of the CL patients had a single lesion (86.0%; n = 43) of an ulcerative type (34.0%; n = 17), mostly on their upper limb (67.9%; n = 34). Lesions were mostly 5-10 mm diameter (59.9%; n = 30) in size with poorly defined margins (72.0%; n = 36). Amongst the diagnostic techniques, microscopic examination of slit skin smear and tissue impression smear were able to discriminate the majority of patients (92.1%; n = 46) for CL. CONCLUSIONS: In order to highlight the true burden of leishmaniasis in the military personnel, cases of leishmaniasis from military institutes should be recognized as a different entity per say and be included in the national figures so as to depict the real magnitude of the disease burden amongst this high-risk group.

Histopathology of Cutaneous Leishmaniasis Caused by Leishmania donovani in Sri Lanka.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that is gaining importance in Sri Lanka and internationally. The clinical presentation, pathology, and method of parasite elimination in CL vary according to the species. Leishmania donovani is the causative organism for leishmaniasis in Sri Lanka. This collaborative cross-sectional study describes the clinicopathological features of cutaneous leishmaniasis among personnel of the tri-forces serving in the North and East of the country. The histology of fifty cases of CL confirmed by at least two methods (slit skin smear, lesion aspirate, tissue impression, and histology) was reviewed. The parasitic load was assessed semiquantitatively. The histological features were correlated with the clinical presentation and organism load. The majority (89.8%; n = 44) presented with a single lesion mostly located in the upper limb (69.4%). The lesion types included papule (34.7%), nodule (32.7%), and an ulcer (30.6%). The evolution time of lesions averaged 31.55 weeks. Epidermal changes were observed in 49 of the biopsies and included hyperkeratosis (90.0%; n = 45), acanthosis (44.0%; n = 22), atrophy (34.0%; n = 17), and interface change (66%; n = 33). Dermal changes were seen in all cases and were characterized by a lymphohistioplasmacytic inflammatory infiltrate of variable intensity with ill-formed granuloma in 19 cases (38%) and well-formed epithelioid granulomas in 22 cases (44%). Focal necrosis was present in 20% (n = 10). Leishmania amastigote forms were observed in 88% (n = 44). Transepidermal elimination (P = 0.025), granuloma (P = 0.027) formation, and type of lesion (P = 0.034) were significantly associated with the organism load. Granuloma formation was associated with a reduction in organism load, indicating that the macrophage activation played an important role in the control of the organism.

Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka.

BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.

Tissue Impression Smears as a Supplementary Diagnostic Method for Histopathology in Cutaneous Leishmaniasis in Sri Lanka.

Cutaneous leishmaniasis (CL) is diagnosed mainly by light microscopy of smears made using lesion material. Histopathology is usually done in atypical presentations or when lesion smears are negative. Tissue impression smears (TIS) made from skin biopsy specimens were compared with histopathology for the diagnosis of CL. Out of the 111 patients included, 83 (74.8%) were positive by either methods. The TIS was positive in 70.3% whereas histopathology was positive in 56.8% of patients. Tissue impression smears can be used as a supplementary diagnostic test that gives sensitive and rapid results when tissue biopsies are used as the source of lesion material for diagnosis of CL.