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.

Risk Factor Analysis of Cutaneous Leishmaniasis in Sri Lanka through a Nationwide Survey.

Leishmaniasis in Sri Lanka was first reported in the early 1990s. Cutaneous leishmaniasis (CL) cases have markedly increased in recent years, demanding due attention from health authorities. The spatial distribution of CL is not homogeneous. This case-control study investigated factors that may contribute to this heterogeneous distribution through a nationwide study. Information on sociodemographic, economic, and environmental characteristics was collected from study participants (cases, n = 303; controls, n = 2,762). All individuals were followed up for 3 years, and signs of CL or associated complications were recorded. Differences in possible risk factors between cases and controls were analyzed. Individuals <18 years old, electricity supply, spending >2 hours outdoors, visiting jungles/water bodies, and living near CL patients were identified as risk factors. Household members of 1.3% of cases, 2.3% of controls residing within a perimeter of 500 m from a patient, and 0.8% of controls living beyond 2 km from a case developed CL. Thus, CL in Sri Lanka appears intertwined with living environment and host behavior. Common environmental factors may be responsible for the higher risk of CL in individuals living in close proximity to CL patients. This may at least partly explain the clustering of CL cases in selected areas of the country.

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.

The use of recombinant K39, KMP11, and crude antigen-based indirect ELISA as a serological diagnostic tool and a measure of exposure for cutaneous leishmaniasis in Sri Lanka.

Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani, a parasite widely known to cause visceral leishmaniasis. Despite the fact that CL is not generally believed to elicit serological immune responses, recent studies show the presence of antibody responses against this atypical form of CL. This study assesses the potential of using recombinant K39 (rK39), KMP11, and crude parasite antigen-based indirect ELISAs as serological diagnostic tools and measures of exposure for CL in Sri Lanka. The study used serum samples from confirmed CL patients (n = 266) and apparently healthy individuals from endemic settings (n = 411). Serum samples from individuals residing in non-endemic areas were used as negative controls. In-house indirect ELISAs were optimized and validated for recombinant antigens. Previously validated crude parasite extract-based indirect ELISA was performed for comparison. The statistical analyses were performed using SPSS v26.0. The rK39 (sensitivity = 71.2%, specificity = 64%) and KMP11 (sensitivity = 79.2%, specificity = 71.4%) based indirect ELISA were shown to be less suitable for the diagnosis of CL, while crude parasite extract-based indirect ELISA (sensitivity = 82.4%, specificity = 85.7%) might be a better method of diagnosis. All 03 ELISAs seemed to be good methods as measures of exposure since correlations were observed between the seropositivity of all 03 ELISAs (rK39: p = 0.037, KMP11: p = 0.007, CrudeAg: p = 0.000) with provincial case incidences. The findings will be important in identifying the disease hotspots in order to design the control measures for CL induced by L. donovani in Sri Lanka.

Unfolded protein response pathway in leishmaniasis: A review.

Alteration in the physiological state of the endoplasmic reticulum (ER) leads to the specific response known as unfolded protein response (UPR) or ER stress response. The UPR is driven by three sensor proteins, namely: Inositol-Requiring Enzyme 1, Protein Kinase RNA-like ER kinase and Activating Transcription Factor 6 to restore ER homeostasis. Pathogenic infection can initiate UPR activation; some pathogens can subvert the UPR to promote their survival and replication. Many intracellular pathogens, including Leishmania, can interact and hijack ER for their survival and replication, triggering ER stress and subsequently ER stress response. This review aims to provide a comprehensive overview of the ER stress response in infections with the Leishmania species.

Assessment of knowledge and perceptions on leishmaniasis: An island-wide study in Sri Lanka.

Cutaneous leishmaniasis (CL) is a notifiable disease in Sri Lanka with increasing case numbers reported from every part of the country. In addition to disease treatment and vector control measures, knowledge and perceptions in a community are key contributors to a successful intervention program. An island-wide survey was carried out to assess the knowledge and perceptions regarding CL across the island, with 252 confirmed CL cases and 2,608 controls. Data was collected by trained personnel, using a pre-tested Case Reporting Form (CRF). Although the percentage who referred to CL by its correct name was low (1.4%), majority stated that it is a fly induced skin disease (79.1%). Knowledge on the symptoms, curability and the name of the vector was high in these communities, but specific knowledge on vector breeding places, biting times and preventive methods were poor. The patients were more knowledgeable when compared to the controls. Differences in the level of knowledge could be identified according to the level of education of the participants as well as across the different areas of the country. The main source of information was through the healthcare system, but the involvement of media in educating the communities on the disease was minimal. While this study population was unaccustomed to the use of repellants or sprays, the use of bed nets was high (77.7% of the participants) in this study population. Although misconceptions and incorrect practices are rare in Sri Lankan communities, promoting health education programs which may improve disease awareness and knowledge on vector and its control will further strengthen the control and prevention strategies.

ELISA-based evaluation of antibody response to Leishmania in a region endemic for cutaneous leishmaniasis.

Leishmaniasis includes several clinical forms. While routine diagnosis of cutaneous leishmaniasis (CL) is by microscopy, an antibody response to CL has been reported in several recent studies. This study evaluated anti-leishmanial immunoglobulin G (IgG) antibody responses as a biomarker of active leishmaniasis and a measure of exposure to Leishmania. Sera from 50 untreated CL patients, 140 patients under treatment and 280 healthy individuals residing in endemic regions collected as part of an epidemiological survey, was analysed with an enzyme-linked immunosorbent assay established in-house using receiver operator characteristic curve at optimized cut-off value. The assay showed high performance as a diagnostic tool in identifying exposure in endemic individuals (sensitivity: 98%, specificity: 90.3%). All patients showed lower antibody levels over time since onset of lesion/s. Antibody levels were higher (p Ë‚ .01) and persisted for a longer period in untreated patients. In patients under treatment, the level of anti-IgG antibodies was negatively correlated with the total duration the patient had been on treatment. The anti-leishmanial IgG response in Leishmania donovani-induced CL is transient and is unlikely to confer protective immunity. Optimized serological assays may be useful in endemic settings for diagnosis and monitoring the treatment response in CL.

Spatiotemporal distribution of cutaneous leishmaniasis in Sri Lanka and future case burden estimates.

BACKGROUND: Leishmaniasis is a neglected tropical vector-borne disease, which is on the rise in Sri Lanka. Spatiotemporal and risk factor analyses are useful for understanding transmission dynamics, spatial clustering and predicting future disease distribution and trends to facilitate effective infection control. METHODS: The nationwide clinically confirmed cutaneous leishmaniasis and climatic data were collected from 2001 to 2019. Hierarchical clustering and spatiotemporal cross-correlation analysis were used to measure the region-wide and local (between neighboring districts) synchrony of transmission. A mixed spatiotemporal regression-autoregression model was built to study the effects of climatic, neighboring-district dispersal, and infection carryover variables on leishmaniasis dynamics and spatial distribution. Same model without climatic variables was used to predict the future distribution and trends of leishmaniasis cases in Sri Lanka. RESULTS: A total of 19,361 clinically confirmed leishmaniasis cases have been reported in Sri Lanka from 2001-2019. There were three phases identified: low-transmission phase (2001-2010), parasite population buildup phase (2011-2017), and outbreak phase (2018-2019). Spatially, the districts were divided into three groups based on similarity in temporal dynamics. The global mean correlation among district incidence dynamics was 0.30 (95% CI 0.25-0.35), and the localized mean correlation between neighboring districts was 0.58 (95% CI 0.42-0.73). Risk analysis for the seven districts with the highest incidence rates indicated that precipitation, neighboring-district effect, and infection carryover effect exhibited significant correlation with district-level incidence dynamics. Model-predicted incidence dynamics and case distribution matched well with observed results, except for the outbreak in 2018. The model-predicted 2020 case number is about 5,400 cases, with intensified transmission and expansion of high-transmission area. The predicted case number will be 9115 in 2022 and 19212 in 2025. CONCLUSIONS: The drastic upsurge in leishmaniasis cases in Sri Lanka in the last few year was unprecedented and it was strongly linked to precipitation, high burden of localized infections and inter-district dispersal. Targeted interventions are urgently needed to arrest an uncontrollable disease spread.

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.

Spatial Epidemiologic Trends and Hotspots of Leishmaniasis, Sri Lanka, 2001-2018.

Leishmaniasis, a neglected tropical disease, is on the decline in South Asia. However, cases of cutaneous leishmaniasis have risen in Sri Lanka since 2001, and the lack of in-depth research on its epidemiologic characteristics hampers control efforts. We analyzed data collected from patients with cutaneous leishmaniasis in Sri Lanka during 2001-2018 to study temporal and geographic trends and identify and monitor disease hotspots. We noted a progression in case rates, including a sharp rise in 2018, showing temporal expansion of disease-prevalent areas and 2 persistent hotspots. The northern hotspot shifted and shrank over time, but the southern hotspot progressively expanded and remained spatially static. In addition, we noted regional incidence differences for age and sex. We provide evidence of temporally progressive and spatially expanding incidence of leishmaniasis in Sri Lanka with distinct geographic patterns and disease hotspots, signaling an urgent need for effective disease control interventions.

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.