Next-generation vaccines for tropical infectious diseases.

Tropical infectious diseases inflict an unacceptable burden of disease on humans living in developing countries. Although anti-pathogenic drugs have been widely used, they carry a constant threat of selecting for resistance. Vaccines offer a promising means by which to enhance the global control of tropical infectious diseases; however, these have been difficult to develop, mostly because of the complex nature of the pathogen lifecycles. Here, we present recently developed vaccine candidates for five tropical infectious diseases in the form of a catalog that have either entered clinical trials or have been licensed for use. We deliberate on recently licensed dengue vaccines, provide evidence why combination vaccination could have a synergistic impact on schistosomiasis, critically appraise the value of typhoid conjugate vaccines, and discuss the potential of vaccines in the efforts to eliminate vivax malaria and hookworms.

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.

Antimony resistance and gene expression in Leishmania: spotlight on molecular and proteomic aspects.

Leishmaniasis is a vector-borne parasitic disease caused by Leishmania parasites with a spectrum of clinical manifestations, ranging from skin lesions to severe visceral complications. Treatment of this infection has been extremely challenging with the concurrent emergence of drug resistance. The differential gene expression and the discrepancies in protein functions contribute to the appearance of 2 distinct phenotypes: resistant and sensitive, but the current diagnostic tools fail to differentiate between them. The identification of gene expression patterns and molecular mechanisms coupled with antimony (Sb) resistance can be leveraged to prompt diagnosis and select the most effective treatment methods. The present study attempts to use comparative expression of Sb resistance-associated genes in resistant and sensitive Leishmania, to disclose their relative abundance in clinical or in vitro selected isolates to gain an understanding of the molecular mechanisms of Sb response/resistance. Data suggest that the analysis of resistance gene expression would verify the Sb resistance or susceptibility only to a certain extent; however, none of the individual expression patterns of the studied genes was diagnostic as a biomarker of Sb response of Leishmania. The findings highlighted will be useful in bridging the knowledge gap and discovering innovative diagnostic tools and novel therapeutic targets.

Histological findings associated with treatment response in cutaneous leishmaniasis: a clinicopathological correlation study.

BACKGROUND: Treatment responses to cutaneous leishmaniasis (CL) observed in Sri Lanka show variability, ranging from quick healing to delayed or failed responses to routine medication. The determinants of these differences in treatment response are not well defined. This study aimed to identify predictive features of treatment response and outcome in localized CL caused by Leishmania donovani, focusing on both clinical and histopathological findings in the patients. METHODS: Tissue sections (n = 103) derived from 3 mm punch biopsies of parasitologically confirmed patients were assessed. Patients were followed up weekly until complete healing of skin lesions and were reviewed at the end of 6 months and 1 year. RESULTS: Healing required 7-21 weekly doses of intralesional sodium stibogluconate (IL-SSG) (mean = 12.2 ± 0.622). Twenty-nine (28.1%) patients were identified as delayed responders. None had recurred at the end of 1 year. The demographic or clinical features (age, gender, lesion type, size, location, and lesion duration) did not significantly influence the treatment response. A heavy parasite load and acanthosis were significant predictors of a delayed response to treatment (P < 0.001). Higher parasite loads were associated with inflammation of the entire dermis (P = 0.008), more intense infiltration of macrophages (p = 0.001), and epidermal atrophy (P = 0.033). Well-formed granulomas were inversely proportional to parasite loads. CONCLUSIONS: Histology findings proved to be better prognostic markers than clinical features for delayed responders to treatment and will aid in targeted patient management when tissue biopsies are performed in the initial diagnosis of CL.

Impact of climatic factors on temporal variability of sand fly abundance in Sri Lanka: Longitudinal study (2018 to 2020) with two-stage hierarchical analysis.

Background: Phlebotomine sand flies serve as vectors for leishmaniasis, a major health concern, but a neglected tropical disease. The risk of vector activity is governed by climatic factors that vary in different geographic zones in the country. Thus, we aimed to quantify the effect of climatic variables on sand fly vector activity in ten sentinel sites across Sri Lanka. Methods: Mean rainfall, ambient temperature, relative humidity, wind speed, soil temperature, evaporation, sunshine hours, and vector densities were recorded at monthly intervals in each location from March 2018 to February 2020. The association between weather variables and sand fly densities was analysed using a two-staged hierarchical procedure; Distributed Lag Non-Linear (DLNM) modelling framework and the DLNM method implemented in the R package dlnm (version number 2.4.6). Results: Moderate rainfall values up to 120 mm per month and increasing RH up to 82 at lag of 0 months along with increasing soil temperature and evaporation rate at lag of 2 months were associated with statistically significant increase in the sand fly activity. These associations were heterogeneous across study settings. Whereas increasing ambient and soil temperature, sunshine hours, evaporation rate appeared to reduce the sand fly activity homogeneously at lag of 0 month in all the study settings. Conclusions: The abundance of sand fly vectors varied in relation to selected climatic variables, either in real-time or with a time lag. This information can be utilized for predicting sand fly densities and for the development of effective strategies to prevent leishmaniasis transmission in specific settings.

First evidence of experimental genetic hybridization between cutaneous and visceral strains of Leishmania donovani within its natural vector Phlebotomus argentipes.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of genus Leishmania, and transmitted by different species of Phlebotomine sand flies. More than 20 species of Leishmania are known to cause disease in humans and other animals. Leishmania donovani species complex is known to have a vast diversity of clinical manifestations in humans, but underlying mechanisms for such diversity are yet unknown. Long believed to be strictly asexual, Leishmania have been shown to undergo a cryptic sexual cycle inside its sandfly vector. Natural populations of hybrid parasites have been associated with the rise of atypical clinical outcomes in the Indian subcontinent (ISC). However, formal demonstration of genetic crossing in the major endemic sandfly species in the ISC remain unexplored. Here, we investigated the ability of two distinct variants of L. donovani associated with strikingly different forms of the disease to undergo genetic exchange inside its natural vector, Phlebotomus argentipes. Clinical isolates of L. donovani either from a Sri Lankan cutaneous leishmaniasis (CL) patient or an Indian visceral leishmaniasis (VL) patient were genetically engineered to express different fluorescent proteins and drug-resistance markers and subsequently used as parental strains in experimental sandfly co-infection. After 8 days of infection, sand flies were dissected and midgut promastigotes were transferred into double drug-selective media. Two double drug-resistant, dual fluorescent hybrid cell lines were recovered, which after cloning and whole genome sequencing, were shown to be full genomic hybrids. This study provides the first evidence of L. donovani hybridization within its natural vector Ph. argentipes.

Cytokine and phenotypic cell profiles in human cutaneous leishmaniasis caused by Leishmania donovani.

BACKGROUND: The innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease. METHODS: Peripheral blood mononuclear cell (PBMC) derived macrophages and dendritic cells from patients with LCL and healthy controls from endemic and non-endemic areas, were stimulated with soluble Leishmania antigen (SLA). Inflammatory mediators produced by macrophages (TNF-α/TGF-ß/IL-10, ELISA; NO, Griess method) and dendritic cells (IL-12p70, IL-10, flowcytometry) and macrophage expression of surface markers of polarization, activation and maturation (flowcytometry) were determined at 24h, 48h and 72h and compared. Study was conducted prospectively from 2015-2019. RESULTS: Patient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-ß and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-ß levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other. CONCLUSIONS: The overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype.

Treatment of Cutaneous Leishmaniasis and Insights into Species-Specific Responses: A Narrative Review.

Cutaneous leishmaniasis (CL) is a complex skin infection that has imposed a heavy burden on many developing countries and is caused by more than 20 Leishmania species. This disease is predominantly associated with disfiguring scars and major social stigma upon infection. The severity of the disease seemingly depends on many factors including the species of parasite, the host, region of endemicity, socio-economic status and the accessibility to health facilities. Despite myriad studies that have been performed on current and novel therapies, the treatment outcomes of CL remain contentious, possibly because of the knowledge gaps that still exist. The differential responses to the current CL therapies have become a major drawback in disease control, and the dearth of information on critical analyses of outcomes of such studies is a hindrance to the overall understanding. On the basis of currently available literature on treatment outcomes, we discuss the most effective doses, drug susceptibilities/resistance and treatment failures of the Leishmania genus for both monotherapy and combination therapy. This review focuses on the available treatment modalities for CL caused by different Leishmania species, with insights into their species-specific efficacies, which would inform the selection of appropriate drugs for the treatment and control of leishmaniasis.

Treatment failure to sodium stibogluconate in cutaneous leishmaniasis: A challenge to infection control and disease elimination.

The first-line treatment for Leishmania donovani-induced cutaneous leishmaniasis (CL) in Sri Lanka is intra-lesional sodium stibogluconate (IL-SSG). Antimony failures in leishmaniasis is a challenge both at regional and global level, threatening the ongoing disease control efforts. There is a dearth of information on treatment failures to routine therapy in Sri Lanka, which hinders policy changes in therapeutics. Laboratory-confirmed CL patients (n = 201) who attended the District General Hospital Hambantota and Base Hospital Tangalle in southern Sri Lanka between 2016 and 2018 were included in a descriptive cohort study and followed up for three months to assess the treatment response of their lesions to IL-SSG. Treatment failure (TF) of total study population was 75.1% and the majority of them were >20 years (127/151,84%). Highest TF was seen in lesions on the trunk (16/18, 89%) while those on head and neck showed the least (31/44, 70%). Nodules were least responsive to therapy (27/31, 87.1%) unlike papules (28/44, 63.6%). Susceptibility to antimony therapy seemed age-dependant with treatment failure associated with factors such as time elapsed since onset to seeking treatment, number and site of the lesions. This is the first detailed study on characteristics of CL treatment failures in Sri Lanka. The findings highlight the need for in depth investigations on pathogenesis of TF and importance of reviewing existing treatment protocols to introduce more effective strategies. Such interventions would enable containment of the rapid spread of L.donovani infections in Sri Lanka that threatens the ongoing regional elimination drive.

Genetic diversity and population structure of Phlebotomus argentipes: Vector of Leishmania donovani in Sri Lanka.

Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on FST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging.

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.

The role of improved housing and living environments in malaria control and elimination.

Malaria risk and endemicity is often associated with the nature of human habitation and living environment. The disappearance of malaria from regions where it had been endemic for centuries, such as coastal areas of southern England, has been attributed, at least in part, to improvement in the quality of housing. Moreover, indigenous malaria transmission ceased throughout England without the necessity to eliminate the vector mosquitoes. The principles of malaria transmission, as formulated following the thinking of the pioneers of malaria epidemiology, Ronald Ross and George Macdonald, show how this may happen. Malaria ceases to be sustainable where its reproduction number, R0, the number of new cases generated on average for each existing case of malaria, falls below 1. In the terms of a Ross/Macdonald analysis the reduced contact between humans and blood-feeding mosquitoes that is achieved through housing that is secure against mosquito entry can have a powerful effect in reducing malaria R0. The island of Sri Lanka, where malaria had been endemic probably for centuries previously, has reported no indigenous cases of malaria since 2012. The disappearance of malaria from Sri Lanka followed an effective attack upon malaria transmission by the Sri Lanka Anti Malaria Campaign. The targeted and enhanced efforts of this campaign launched in 1999, drove the malaria R0 below 1 for most of the period up to 2012, leading to a nearly continuous decline in malaria cases until their extinction. The decades leading up to the launch of these efforts were ones of general improvement of living environment and notably in the quality of housing stock. Studies in the late 1980s had shown that quality of housing in a highly malarious district of Sri Lanka was a strong determinant of malaria risk. Through its effects on malaria R0, improved housing is likely to have facilitated the malaria control and cessation of indigenous malaria transmission in Sri Lanka and that it will help reduce the risk of the re-introduction of malaria to the island.

Atypical Presentation of Post-Kala-Azar Dermal Leishmaniasis in Bhutan.

This article describes an atypical case of post-kala-azar dermal leishmaniasis associated with complications due to delayed diagnosis and poor case management. The grave consequences of the prolonged disease process that continued for over 2 decades with eventual healing included facial disfigurement, visual impairment, and mental distress both to the patient and the family. The persistent infection within the skin over a lengthy period with likely increased risk of infection spread in the community highlights its potential negative impact on the ongoing leishmaniasis elimination program in the Indian subcontinent. Bhutan is a member of the leishmaniasis elimination network in Asia, and the government continues to invest in maintenance of the national healthcare system. The case study reveals the gaps in the healthcare system with hardships faced by a patient to access quality healthcare and poor patient outcome used as proxy indicators. It also points to the need to enhance access to healthcare to ensure early diagnosis and effective treatment for leishmaniasis patients including those who live in remote areas, in order to achieve the planned disease elimination targets. It also points towards the key challenges faced by a resource poor nation such as Bhutan in achieving universal health coverage and reaching the set goals for disease elimination. The findings underscore the need for a careful review of the national health care system and to address the deficiencies.

Use of a Plasmodium vivax genetic barcode for genomic surveillance and parasite tracking in Sri Lanka.

BACKGROUND: Sri Lanka was certified as a malaria-free nation in 2016; however, imported malaria cases continue to be reported. Evidence-based information on the genetic structure/diversity of the parasite populations is useful to understand the population history, assess the trends in transmission patterns, as well as to predict threatening phenotypes that may be introduced and spread in parasite populations disrupting elimination programmes. This study used a previously developed Plasmodium vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics of P. vivax parasite isolates from Sri Lanka and to assess the ability of the SNP barcode for tracking the parasites to its origin. METHODS: A total of 51 P. vivax samples collected during 2005-2011, mainly from three provinces of the country, were genotyped for 40 previously identified P. vivax SNPs using a high-resolution melting (HRM), single-nucleotide barcode method. Minor allele frequencies, linkage disequilibrium, pair-wise FST values, and complexity of infection (COI) were evaluated to determine the genetic diversity. Structure analysis was carried out using STRUCTURE software (Version 2.3.4) and SNP barcode was used to identify the genetic diversity of the local parasite populations collected from different years. Principal component analysis (PCA) was used to determine the clustering according to global geographic regions. RESULTS: The proportion of multi-clone infections was significantly higher in isolates collected during an infection outbreak in year 2007. The minor allele frequencies of the SNPs changed dramatically from year to year. Significant linkage was observed in sample sub-sets from years 2005 and 2007. The majority of the isolates from 2007 consisted of at least two genetically distinct parasite strains. The overall percentage of multi-clone infections for the entire parasite sample was 39.21%. Analysis using STRUCTURE software (Version 2.3.4) revealed the high genetic diversity of the sample sub-set from year 2007. In-silico analysis of these data with those available from other global geographical regions using PCA showed distinct clustering of parasite isolates according to geography, demonstrating the usefulness of the barcode in determining an isolate to be indigenous. CONCLUSIONS: Plasmodium vivax parasite isolates collected during a disease outbreak in year 2007 were more genetically diverse compared to those collected from other years. In-silico analysis using the 40 SNP barcode is a useful tool to track the origin of an isolate of uncertain origin, especially to differentiate indigenous from imported cases. However, an extended barcode with more SNPs may be needed to distinguish highly clonal populations within the country.

Insecticide susceptibility of the sand fly leishmaniasis vector Phlebotomus argentipes in Sri Lanka.

BACKGROUND: Leishmania donovani-induced and sand fly-transmitted leishmaniasis is a growing health problem in Sri Lanka. Limited knowledge on biological and behavioral characteristics of probable vector Phlebotomus argentipes hinders disease control. Here, insecticide susceptibility patterns of P. argentipes were investigated with exploration of probable underlying resistance mechanisms. METHODS: Adult sand flies were collected using standard cattle baited net traps and CDC light traps from selected sites in four districts. Adult F1 progeny of P. argentipes were exposed to different concentrations of DDT, malathion, deltamethrin and propoxur using WHO susceptibility bioassay kits. Post-1-h knockdown and post-24-h mortality were recorded and analyzed. Metabolic enzyme activity and the sensitivity of the acetylcholinesterase target-site were determined by biochemical assays using wild-caught flies. Extracted fly DNA samples were tested for the presence of knockdown-resistance (kdr) type mutations. RESULTS: The LC100 values for DDT, malathion, propoxur and deltamethrin were 0.8-1.5%, 0.9-2.0%, 0.017-0.03% and 0.007% respectively. Insecticide-susceptibility levels were higher than the discriminating dosages established for Aedes mosquitoes, except for malathion. The lowest susceptibility levels (except for deltamethrin) were detected in the Mamadala population, whereas the highest levels were detected in the Mirigama population. The percentage of knocked-down sand flies was < 75% at any tested concentration, including those, which exhibited 100% mortality after 24 h. Elevated activity levels of glutathione S-transferase (3%, 7%, 12.5% and 14%) and esterase (2%, 5%, 5.5% and 6.5%) were detected in flies that originated from Mirigama, Pannala, Thalawa and Mamadala respectively, while monooxygenase quantities remained below the cut-off level. Ten to 34.5% of flies were heterozygous for acetylcholinesterases target-site insensitivity, associated with organophosphate and carbamate resistance. Pyrethroid-resistance-associated L1014F kdr-type mutation in the voltage gated sodium channel gene was detected in 30/53 flies. CONCLUSIONS: Populations of P. argentipes in Sri Lanka are largely susceptible to common insecticides, except for malathion (used extensively in the past for malaria control). Their insecticide susceptibility appears negatively associated with past malaria endemicity of the study sites, with signs of early insecticide tolerance. Presence of insecticide target site insensitivity in a notable proportion of flies and enhanced insecticide metabolizing enzyme activities imply potential future challenges for leishmaniasis control, with a call for urgent proactive measures for its containment.

Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay.

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu's phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10-500 µg/ml (1-50 µg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

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.

High resolution melting based method for rapid discriminatory diagnosis of co-infecting Leptomonas seymouri in Leishmania donovani-induced leishmaniasis.

Leishmania donovani, a protozoan parasite of family Trypanosomatidae, causes fatal visceral leishmaniasis (VL) in the Indian subcontinent and Africa and cutaneous leishmaniasis (CL) in Sri Lanka. Another member of Trypanosomatidae, Leptomonas seymouri, resembling Leishmania was discovered recently to co-exist with L. donovani in the clinical samples from India and Sri Lanka and therefore, interfere with its investigations. We earlier described a method for selective elimination of such co-existing L. seymouri from clinical samples of VL exploiting the differential growth of the parasites at 37 °C in vitro. Here, we explored ways for a rapid discriminatory diagnosis using high resolution melting (HRM) curves to detect co-occurring L. seymouri with L. donovani in clinical samples. Initial attempt with kDNA-minicircle (mitochondrial DNA) based HRM did not display different Tm values between L. donovani and L. seymouri. Surprisingly, all of their minicircle sequences co-existed in similar clades in the dendrogram analysis, although the kDNA sequences are known for its species and strain specific variations among the Trypanosomatids. However, an HRM analysis that targets the HSP70 gene successfully recognized the presence of L. seymouri in the clinical isolates. This discovery will facilitate rapid diagnosis of L. seymouri and further investigations in to this elusive organism, including the clinico-pathological implications of its co-existence with L. donovani in patients.

First Evidence for Two Independent and Different Leishmaniasis Transmission Foci in Sri Lanka: Recent Introduction or Long-Term Existence?

Cutaneous leishmaniasis caused by a genetic variant of L. donovani is being reported from Sri Lanka since year 2001. Patients presented from different geographical locations (600 patients from North or South and a minority of cases from other foci, 2001-2013) were studied. Analysis revealed two different sociodemographic and clinical profiles of leishmaniasis in Northern and Southern Sri Lanka. Also, the same different profiles were present in these foci since the onset of the recent outbreak and had independently propagated within each focus over the time. A profile of 14 parameters identified in the Northern focus was further examined with regard to other locations. Northwestern (10/14) and Central parts (9/14) of the island were more similar to Northern focus (14/14). Infection would have originated in one focus and spread to other 2 in Northern Sri Lanka. Southern focus was different from and appeared older than all others (2/14). Western focus that accommodates a large transient population had a mixed picture of North and South features (4/14). Lesions in North showed a slow progression and a nonulcerative nature (128/185, 69.2%), while those in South showed a rapid progression and less nonulcerative lesions (193/415, 46.5%). Clinical analysis favoured a parasite aetiology (considerable strain differences) rather than a host aetiology (age, gender, or genetics). Both foci demonstrated a biannual seasonal variation since the onset of the epidemic. Two peaks were observed during the early and latter parts of the year. Furthermore, long-term existence and recent spatiotemporal expansion and detection of leishmaniasis in this country rather than a recent introduction and establishment were indicated by these findings. Vigorous antimalarial activities that existed in Sri Lanka until few decades ago, lack of professional awareness, and more recent military activities that brought human population in close contact with a sylvatic cycle would have played a role in silent propagation of Leishmania parasites and subsequent increment in human cases, respectively, in this country.