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.

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.

Host genetic polymorphisms and serological response against malaria in a selected population in Sri Lanka.

BACKGROUND: Antibodies against the merozoite surface protein 1-19 (MSP1-19) and the apical membrane antigen 1 (AMA1) of the malaria parasite (Plasmodium vivax) are proven to be important in protection against clinical disease. Differences in the production/maintenance of antibodies may be due to many factors including host genetics. This paper discusses the association of 4 anti-malarial antibodies with selected host genetic markers. METHODS: Blood was collected from individuals (n = 242) with a history of malaria within past 15 years for DNA and serum. ELISA was carried out for serum to determine the concentration of anti-malarial antibodies MSP1-19 and AMA1 for both vivax and falciparum malaria. 170 SNPs related to malaria were genotyped. Associations between seropositivity, antibody levels and genetic, non-genetic factors were determined. RESULTS: Age ranged 13-74 years (mean age = 40.21 years). Majority were females. Over 90% individuals possessed either one or more type(s) of anti-malarial antibodies. Five SNPs were significantly associated with seropositivity. One SNP was associated with MSP1-19_Pv(rs739718); 4 SNPs with MSP1-19_Pf (rs6874639, rs2706379, rs2706381 and rs2075820) and1 with AMA1_Pv (rs2075820). Eleven and 7 genotypes (out of 15) were significantly associated with either presence or absence of antibodies. Three SNPs were found to be significantly associated with the antibody levels viz. rs17411697 with MSP1-19_Pv, rs2227491 with AMA1_Pv and rs229587 with AMA1_Pf. Linkage of the markers in the two groups was similar, but lower LOD scores were observed in seropositives compared to seronegatives. DISCUSSION AND CONCLUSIONS: The study suggests that several SNPs in the human genome that exist in Sri Lankan populations are significantly associated with anti-malarial antibodies, either with generation and/or maintenance of antibodies for longer periods, which can be due to either individual polymorphisms or most probably a combined effect of the markers.

Effectiveness of a serological tool to predict malaria transmission intensity in an elimination setting.

BACKGROUND: Sri Lanka achieved the WHO certificate as a malaria free country in September 2016, thus monitoring of malaria transmission using sensitive and effective tools is an important need. Use of age-specific antibody prevalence as a serological tool to predict transmission intensity is proven to be a cost effective and reliable method under elimination settings. This paper discusses the correlation of four anti-malarial antibodies against vivax and falciparum malaria with the declining transmission intensities in two previously high malaria endemic districts i.e. Kurunegala and Moneragala of Sri Lanka. METHODS: Sera was collected from 1,186 individuals from the two districts and were subjected to standard ELISA together with control sera from non-immune individuals to obtain Optical Density (OD) values for four anti-malarial antibodies i.e. anti-MSP1 and anti-AMA1 for both Plasmodium vivax and Plasmodium falciparum. The sero-positive samples were determined as mean OD + 3SD of the negative controls. The sero-prevalence was analyzed against the demographic characteristics of the population. A simple reversible catalytic model was fitted into sero-prevalence data to predict the sero-conversion and sero-reversion rates. RESULTS: Over 60% of the population was sero-positive for one or more antibodies except young children (<10 years). The sero-prevalence was zero in young children and very low in young adults when compared to the older age groups. The model developed for falciparum malaria that assumed the presence of a change in transmission was not significant in the Kurunegala district although significant reduction in transmission was observed when the model was used for P. vivax antibody data in that district. In Moneragala district however, all the serological markers indicated a change in transmission that has occurred approximately 15 years ago. CONCLUSIONS: Assessment of MSP1 and AMA1 anti-malarial antibodies of P. vivax and P. falciparum proved to be useful indicators in predicting transmission under elimination settings as prevailed in Sri Lanka. The sero-conversion rates for the two districts studied are shown to be very low or zero indicating the absence of active and/or hidden transmission confirming a "true" state of elimination at least, in the two study districts in Sri Lanka.

G6PD gene variants and its association with malaria in a Sri Lankan population.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that plays an important role in many cellular functions. Deficiency of this enzyme results from point mutations in the coding region of the G6PD gene. G6PD-deficiency is important in malaria, as certain anti-malarial drugs could induce haemolysis in such patients and mutations in this gene may influence the susceptibility or resistance to the disease. Detailed information on genetic variations in the G6PD gene for Sri Lankan populations is yet to be revealed. This study describes a set of G6PD mutations present in a Sri Lankan population and their association with uncomplicated malaria. METHODS: DNA was extracted from 1,051 individuals. Sixty-eight SNPs in the region of the G6PD gene were genotyped. A database created during the 1992-1993 malaria epidemic for the same individuals was used to assess the associations between the G6PD SNPs and parasite density or disease severity of uncomplicated malaria infections. Linkage disequilibrium for SNPs and haplotype structures were identified. RESULTS: Seventeen genetic variants were polymorphic in this population. The mutant allele was the major allele in 9 SNPs. Common G6PD variants already described in Asians or South-Asians seemed to be absent or rare in this population. Both the severity of disease in uncomplicated malaria infections and parasitaemia were significantly lower in males infected with Plasmodium falciparum carrying the ancestral allele of rs915942 compared to those carrying the mutant allele. The parasite density of males infected with P. falciparum was significantly lower also in those who possessed the mutant alleles of rs5986877, rs7879049 and rs7053878. Two haplotype blocks were identified, where the recombination rates were higher in males with no history of malaria when compared to those who have experienced the disease in the past. CONCLUSIONS: This is the most detailed survey of G6PD SNPs in a Sri Lankan population undertaken so far that enabled novel description of single nucleotide polymorphisms within the G6PD gene. A few of these genetic variations identified, demonstrated a tendency to be associated with either disease severity or parasite density in uncomplicated disease in males. Known G6PD gene polymorphisms already described from elsewhere were either absent or rare in the local study population.

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.

Diagnostic Tools for Cutaneous Leishmaniasis Caused by Leishmania donovani: A Narrative Review.

Leishmaniasis, a neglected tropical disease, encompasses a spectrum of clinical conditions and poses a significant risk of infection to over one billion people worldwide. Visceral leishmaniasis (VL) in the Indian sub-continent (ISC), where the causative parasite is Leishmania donovani, is targeted for elimination by 2025, with some countries already reaching such targets. Other clinical phenotypes due to the same species could act as a reservoir of parasites and thus pose a challenge to successful control and elimination. Sri Lanka has consistently reported cutaneous leishmaniasis (CL) due to L. donovani as the primary disease presentation over several decades. Similar findings of atypical phenotypes of L. donovani have also been reported from several other countries/regions in the Old World. In this review, we discuss the applicability of different methods in diagnosing CL due to L. donovani and a comprehensive assessment of diagnostic methods spanning clinical, microscopic, molecular, and immunological approaches. By incorporating evidence from Sri Lanka and other regions on L. donovani-related CL, we thoroughly evaluate the accuracy, feasibility, and relevance of these diagnostic tools. We also discuss the challenges and complexities linked to diagnosing CL and review novel approaches and their applicability for detecting CL.

Genetic polymorphisms associated with anti-malarial antibody levels in a low and unstable malaria transmission area in southern Sri Lanka.

BACKGROUND: The incidence of malaria in Sri Lanka has significantly declined in recent years. Similar trends were seen in Kataragama, a known malaria endemic location within the southern province of the country, over the past five years. This is a descriptive study of anti-malarial antibody levels and selected host genetic mutations in residents of Kataragama, under low malaria transmission conditions. METHODS: Sera were collected from 1,011 individuals residing in Kataragama and anti-malarial antibodies and total IgE levels were measured by a standardized ELISA technique. Host DNA was extracted and used for genotyping of selected SNPs in known genes associated with malaria. The antibody levels were analysed in relation to the past history of malaria (during past 10 years), age, sex, the location of residence within Kataragama and selected host genetic markers. RESULTS: A significant increase in antibodies against Plasmodium falciparum antigens AMA1, MSP2, NANP and Plasmodium vivax antigen MSP1 in individuals with past history of malaria were observed when compared to those who did not. A marked increase of anti-MSP1(Pf) and anti-AMA1(Pv) was also evident in individuals between 45-59 years (when compared to other age groups). Allele frequencies for two SNPs in genes that code for IL-13 and TRIM-5 were found to be significantly different between those who have experienced one or more malaria attacks within past 10 years and those who did not. When antibody levels were classified into a low-high binary trait, significant associations were found with four SNPs for anti-AMA1(Pf); two SNPs for anti-MSP1(Pf); eight SNPs for anti-NANP(Pf); three SNPs for anti-AMA1(Pv); seven SNPs for anti-MSP1(Pv); and nine SNPs for total IgE. Eleven of these SNPs with significant associations with anti-malarial antibody levels were found to be non-synonymous. CONCLUSIONS: Evidence is suggestive of an age-acquired immunity in this study population in spite of low malaria transmission levels. Several SNPs were in linkage disequilibrium and had a significant association with elevated antibody levels, suggesting that these host genetic mutations might have an individual or collective effect on inducing or/and maintaining high anti-malarial antibody levels.

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.

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.

Toxocara seropositivity in Sri Lankan children with asthma.

BACKGROUND: Toxocariasis occurs in humans due to infection with Toxocara canis or T. cati, the nematode parasites of dogs and cats, respectively. The relationship between toxocariasis and asthma is complex, with some studies demonstrating that children with asthma were more likely to be Toxocara seropositive as compared to non-asthmatic children, and other studies indicating no such significant relationship. The aim of the present study was to investigate Toxocara seropositivity and its association with asthma in a selected group of Sri Lankan children. METHODS: Two groups of children were studied: group 1 included 100 children with confirmed bronchial asthma who were on regular inhaler steroid treatment for asthma; group 2 included 96 children who did not have physician-diagnosed asthma or upper respiratory tract infections, attending the same hospital. Diagnosis of Toxocara seropositivity was based on IgG Toxocara Microwell Serum Elisa Kits. Enzyme-linked immunosorbent assay was regarded as positive for a reading of 0.3 optical density units. Stool samples were examined for helminth ova. RESULTS: Toxocara seropositivity in children with asthma was 29% and this was significantly more than Toxocara seropositivity among non-asthmatic children (P < 0.001). Toxocara seropositivity was identified as a significant risk factor of asthma in a univariate model. Eosinophilia was seen in a significantly higher proportion of non-asthmatic and asthmatic children who were Toxocara seropositive. Toxocara seropositivity, however, was not identified as a significant risk factor in a multivariate model. CONCLUSIONS: The analysis confirmed previously identified risk factors for asthma but there was no association between the helminth parasitic infection, toxocariasis and bronchial asthma in children.