Dermotropic Leishmania donovani in Sri Lanka: visceralizing potential in clinical and preclinical studies.

The visceralizing potential of apparently dermotropic Leishmania donovani in Sri Lanka (L. donovani-SL) was investigated through long-term follow-up of cutaneous leishmaniasis (CL) patients and in vivo and in vitro experimental infection models. CL patients (n = 250) treated effectively with intra-lesional antimony therapy were followed-up six monthly for 4 years. There was no clinical evidence of visceralization of infection (VL) during this period. Infection of BALB/c mice with L. donovani-SL (test) through intra-dermal route led to the development of cutaneous lesions at the site of inoculation with no signs of systemic dissemination, in contrast to the observations made in animals similarly infected with a visceralizing strain of L. donovani-1S (control). Cytokine (IL-10, IFN-γ) release patterns of splenocytes and lymph node cell cultures derived from mice primed with experimental infections (with either test or control parasites) revealed significantly high IFN-γ response associated with test mice with CL, while prominent IL-10 levels were observed in association with control mice with VL. Furthermore, diminished infection efficiency, intracellular growth and survival of L. donovani-SL parasites compared with L. donovani-1S were evident through in vitro macrophage infection experiments. These studies confirm, for the first time, the essential dermotropic nature of L. donovani-SL suggesting natural attenuation of virulence of local parasite strains.

Emergence of visceral leishmaniasis in Sri Lanka: a newly established health threat.

BACKGROUND: Sri Lanka is a new focus of human cutaneous leishmaniasis caused by a genetic variant of usually visceralizing parasite Leishmania donovani. Over 3000 cases have been reported to our institution alone, during the past two decades. Recent emergence of visceral leishmaniasis is of concern. METHODS: Patients suspected of having visceral leishmaniasis (n = 120) fulfilling at least two of six criteria (fever > 2 weeks, weight loss, tiredness affecting daily functions, splenomegaly, hepatomegaly and anemia) were studied using clinic-epidemiological, immunological and haematological parameters. Seven cases (four progressive, treated (group A) and 3 non- progressive, potentially asymptomatic and observed (group B) were identified. Clinical cases were treated with systemic sodium stibogluconate or amphotericin B and all were followed up at the leishmaniasis clinic of University of Colombo for 3 years with one case followed up for 9 years. RESULTS: All treated cases responded well to anti leishmanial treatment. Relapses were not noticed. Clinical features subsided in all non-progressive cases and did not develop suggestive clinical features or change of laboratory parameters. Visceral leishmaniasis cases have been originated from different districts within the country. Majority had a travel history to identified local foci of cutaneous leishmaniasis. CONCLUSION: Visceral leishmaniasis is recognized as an emerging health threat in Sri Lanka. At least a proportion of locally identified strains of L. donovani possess the ability to visceralize. Apparent anti leishmanial sensitivity is encouraging. Timely efforts in disease containment will be important in which accurate understanding of transmission characteristics, increased professional and community awareness, improved diagnostics and availability of appropriate treatment regimens.

In situ immunopathological changes in cutaneous leishmaniasis due to Leishmania donovani.

Cutaneous leishmaniasis in Sri Lanka is a newly established parasitic disease caused by the usually visceralizing Leishmania donovani. Skin lesions manifest as non-itchy, non-tender papules, nodules or ulcers. In situ cytokine expression provides clues for immunopathogenesis of this localized form of disease. Skin biopsies from 58 patients were analyzed for histological appearance and in situ cytokine expression of T-helper 1 (Th1) and T-helper 2 (Th2) cytokines, namely interferon (IFN)-γ, interleukin (IL)-12A, tumor necrosis factor (TNF)-α, IL-4 and IL-10 by real-time RT-PCR. Significant up-regulation of the Th1 cytokine IFN-γ and down-regulation of the Th2 cytokine IL-4 were seen in patients compared to healthy controls. Significantly elevated tissue expression of IFN-γ and TNF-α was seen in lesions that presented later than 6 months from the time of onset, while IL-4 expression was more prominent in lesions that responded poorly to antimony therapy. A prominent Th1 response appears to support resolving of lesions, whereas a Th2-biased milieu tends to favor poor responsiveness to antimony and delayed lesion healing in L. donovani infections in Sri Lanka.

Loop-mediated isothermal amplification assay as a sensitive diagnostic tool for Leishmania donovani infections in Sri Lanka.

INTRODUCTION: Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani MON 37. Confirmation of diagnosis is done through microscopy, either directly or after in vitro culture. Molecular diagnostic methods are sensitive, but require well established laboratories. Loop mediated isothermal amplification assay (LAMP) is rapid, specific for parasite speciesspecific DNA amplification, and requires only basic laboratory equipment. The aim of the study was to determine the potential utility of LAMP to diagnose leishmaniasis. METHODS: Thirty one patients clinically diagnosed as CL were enrolled in the study. Light microscopy, a widely used and universally accepted method was used as the reference standard for confirmation of diagnosis. RESULTS: LAMP was positive for 19/23 microscopically positive patients, yielding a sensitivity of 82.6%. Specificity of the LAMP assay was 100% and the positive and negative predictive values were 100% and 66% respectively. The average time taken for the LAMP assay was 1 hour and 40 minutes and the cost per sample was about SLR 2 000, which was approximately half the time and cost of a nested PCR (polymerase chain reaction). CONCLUSIONS: LAMP could be considered a potentially useful diagnostic tool for leishmaniasis.

Characterisation of cutaneous leishmaniasis in Matara district, southern Sri Lanka: evidence for case clustering.

Leishmaniasis is a neglected tropical disease transmitted by Phlebotomus spp. sand flies. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani. Transmission patterns are different in Southern and Northern Sri Lanka. Current study examined the prevalence, risk factors and distribution of CL in Matara District, Southern Sri Lanka. Total of 2260 individuals from four District Secretariat divisions (DSDs) were screened by house to house surveys using an interviewer administered questionnaire. The study population had an age range of 1-90 years (median = 43 ± 17.31), low monthly income ( < 20 000 LKR, 52.8%) and a male to female ratio of 1 : 2. Thirty eight patients were diagnosed by light microscopy, culture and/or PCR with a disease prevalence of 1.68%. Spatial mapping provided evidence for significant case clustering, which tended to be more prominent with proximity to forest areas. The risk factors identified were un-plastered brick walls, absence or low usage of protective measures against insect bites, low income and excessive time (>4 hours/day) spent outdoors. However, exposure of limbs while outdoors, unawareness about the disease, type of occupation, common water source as the mode of water supply and presence of animal shelters within 200 m were not associated with the risk of acquiring the disease. Peri-domestic transmission is likely to contribute to the observed case clustering with all age groups at risk of acquiring the infection. Human behavioural habits coinciding with that of the vector, sand fly are likely to enable host-vector contact promoting its spread. Appropriate vector control measures, improvement of housing conditions, public education regarding preventive measures are required to contain the spread of disease.

Use of a clinical tool for screening and diagnosis of cutaneous leishmaniasis in Sri Lanka.

Cutaneous leishmaniasis (CL) was first detected in Sri Lanka in 1992.Local disease is caused by a genetically different variant of Leishmania donovani. Early case detection and management is the mainstay of L. donovani control. High degree of clinical suspicion is critical but a clinical diagnostic tool is not available for leishmaniasis. Current study described, for the first time, a two-staged clinical algorhythm that facilitates screening of CL in Sri Lanka by primary health care worker in stage 1 and management by medical professional in stage 2.Selected clinical markers of 400 patients suspected of CL were analysed retrospectively with laboratory confirmation of leishmaniasis. Ten clinical markers predicted CL with a over 90% accuracy. Subsets of markers showed high levels of sensitivities (60-97.2%) and/or significant association with positive laboratory results as compared to negative lesions [typical onset (acne-form, painless non-itchy), (P = 0.026), size up to 2 cm (P = 0.046), well-defined edges (P = 0.002), regular edges (P = 0.018), rounded shape (P = 0.030), and lesions at 5-8 months (P = 0.052)]. Five of them (typical onset, number up to 2, small size, rounded edges, and rounded shape) also had > 70% sensitivity levels as compared to laboratory findings. Typical onset had the highest sensitivity of 97% and a PPV of 72%. Lesions at 5-8 months duration having defined edges (P = 0.013, specificity 89.7%, PPV 83.1) or having regular edges (P = 0.006, specificity 86.2%, PPV 82.4%) were also predictive of CL. Most of early laboratory-confirmed ( < 12 months) lesions remained < 3 cm (sensitivity > 67%, PPV > 70%) and had defined edges (sensitivity of 52-71%, specificity 46.7-68.8%), (PPV 75.1-86%). Four clinical markers served as good diagnostic markers in both early ( ≤ 4) and late (>12 months) lesions, viz. typical onset (91.3-98.4%), presence of ≤ 2 lesions (sensitivity 82.6-94.7%), size ≤ 2 cm (66.9-73.7%), and regular edges (68.6-76.3%). Reliability of clinical markers generally declined in chronic lesions. However, small lesions of over 12 months were highly indicative of CL (sensitivity of 66%, specificity 66.7%). None of the single/combination markers, however, were 100% sensitive or specific, highlighting the undeniable usefulness of laboratory confirmation, in diagnosis. Decision-making algorithm used 10 basic clinical features for screening and seven specific clinical markers for clinical handling and referral for investigations.

Clinical and epidemiological studies on the cutaneous leishmaniasis caused by Leishmania (Leishmania) donovani in Sri Lanka.

Sri Lanka is the newest reported focus of human leishmaniasis within the Indian subcontinent. Over the last 8 years, more than 2000 cases of cutaneous leishmaniasis (CL), apparently caused by Leishmania donovani (a species usually associated with the visceral form of the disease), have been passively identified in the country. The clinical profiles of 401 suspected cases of CL in Sri Lanka were recently explored and some of the cases' immunological responses were investigated, in antibody-detection assays based on the rk39 antigen. These studies were followed by cross-sectional surveys, involving active case detection, in three areas of Sri Lanka, two of them known to be at relatively high risk for CL, with the aims of estimating the local prevalences of the disease and identifying the main risk factors for its acquisition. This appears to be the first detailed report on the prevalence, risk factors and human serological response associated with human leishmaniasis in Sri Lanka. Although the data collected indicated that the transmission of the parasite causing CL was mostly outdoor (and possibly zoonotic) in the north of the country, most of the transmission in the south seemed to be peridomestic. The CL was found to affect a wide age range, in both male and female subjects. Curiously, the 24 cases of CL that were investigated in the rk39 assays gave negative results whereas the single cases of mucosal or visceral leishmaniasis that were studied were found positive for antibodies reacting with the rk39 antigen. More programmes of active case detection need to be launched across Sri Lanka before the true national burden posed by human leishmaniasis can be accurately evaluated. General awareness of leishmaniasis needs to be raised. Hopefully, continued research and disease monitoring will allow the effective control of leishmaniasis in Sri Lanka.

A simple, cost effective method for isolation and transportation of Leishmania parasites.

Isolation and propagation of Leishmania parasites using conventional culture methods are difficult, especially under field conditions. Transportation of live parasites requires the maintenance of low temperatures, which increases the cost considerably. The present study demonstrates that monophasic micro capillary culture is a simpler, more economical and better alternative to using conventional Evan's modified Tobie's medium to isolate and transport the Leishmania parasite over long distances without the use of temperature control methods.

Is leishmaniasis in Sri Lanka benign and be ignored?

Cutaneous leishmaniasis is now an endemic disease in Sri Lanka. Many studies have focussed on various aspects of this disease but the knowledge, particularly on epidemiological and vector aspects is still poor and the awareness among the general public and even medical/paramedical personnel regarding this disease remains grossly inadequate. The steady increase in the numbers and spread of cutaneous leishmaniasis cases in Sri Lanka and the very close similarity (genotypic and phenotypic) between the local parasite Leishmania donovani MON-37 and the parasite causing visceral leishmaniasis in India (L. donovani MON-2), considered together with the more recent case reports of autochthonous cases of visceral disease in this country, calls for urgent action for setting up of a surveillance programme to estimate the true disease burden and to implement an organized control strategy, combined with operational and epidemiological research to aid control efforts to avert a potentially major catastrophe of more virulent form of leishmaniasis, particularly the visceral type becoming endemic in Sri Lanka.

Studies on prevalence of anopheline species and community perception of malaria in Jaffna district, Sri Lanka.

BACKGROUND & OBJECTIVES: Over two decades of civil unrest and the conflict situation have had detrimental effects on vector control activities and management of malaria in Jaffna district which is an endemic region for malaria in Sri Lanka. With the background that only a few small-scale studies on malaria and its vectors have been reported from this district, a study was designed to explore the current status of malaria in the Jaffna district in relation to vector and community aspects. METHODS: Adults and larvae of anopheline mosquitoes were collected monthly from selected endemic localities. Species prevalence of the collected mosquitoes was studied while the collected adults of Anopheles subpictus, a potential vector in the district, was screened for sibling species composition based on morphological characteristics and exposed to common insecticides using WHO bioassay kits. Knowledge, attitude and practices (KAP) of the community were tested using a pre-tested structured questionnaire in high-risk and low-risk localities in the district. RESULTS: The anopheline mosquito species distribution in the district was--An. culicifacies (0.5%), An. subpictus (46%), An. varuna (4%), An. nigerrimus (44%) and An. pallidus (5.5%). Among the collected larvae the percent prevalence of An. culicifacies was 13% and other species follows as: An. subpictus (71%), An. varuna (4%), An. nigerrimus (10%) and An. pallidus (2%). Sibling species B, C and D of An. subpictus were present in the district with the predominance of B in both coastal and inland areas, while all members showed both indoor and outdoor resting characteristics, they were highly resistant to DDT (4%) and highly susceptible to malathion (5%). KAP study in the district showed a reasonable level of knowledge, positive attitude and practices towards malaria. CONCLUSION: An. subpictus, the reported major vector of Jaffna and a well-established secondary vector of malaria in the country, continues to be the predominant anopheline species. The distribution of sibling species of An. subpictus complex in the Jaffna district, revealed for the first time, has implications for future studies on its bionomics and malaria transmission pattern in this area and the planning of control strategies for this region. The community perception of disease, which revealed a satisfactory knowledge indicates the potential for better community participation in future malaria control activities in this region. As potential vectors are still present, health authorities need to be vigilant to prevent any future epidemics of malaria.

Costs and effectiveness of application of Poecilia reticulata (guppy) and temephos in anopheline mosquito control in river basins below the major dams of Sri Lanka.

In this study we examined the costs and effectiveness of using larvivorous fish, Poecilia reticulata, and a chemical larvicide, temephos, in anopheline mosquito control in the riverbeds below the major dams in Sri Lanka. Five riverbeds below the dams, namely Laxapana, Kotmale 1, Kotmale 2, Nilambe, Rantembe and Victoria, were selected. Riverbed pools in Laxapana and Kotmale 1 were treated with P. reticulata; Rantembe and Victoria were treated with temephos; and Kotmale 2 and Nilambe were kept as controls. In each area, the anopheline larval density, before and after application of fish/temephos, was estimated. The cost of application of fish/temephos was estimated by activities involved for each treatment. After intervention, there was a significant reduction in anopheline larval density in the fish-treated areas compared with the temephos-treated and control areas. Application of P. reticulata was 2.67 times less costly than that of temephos. The cost of fish application can be further reduced if the community is involved in the application.

Larvivorous potential of the guppy, Poecilia reticulata, in anopheline mosquito control in riverbed pools below the Kotmale dam, Sri Lanka.

The larvivorous potential of Poecilia reticulata was studied in the laboratory and in the field. In the laboratory, the number of anopheline larvae consumed per fish per day and the number of larvae in fecal matter per fish were determined. In the field study, 29 of 60 selected riverbed pools in Kotmale oya, below the Kotmale dam, were stocked with P reticulata, whereas the rest served as controls. Anopheline larval surveys were carried out starting from 1 day prior to stocking of fish and on selected days subsequently. P. reticulata consumed an average of 117 +/- 32.33 larvae per fish per day. Fecal matter of P. reticulata contained an average of 2.7 +/- 2.68 larvae per fish . There was a significant reduction in the number of pools positive for anopheline larvae (P < .001) after stocking fish and the number of larvae per pool (P < .001) and per 100 dips (P < .001) as compared with controls.

Cutaneous leishmaniasis in southern Sri Lanka.

Cutaneous leishmaniasis is an established disease in Sri Lanka. The majority of cases have been reported from the North-Central Province, with the southern parts of the country considered less affected. However, during 2004, when the services of a dermatologist were available, a considerable number of patients were referred from the Southern Province, which formed the basis for this study. The clinical pattern, detailed geographical distribution within the Southern Province and periodicity of the cases were studied over a period of 12 months. Of the 113 patients diagnosed, the highest number was within the 10-19 years age group. Most patients were from densely populated rural areas around Matara, a large town within this province. There was a notable increase in the number of cases presenting during February-March and August-September, which are periods following monsoonal rains. Exposed areas of the skin were commonly affected, with the majority of patients having single lesions. Females and males were equally affected. This is in contrast to our previous findings in the North-Central Province where the majority of patients were male soldiers with multiple lesions.

Impact of the 2004 mass drug administration for the control of lymphatic filariasis, in urban and rural areas of the Western province of Sri Lanka.

Lymphatic filariasis is targeted to be eliminated globally, at least as a public-health problem, by 2020. The comprehensive strategy for achieving this goal includes the interruption of the transmission of the causative parasites, by drastically reducing the prevalences of microfilaraemia in at-risk communities. In a descriptive, comparative, cross-sectional and community-based study, the impact of the 2004 mass drug administration (MDA) on filarial infection, in selected areas of the Western province of Sri Lanka, has now been assessed 1-2 and 11 months after the administration of the diethylcarbamazine-albendazole combination employed. Using the cluster-sampling method, urban study populations were selected in the Colombo districts and rural ones were selected in the Gampaha district. After obtaining informed written consent, 2 ml venous blood were collected, between 20.00 and 24.00 hours, from each subject. Personal details and drug compliance in the 2004 MDA were recorded. The samples of 'night' blood were checked for microfilariae, using membrane filtration, and for filarial antigenaemia, using commercial (NOW) immunochromatographic test kits. Eighty-four (4.10%) of the 2034 subjects examined 1-2 months after the 2004 MDA but only four (0.20%) of the 1974 subjects checked 11 months after the MDA were found antigenaemic and/or microfilaraemic (P<0.001). Between the two follow-ups, the overall prevalences of both antigenaemia (4.03% v. 0.15%; P<0.001) and microfilaraemia (0.20% v. 0.05%; P=0.38) fell, although only the reduction in antigenaemia was statistically significant. The prevalence of infection (as indicated by antigenaemia and/or microfilaraemia) fell significantly within each of the two study districts (P<0.001). Although, when the prevalence of infection was high, drug compliance appeared to be an important determinant of the reduction of antigenaemia (P=0.04), the 20% difference in compliance between urban and rural areas had no apparent effect on the corresponding prevalences of infection recorded at either follow-up.

Anopheline breeding in river bed pools below major dams in Sri Lanka.

Anopheline mosquito larval surveys were carried out from September 2000 to August 2002 in Mahaweli and Kelani river beds, below five major dams in the wet and intermediate zones of Sri Lanka, to study the prevalence of anopheline species in these areas. In each study site, all permanent and semi-permanent pools were surveyed fortnightly by dipping at 6 dips/m(2) surface area of water. Larvae were collected in separate containers, staged and identified at their third and fourth stages. During each survey, the surface area and depth of pools were recorded and each reading was considered as an individual observation. River bed pools below the dams contained stagnant clean water with a little or no aquatic vegetation. The majority of pools were < or =1m(2) in surface area and < or =75 cm in depth. Anopheline mosquito breeding was seen throughout the year in each study site. The average percentage of pools positive for anopheline larvae, the number of larvae per 100 pools and 100 dips were 14.85%, 32.34 and 9.29, respectively. Thirteen anopheline species, including 10 potential vectors, namely, An. barbirostris, An. culicifacies, An. jamesii, An. maculatus, An. nigerrimus, An. peditaeniatus, An. subpictus, An. tessellatus, An. vagus and An. varuna were found breeding in the river bed pools.

Larvivorous potential of fish species found in river bed pools below the major dams in Sri Lanka.

Malaria in Sri Lanka is closely associated with the river systems in the country. Construction of major dams across rivers has resulted in year-round pooling and anopheline mosquito breeding in river beds below the dams. This study was carried out to determine the larvivorous activity of fish found in river bed pools below the dams to identify the potential fish species for anopheline larval control in these areas. Fish species in five river beds below the dams, namely, Laxapana, Kotmale, Nilambe, Victoria, and Rantembe, were identified in June 2000. The larvivorous potential of the 12 fish species collected was determined in the laboratory based on the number of larvae consumed per fish within 10 min and an hour. Danio malabaricus Jerdon, Oreochromis mossambicus Peters, Oreochromis niloticus L., and Poecilia reticulata Peters consumed nine or more larvae per fish within 10 min. Aplocheilus dayi Steindachner and Rasbora daniconius Hamilton consumed nine and eight larvae, respectively, within 1 h. O. mossambicus, O. niloticus, and P. reticulata consumed 10 larvae per fish within 10 min. Despite some species having a high larvivorous potential in the laboratory, field studies are required to determine the feasibility of using these fish for anopheline mosquito control in the river bed pools below the dams.

Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience.

Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.

Effects of climatic, socio-economic and behavioural factors on the transmission of hookworm (Necator americanus) on two low-country plantations in Sri Lanka.

The climatic, socio-economic and behavioural factors influencing hookworm (Necator americanus) infection in Sri Lanka were explored between February 2000 and June 2001. In February 2000, a single stool sample was collected from each of the 477 subjects investigated, who were aged 2-74 years (median = 13 years) and lived on the 'lowcountry' Maliboda and Ayr plantations. The 'baseline' prevalence (28.5%) and intensity of hookworm infection (0- 4828.5 eggs/g faeces, with a mean of 128.4 eggs/g) were then determined by examining these initial samples, as Kato-Katz smears. Subsequently, each participant was treated with a single, 500-mg dose of mebendazole and then followed-up, at monthly intervals, for the next 15 months. Whenever a subject was found smear-positive for hookworm eggs at one of the monthly follow-ups, he or she was treated again with mebendazole. This approach allowed the monthly incidence of hookworm infection to be determined for each subject, assuming that subjects became smear-positive approximately 6 weeks post-infection. During the study period, rainfall and mean temperature were recorded daily and then converted to monthly values so that the relationship between the incidence of infection over each month and the rainfall and mean temperature over the same period could be explored. In addition, potentially relevant data on the socio-economic status and behaviour of each subject were collected, in questionnaire-based interviews with the adult subjects and the caregivers of the children investigated. Odds ratios (OR) and their 95% confidence intervals (CI) were then calculated for each factor that might increase the risk of hookworm infection. The monthly incidence of hookworm infection showed three peaks -- in September 2000 (21.3%), January 2001 (20.8%) and May 2001 (17.5%) -- at Maliboda, and two peaks -- in September 2000 (25.0%) and February 2001 (29.2%) -- at Ayr. With the data for all subjects combined, incidence showed a statistically significant correlation with mean temperature (r = -0.468; P = 0.018). The results of a multivariate analysis also revealed that mean temperature was significant (beta = -5.296; P = 0.01) in hookworm incidence. Bathing and washing with water from rock-pools formed by waterfalls (OR = 1.33; CI = 1.35-4.01), the use of wells (OR = 2.35; CI = 1.29-4.30), and a lack of toilets (OR = 1.60; CI = 1.01-2.53) each appeared to increase the risk of hookworm infection significantly. Those living on the two study plantations, and perhaps many other similar plantations in Sri Lanka, would clearly benefit from improved access to the public water supply (especially to pipe-borne water) and toilets.