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.

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.

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.