Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

Diagnosing human cutaneous leishmaniasis using fluorescence in situ hybridization.

Cutaneous leishmaniasis (CL) is endemic in Sri Lanka. Giemsa-stained slit-skin-smears (SSS-Giemsa) and histology are routinely used in diagnosis with a sensitivity of 40-70%. PCR currently has limited accessibility. Therefore, we assessed the sensitivity and specificity of a previously described fluorescence in situ hybridization assay, on skin smears and biopsy samples to overcome the limitations encountered with routine diagnostic methods.Samples from a total of 123 suspected CL patients were collected and subjected to SSS-Giemsa, fluorescence in situ hybridization (FISH) on slit skin smears (SSS-FISH), formalin-fixed-paraffin-embedded-tissues stained with Hematoxylin & Eosin staining (FFPE-H&E) and FISH on formalin-fixed-paraffin-embedded-tissues (FFPE-FISH). Negative controls of 61 patient samples were collected from a CL non-endemic area and subjected to the same procedures. The gold standard PCR was used as a comparator. For FISH, two previously described cyanine 3 tagged Leihsmania genus-specific probes were used.Compared to PCR, SSS-Giemsa, SSS-FISH, FFPE-H&E, and FFPE-FISH had sensitivities of 76.5%, 79.1%, 50.4% and 80.9%, respectively. Routine diagnostic tests (SSS-Giemsa and FFPE-H&E) had a specificity of 100%. SSS-FISH and FFPE-FISH had specificities of 96.7% and 93.4%, respectively. FFPE-FISH had a statistically significant higher diagnostic performance than FFPE-H&E (p < 0.001). The relative performance of SSS-Giemsa, SSS-FISH and FFPE-FISH was similar (p > 0.05 for all comparisons).We conclude that FFPE-FISH is a more accurate diagnostic tool than FFPE-H&E. SSS-FISH did not have an additional advantage over SSS-Giemsa in diagnosis. However, SSS-FISH could be recommended as a minimally invasive method in studies assessing wound healing where immunological probes are used.

Should chemoprophylaxis be a main strategy for preventing re-introduction of malaria in highly receptive areas? Sri Lanka a case in point.

BACKGROUND: Imported malaria cases continue to be reported in Sri Lanka, which was declared 'malaria-free' by the World Health Organization in September 2016. Chemoprophylaxis, a recommended strategy for malaria prevention for visitors travelling to malaria-endemic countries from Sri Lanka is available free of charge. The strategy of providing chemoprophylaxis to visitors to a neighbouring malaria-endemic country within the perspective of a country that has successfully eliminated malaria but is highly receptive was assessed, taking Sri Lanka as a case in point. METHODS: The risk of a Sri Lankan national acquiring malaria during a visit to India, a malaria-endemic country, was calculated for the period 2008-2013. The cost of providing prophylaxis for Sri Lankan nationals travelling to India for 1, 2 and 4 weeks was estimated for that same period. RESULTS: The risk of a Sri Lankan traveller to India acquiring malaria ranged from 5.25 per 100,000 travellers in 2012 to 13.45 per 100,000 travellers in 2010. If 50% of cases were missed by the Sri Lankan healthcare system, then the risk of acquiring malaria in India among returning Sri Lankans would double. The 95% confidence intervals for both risks are small. As chloroquine is the chemoprophylactic drug recommended for travellers to India by the Anti Malaria Campaign of Sri Lanka, the costs of chemoprophylaxis for travellers for a 1-, 2- and 4-weeks stay in India on average are US$ 41,604, 48,538 and 62,407, respectively. If all Sri Lankan travellers to India are provided with chemoprophylaxis for four weeks, it will comprise 0.65% of the national malaria control programme budget. CONCLUSIONS: Based on the low risk of acquiring malaria among Sri Lankan travellers returning from India and the high receptivity in previously malarious areas of the country, chemoprophylaxis should not be considered a major strategy in the prevention of re-introduction. In areas with high receptivity, universal access to quality-assured diagnosis and treatment cannot be compromised at whatever cost.

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Importance of active case detection in a malaria elimination programme.

BACKGROUND: With the aim of eliminating malaria from Sri Lanka by 2014, the Anti-Malaria Campaign of Sri Lanka (AMC) sought the support of Tropical and Environmental Disease and Health Associates Private Limited (TEDHA), a private sector organization. In 2009, TEDHA was assigned 43 government hospitals in the district of Mannar in the Northern Province and in districts of Trincomalee, Batticaloa and Ampara in the Eastern Province to carry out malaria surveillance to complement the surveillance activities of the AMC. Passive case detection (PCD), activated passive case detection (APCD) and active case detection (ACD) for malaria have been routinely carried out in Sri Lanka. METHODS: The active case detection programme of TEDHA involves screening of populations irrespective of the presence of fever or any other signs or symptoms of malaria to detect infections and residual parasite carriers. ACD is done by TEDHA in a) high risk populations through mobile malaria clinics including armed forces personnel and b) pregnant females who visit antenatal clinics for asymptomatic malaria infections during the first trimester of pregnancy. Populations are selected in consultation with the Regional Malaria Officer of the AMC thus avoiding any overlap with the population screened by the government. RESULTS: TEDHA screened 387,309 individuals in the four districts for malaria by ACD including high risk groups and pregnant women between January 2010 and December 2012. During this period seven individuals were diagnosed with Plasmodium vivax infections and one individual was detected with a mixed infection of P. vivax and Plasmodium falciparum. All eight cases were detected by ACD carried out by mobile malaria clinics among high risk groups in the Mannar district. CONCLUSION: The progress made by Sri Lanka in the malaria elimination drive is largely due to increased surveillance and judicious use of control methods which has resulted in zero indigenous malaria cases being reported since October 2012. ACD played a major role in interrupting malaria transmission in the country.