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.