BackgroundCutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani. This study assessed the diagnostic value of a new rapid diagnostic immunochromatographic strip (CL-Detect™ IC-RDT), that captures the peroxidoxin antigen of Leishmania amastigotes.Methodology/Principal findingsWe sampled 74 clinically suspected CL lesions, of which 59 (79.7%) were positive by PCR, 43 (58.1%) by Giemsa stained slit skin smear (SSS) and 21 (28.4%) by the new IC-RDT. All samples which were positive either by SSS or IC-RDT or both were positive by PCR. The sensitivities of the IC-RDT and SSS compared to PCR were 36% and 73%, respectively. Fifteen patients from this endemic region were negative by all three tests. Twenty two clinically non-CL skin lesions from a CL non-endemic region were also negative by all three methods. Specificity and PPV of both IC-RDT and SSS compared to PCR were 100%; the NPVs of IC-RDT and SSS were 37% and 58%, respectively. The median parasite grading of the 59 PCR positive samples was 2+ (1–10 parasites/100 HPFs) and IC-RDT positive lesions was 3+ (1–10 parasites /10HPFs). The duration of the lesion was not associated with IC-RDT positivity.Conclusions/SignificanceThe median parasite grade of Sri Lankan CL lesions is low. The low sensitivities of SSS and CL Detect™ IC-RDT may be due to low parasite counts or low expression of peroxidoxin antigen in amastigotes of the Sri Lankan L. donovani strain. Our results indicate that negative SSS has to be combined with PCR for confirmation of CL in Sri Lanka. The current commercially available IC-RDT is not suitable to diagnose CL in Sri Lanka; an IC-RDT with improved sensitivity to detect L. donovani would be a valuable addition in the diagnostic tool kit for Sri Lanka.

This study showed that intralesional SSG has the best response against CL, while intralesional metronidazole was an effective alternative treatment.

Cutaneous leishmaniasis (CL) is caused byLeishmania donovani in Sri Lanka. Pentavalent antimonials (e.g. sodium stibogluconate; SSG) remain first line drugs for CL with no new effective treatments emerging. We studied whole blood and lesion transcriptomes from Sri Lankan CL patients at presentation and during SSG treatment. From lesions but not whole blood, we identified differential expression of immune-related genes, including immune checkpoint molecules, after onset of treatment. Using spatial profiling and RNA-FISH, we confirmed reduced expression of PD-L1 and IDO1 proteins on treatment in lesions of a second validation cohort and further demonstrated significantly higher expression of these checkpoint molecules on parasite-infected compared to non-infected lesional CD68 + monocytes / macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure (HR = 4.88) and occurred in parallel with reduction in parasite load. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition on T cells, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as a predictor of rapidity of clinical response to SSG treatment in Sri Lanka and support further evaluation of PD-L1 as a host directed therapeutic in leishmaniasis.

21 22 23 24 2 Cutaneous leishmaniasis (CL) is a disfiguring disease caused by infection with 25 Leishmania parasites and is characterised by parasitism of the dermis and chronic 26 inflammation. Whilst T cell responses to Leishmania are essential for both parasite 27 clearance and disease resolution they also drive inflammation, and clinical presentation 28 reflects the balance of these opposing activities 1 . Pentavalent antimonials (e.g. sodium 29 stibogluconate; SSG) remain the first line drugs for CL, even though treatment may be 30 protracted and painful. Although evidence from animal models indicates that an 31 effective clinical response to antimonials requires immune-drug synergy 2 , little is known 32 about how this operates in human disease. Here, we studied formalin fixed paraffin 33 embedded (FFPE) skin biopsies from patients in Sri Lanka with CL, at presentation 34 and during intra-lesional SSG treatment. Immune-targeted transcriptomics in a test 35 patient cohort indicated heightened immune checkpoint pathway expression at 36 presentation. We confirmed reduced PD-L1 and IDO1 protein expression on treatment 37 in a second validation cohort, using digital spatial profiling and quantitative 38 immunohistochemistry. PD-L1 and IDO1 expression on CD68 + monocytes / 39 macrophages was positively correlated with the degree of intracellular parasitism, as 40 determined by parasite-specific RNA FISH. Our data support a model whereby the 41 initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition of T 42 cell immunity, thus facilitating immune-drug synergism and clinical cure. We suggest a 43 need to evaluate shorter course SSG treatment and/or the use of checkpoint inhibition 44 as an adjunct host directed therapy (HDT) in CL. 45 46 One billion people are thought to be at risk of leishmaniasis, a group of diseases caused by 47 infection with protozoan parasites of the genus Leishmania and transmitted by phlebotomine 48 sand flies 3-5 . Approximately 600,000 -1 million new cases of CL occur, with a broad global 49 3 distribution, often leading to stigma and reduced life chances and placing a burden on health 50 services 6,7 . Treatment options for CL have changed little in over 70 years, since pentavalent 51antimonial drugs were first introduced, and there are scant new treatments on the horizon 8,9 . 52 Sri Lanka is endemic for CL 10 with the first autochthonous case being reported in 1992 11 . 53Sri Lankan CL is caused by Leishmania donovani zymodeme MON-37 12-14 , usually 54 associated with visceral leishmaniasis. Current treatment for CL in Sri Lanka involves 55 weekly intra-lesional or daily intra-muscular administration of SSG, with or without 56 cryotherapy, based on the site and size of the lesion and response to treatment. Cure often 57 takes many months, and some patients may fail to respond completely or withdraw from 58 treatment 15 . It is widely proposed that immune-drug synergy is required for fully effective 59 treatment in leishmaniasis and that host directed...

Cutaneous leishmaniasis (CL) is caused by an intracellular protozoan parasite transmitted through a sand fly bite. A review of clinical trials examining different treatment modalities for CL found studies to be deficient in design, execution, analysis and reporting. Core outcome sets (COS), including agreed domains and measures, have been developed for several dermatological disorders. The aims of this study were to reach consensus on the most important core outcome domains to measure when assessing CL, agree on parameters to measure the domains and develop a clinical tool to assess CL. Potential outcome domains for CL were identified through a collaborative international approach involving virtual discussions and e-consultations with stakeholders, including teams caring for CL from Sri Lanka, India, Brazil and the UK. A subsequent judgement process was then undertaken, using the multidisciplinary stakeholder panel of international experts, adhering to nominal group technique to reach final agreement on the core outcome domains. Clinicians presented cases representing the clinical and psychosocial impact of CL to ensure the patient’s perspective was considered. Established core outcome domains included signs and symptoms (objective and subjective), treatment efficacy, treatment impact, clinical sequelae (scarring and pigment) and health-related quality of life (HRQoL). For each core outcome domain, a review of previous approaches to measure the domain was considered. Items to measure the domains were then evaluated by multiple rounds until the panel of experts reached consensus. It was agreed that ‘signs and symptoms’ should capture clinical morphology, diameter and induration of an index lesion with the aid of a palpability score; ‘treatment efficacy and impact’ should capture patient and investigator views through visual analogue scores, percentage change in size of the lesion and re-epithelialization compared with baseline. Sequelae assessment rated pigment, as well as atrophic and hypertrophic/keloid scars. Two open-ended questions were included as a means of capturing some aspects of HRQoL. CutLeishCOM was then generated to capture demographic details, reflect the core outcome domains and assess outcome measurements in each patient. CutLeishCOM has been validated in a small cohort in this study; further validation is required. A specific patient-reported outcome measure should also be considered to ensure adverse effects from treatments, and negative impacts are captured when assessing CL, thus enabling a patient-focused and empathic approach to management.