Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia

Cutaneous leishmaniasis, L. aethiopica, clinical outcomes measure, scar assessment, cost effectiveness, patient reported outcome measures, Ethiopia ALL Metrics - Views Downloads How to cite this article Mohammed AB, Mohammed FS, Zewdu FT et al. Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.13432.2) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente Select a format first ▬ ✚ Study Protocol Revised Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations] Amel Beshir Mohammed1,2, Fewzia Shikur Mohammed1,2, Feleke Tilahun Zewdu3,4, [...] Shimelis Doni Nigusse https://orcid.org/0000-0003-0455-2518 1, Yohannes Hailemichael https://orcid.org/0000-0002-4831-6077 5, Teklu Cherkose https://orcid.org/0000-0003-2344-5435 5, Abebaw Yeshambel Alemu https://orcid.org/0000-0001-5722-250X 4-6, Eshetu Molla https://orcid.org/0000-0001-8262-0622 5, Kidist Bobosha5, Vanessa Yardley https://orcid.org/0009-0005-3744-7003 7, Iris Mosweu8, Mirgissa Kaba9, Catherine Pitt8, Elizabeth Allen10, Saba Maria Lambert https://orcid.org/0000-0003-4376-5495 1,11, Michael Marks https://orcid.org/0000-0002-7585-4743 11-13, Stephen L. Walker https://orcid.org/0000-0002-2034-8376 11,13, Endalamaw Gadisa https://orcid.org/0000-0002-5179-6278 5, SHARP collaborationAmel Beshir Mohammed1,2, Fewzia Shikur Mohammed1,2, [...] Feleke Tilahun Zewdu3,4, Shimelis Doni Nigusse https://orcid.org/0000-0003-0455-2518 1, Yohannes Hailemichael https://orcid.org/0000-0002-4831-6077 5, Teklu Cherkose https://orcid.org/0000-0003-2344-5435 5, Abebaw Yeshambel Alemu https://orcid.org/0000-0001-5722-250X 4-6, Eshetu Molla https://orcid.org/0000-0001-8262-0622 5, Kidist Bobosha5, Vanessa Yardley https://orcid.org/0009-0005-3744-7003 7, Iris Mosweu8, Mirgissa Kaba9, Catherine Pitt8, Elizabeth Allen10, Saba Maria Lambert https://orcid.org/0000-0003-4376-5495 1,11, Michael Marks https://orcid.org/0000-0002-7585-4743 11-13, Stephen L. Walker https://orcid.org/0000-0002-2034-8376 11,13, Endalamaw Gadisa https://orcid.org/0000-0002-5179-6278 5, SHARP collaboration PUBLISHED 03 Dec 2024 Author details Author details 1 ALERT Hospital, Addis Ababa, Ethiopia 2 Department of Dermatovenereology, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia 3 Boru Meda Hospital, Boru Meda, Ethiopia 4 Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia 5 Department of Malaria and Neglected Tropical Disease Research, Armauer Hansen Research Institute, Addis Ababa, Ethiopia 6 Department of Paediatrics and Neonatal Health Nursing, College of Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia 7 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 8 Department of Global Health and Development, London School of Hygiene & Tropical Medicine, London, UK 9 School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia 10 Department of Medical Statistics, Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK 11 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 12 University College London, London, UK 13 Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, London, UK 2 Department of Dermatovenereology, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia 3 Boru Meda Hospital, Boru Meda, Ethiopia 4 Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia 5 Department of Malaria and Neglected Tropical Disease Research, Armauer Hansen Research Institute, Addis Ababa, Ethiopia 6 Department of Paediatrics and Neonatal Health Nursing, College of Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia 7 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 8 Department of Global Health and Development, London School of Hygiene & Tropical Medicine, London, UK 9 School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia 10 Department of Medical Statistics, Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK 11 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 12 University College London, London, UK 13 Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, London, UK Amel Beshir Mohammed Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Fewzia Shikur Mohammed Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Feleke Tilahun Zewdu Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Shimelis Doni Nigusse Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Yohannes Hailemichael Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation Teklu Cherkose Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Abebaw Yeshambel Alemu Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Eshetu Molla Roles: Investigation, Writing – Original Draft Preparation Roles: Investigation, Writing – Original Draft Preparation Kidist Bobosha Roles: Conceptualization, Writing – Original Draft Preparation Roles: Conceptualization, Writing – Original Draft Preparation Vanessa Yardley Roles: Conceptualization, Writing – Original Draft Preparation Roles: Conceptualization, Writing – Original Draft Preparation Iris Mosweu Roles: Investigation, Methodology, Writing – Review & Editing Roles: Investigation, Methodology, Writing – Review & Editing Mirgissa Kaba Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Catherine Pitt Roles: Conceptualization, Methodology, Writing – Review & Editing Roles: Conceptualization, Methodology, Writing – Review & Editing Elizabeth Allen Roles: Conceptualization, Formal Analysis, Writing – Review & Editing Roles: Conceptualization, Formal Analysis, Writing – Review & Editing Saba Maria Lambert Roles: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Michael Marks Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Stephen L. Walker Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Endalamaw Gadisa Roles: Conceptualization, Data Curation, Project Administration, Writing – Review & Editing Roles: Conceptualization, Data Curation, Project Administration, Writing – Review & Editing OPEN PEER REVIEW REVIEWER STATUS Cutaneous leishmaniasis (CL) is a skin neglected tropical disease, with an estimated 40,000 new cases each year in Ethiopia. CL causes ulcers, nodules, and plaques on the skin, and in some instances the destruction of the nasopharyngeal mucosa and cartilage. Some CL lesions may heal spontaneously, whilst other lesions may require therapies which are associated with discomfort, adverse effects, prolonged treatment, and a frequent lack of a complete response. Scarring, a sequela of CL, causes permanent disfigurement and is associated with stigma linked with a reduction in health-related quality of life. The choice of treatment for CL is based upon factors including the causative species; the number, extent, size, and location of lesions; and the availability of treatments. The development of robust evidence for CL treatment is hindered by a lack of validated and appropriate outcome measures and few data to support hypothesis-generation and trial design. There is a paucity of prospective data with well-defined treatment outcomes for CL caused by L. aethiopica. The overall aim of this study is to improve the understanding of the health and economic burden of CL. We have designed an observational, multi-centre cohort study to examine treatment outcomes for CL in Ethiopia which includes clinical outcomes, laboratory outcomes, patient reported outcome measures, scar assessments and cost effectiveness. We aim to recruit up to 750 participants across two hospital sites. We present here the protocol for this cohort study with a 12-month follow up period for each participant. These data will inform the design of randomized controlled trials to evaluate new treatment strategies, with appropriate economic evaluations. This will help improve evidence-based guidelines and support evidence-led policy decisions, not only in Ethiopia but also globally. Plain English summary Cutaneous leishmaniasis (CL) is a skin neglected tropical disease, with an estimated 40,000 new cases each year in Ethiopia. CL is transmitted by the bite of a sand fly on exposed areas, often the face, resulting in ulcers, nodules, and plaques on the skin, and in some instances the destruction of the nasopharyngeal mucosa and cartilage. Some CL lesions may heal spontaneously, whilst other lesions may require therapies which are associated with discomfort, adverse effects, prolonged treatment, and a frequent lack of a complete response. Scarring, a sequela of CL, causes permanent disfigurement and is associated with stigma linked with a reduction in health-related quality of life. The choice of treatment for CL is based upon factors including the causative species; the number, extent, size, and location of lesions; and the availability of treatments. The development of robust evidence for CL treatment is hindered by a lack of validated and appropriate outcome measures and few data to support hypothesis-generation and trial design. There is a paucity of prospective data with well-defined treatment outcomes for CL caused by L. aethiopica. We therefore designed an observational, multi-centre cohort study to examine treatment outcomes for CL in Ethiopia which includes clinical outcomes, laboratory outcomes, patient reported outcome measures, scar assessments and cost effectiveness. These essential data will inform the design of randomized controlled trials to evaluate new treatment strategies, with appropriate economic evaluations. This will help improve evidence-based guidelines and support evidence-led policy decisions, not only in Ethiopia but also globally. Cutaneous leishmaniasis (CL) is a skin neglected tropical disease, with an estimated 40,000 new cases each year in Ethiopia. CL is transmitted by the bite of a sand fly on exposed areas, often the face, resulting in ulcers, nodules, and plaques on the skin, and in some instances the destruction of the nasopharyngeal mucosa and cartilage. Some CL lesions may heal spontaneously, whilst other lesions may require therapies which are associated with discomfort, adverse effects, prolonged treatment, and a frequent lack of a complete response. Scarring, a sequela of CL, causes permanent disfigurement and is associated with stigma linked with a reduction in health-related quality of life. The choice of treatment for CL is based upon factors including the causative species; the number, extent, size, and location of lesions; and the availability of treatments. The development of robust evidence for CL treatment is hindered by a lack of validated and appropriate outcome measures and few data to support hypothesis-generation and trial design. There is a paucity of prospective data with well-defined treatment outcomes for CL caused by L. aethiopica. We therefore designed an observational, multi-centre cohort study to examine treatment outcomes for CL in Ethiopia which includes clinical outcomes, laboratory outcomes, patient reported outcome measures, scar assessments and cost effectiveness. These essential data will inform the design of randomized controlled trials to evaluate new treatment strategies, with appropriate economic evaluations. This will help improve evidence-based guidelines and support evidence-led policy decisions, not only in Ethiopia but also globally. Cutaneous leishmaniasis, L. aethiopica, clinical outcomes measure, scar assessment, cost effectiveness, patient reported outcome measures, Ethiopia Corresponding Author(s) Saba Maria Lambert ([email protected]) Grant information: This project is funded by the National Institute for Health Research (NIHR) under its Research and Innovation for Global Health Transformation (RIGHT) Programme (Grant Reference Number NIHR200125). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © Crown copyright, 2024 Mohammed AB et al.. This open access work is licensed under the Open Government Licence v3.0 How to cite: Mohammed AB, Mohammed FS, Zewdu FT et al. Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.13432.2) First published: 05 Oct 2023, 3:49 (https://doi.org/10.3310/nihropenres.13432.1) Latest published: 03 Dec 2024, 3:49 (https://doi.org/10.3310/nihropenres.13432.2) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The revised version includes changes following comments by our two reviewers in order to enhance the clarity and flow of the paper. Sentence construction in the introduction has been improved. Under methods, a clarification has been added under the headings "Study participants" and "Overview of the data collection", with an in-depth description of immunology samples as suggested by reviewers. The sample size section has been more clearly defined. In the data analysis section, details of software to be used for data analysis and statistical tests that will be used were expanded for each part of the study objectives in order the clarify the analysis methods. This large cohort study has multiple parts with multiple methodologies: • Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative). The development of a severity scale. • Psychosocial: patient’s treatment experience using ethnographic methods (qualitative) • Economic burden of CL: Serial economic evaluations (quantitative) • Immunological investigations on skin and blood samples – quantitative laboratory research The revised version includes changes following comments by our two reviewers in order to enhance the clarity and flow of the paper. Sentence construction in the introduction has been improved. Under methods, a clarification has been added under the headings "Study participants" and "Overview of the data collection", with an in-depth description of immunology samples as suggested by reviewers. The sample size section has been more clearly defined. In the data analysis section, details of software to be used for data analysis and statistical tests that will be used were expanded for each part of the study objectives in order the clarify the analysis methods. This large cohort study has multiple parts with multiple methodologies: • Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative). The development of a severity scale. • Psychosocial: patient’s treatment experience using ethnographic methods (qualitative) • Economic burden of CL: Serial economic evaluations (quantitative) • Immunological investigations on skin and blood samples – quantitative laboratory research In the data analysis section, details of software to be used for data analysis and statistical tests that will be used were expanded for each part of the study objectives in order the clarify the analysis methods. This large cohort study has multiple parts with multiple methodologies: • Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative). The development of a severity scale. • Psychosocial: patient’s treatment experience using ethnographic methods (qualitative) • Economic burden of CL: Serial economic evaluations (quantitative) • Immunological investigations on skin and blood samples – quantitative laboratory research See the authors' detailed response to the review by Talal Alharazi See the authors' detailed response to the review by Najia Ahmed Up to a million new cases of cutaneous leishmaniasis (CL) occur each year, primarily in the world’s poorest communities1. CL is the most common form of leishmaniasis, which is caused by infection with protozoa of the genus Leishmania, acquired via the bite of a female phlebotomine sandfly. Infection leads to the development of ulcers, nodules, and plaques on the skin. Depending on the infecting Leishmania species, spontaneous resolution of infection can take many months or years and medical therapies are associated with discomfort, adverse effects, prolonged treatment, and a frequent lack of complete response (World Health Organization). Lesions often heal with lifelong scarring or destruction of the nasopharyngeal mucosa causing permanent disfigurement, and associated stigma, and overall psychological morbidity, contributing to a reduction in health-related quality of life (HRQoL)2. These multidimensional morbidities and the often-arduous process of treatment-seeking also impose a substantial economic burden in terms of both costs of care and income loss, which can exacerbate the stress and social impact associated with CL and inhibit access to care3–5. The choice of treatment for CL is based upon several factors including the causative species; the number, extent, size and location of lesions; and the availability of treatments. Small lesions not located near a joint or facial structure may be allowed to self-heal or be treated with a topical agent, intra-lesional injection, cryotherapy, or heat therapy. Localised treatments for CL are associated with irritation, pain, discomfort, and in the case of cryotherapy, long-term skin changes6. Large or widespread lesions and those involving the mucosa require systemic treatment7,8. Despite the large number of cases of CL each year, the evidence for determining the best treatment modality remains limited. In Ethiopia, CL is a growing public health problem, with an estimated 40,000 new cases each year. Several Leishmania species cause CL in Ethiopia; most infections are thought to be due to L. aethiopica9, but L. donovani, L. major and L. tropica are also present. L. aethiopica is associated with three distinct clinical forms of CL; localised CL (LCL); mucocutaneous leishmaniasis (MCL); and diffuse CL (DCL), which is the most disfiguring and difficult to treat9. Lesions caused by L. aethiopica most commonly occur on the face10. The diverse range of clinical phenotypes and presentations makes early diagnosis by community-based health workers difficult. Standard CL treatment in Ethiopia is with intralesional or parenteral sodium stibogluconate (SSG), which may be combined with cryotherapy using liquid nitrogen. There is a particular paucity of prospective data with well-defined treatment outcomes for CL caused by L. aethiopica. A systematic review of treatment for CL caused by L. aethiopica identified two randomised trials with a total of 26 participants and two prospective, non-randomised studies11. All four studies were assessed to have methodological limitations by the authors. One of the small, randomised trials compared itraconazole with placebo (n=14 participants)12 and the other compared rifampicin, isoniazid and amithiozone with pentamidine (n=12 participants)13. The much larger prospective study (n=123) reported improvement in various forms of CL: 103 (81%) of participants were cured with liquid nitrogen cryotherapy and 85% of the 20 participants treated with intramuscular SSG (20 mg/kg up to a maximum of 850mg daily) for 30 days were cured14. Another Ethiopian study reported that 21% of participants (n= 167) had “a positive outcome” with intralesional pentavalent antimonial drug meglumine antimoniate (MA) and 47% had “a positive outcome” with systemic MA (20 mg/kg)15. The development of robust evidence for CL treatment is hindered by a lack of validated and appropriate outcome measures and few data to support hypothesis-generation and trial design. A Cochrane review demonstrated that existing studies used heterogeneous outcome measures which mostly lacked validity, hindering comparison of study results16. A consensus document on “harmonized” clinical research methodologies for LCL (but not MCL or DCL) was published by an international group of experts following meetings organised by the World Health Organization (WHO) and the Drugs for Neglected Diseases initiative17. This publication highlighted the importance of laboratory confirmation with identification of Leishmania species in future trials and defined “initial” cure for non-American CL as more than 50% re-epithelialization (if an ulcer) or flattening (if a non-ulcerated lesion) at Day 42; and as 100% re-epithelialization or flattening at Day 90. “Final” cure was defined as 100% re-epithelialization or flattening at Day 180. More recently, open label studies, incorporating these expert-agreed outcomes, were conducted on treatment for CL due to L. aethiopica. One study reported physician-determined cure rates of 59.7% for intralesional SSG18 at Day 90, whilst another study reported physician-determined cure rates of 12.5% and 72.7% for miltefosine19 at two study sites at Day 90. The “harmonized” clinical research methodologies for LCL provided a significant step forward but several gaps remain. No agreed methodologies are available for assessing flattening or re-epithelisation and it remains unclear if outcomes should be measured from the start or end of a course of treatment. The agreed outcome measures are entirely clinical and do not include measures to reflect the outcomes and experiences of affected individuals. The treatment preferences of individuals may be influenced by the frequency of adverse events and the extent to which healing improves their health and psychosocial wellbeing. Appropriate patient reported outcome measures (PROMs) therefore need to be identified and collected alongside clinical measures. Finally, the consensus document recognised that scarring should be assessed as a long-term outcome but did not propose a method for doing so. Given the scarcity of resources for health, and for the treatment of CL in particular, treatment recommendations should also be informed by an understanding of costs and efficiency. However, cost-effectiveness analyses of alternative treatment strategies for non-American CL have only been conducted in Asia20–23, none have addressed infection with L. aethiopica, and only one has assessed effectiveness using a multidimensional outcome metric20, which is essential for comparing the value of investments across intervention types and diseases. No studies of the economic burden of CL or treatment costs in Africa have been published. The Ethiopian Federal Ministry of Health (FMOH) has identified an urgent need for the evaluation of treatments alternatives for all forms of CL. Such evaluation requires better understanding of outcomes, and cost-effectiveness data. We therefore designed an observational, multi-centre cohort study of CL in Ethiopia24. The overall aim of this study is to improve the understanding of the health, psychosocial and economic burden of CL, and to lay the groundwork for well-designed, randomized controlled trials and economic evaluations of alternative CL treatments in Ethiopia. 1. To assess clinical outcomes in LCL, MCL and DCL in a prospective cohort 2. To assess clinical parameters in LCL, MCL and DCL in a prospective cohort 3. To assess the change in HRQoL associated with CL before and after treatment. 4. To develop a severity scale for CL 5. To understand the lived experiences of individuals with CL attending a referral centre by conducting in-depth interviews 6. To assess household costs of CL, health care provider and societal costs of treating CL 7. To characterise how cell-mediated immune responses associate with treatment responses in CL due to L. aethiopica and Human Leucocyte Antigen (HLA) variation correlate with different clinical phenotypes. Patients with CL were involved in the validation of the translations of PROMs through focus group discussions, cultural adaptation and piloting. The study will enrol individuals receiving treatment for CL at two centres: ALERT Hospital, Addis Ababa and Boru Meda Hospital, South Wollo Zone, Amhara Region. ALERT Hospital serves as both a general hospital for patients in Addis Ababa and a regional and national specialized referral centre for individuals with CL and other skin conditions. The hospital has nine dermatologists and a 20-bed unit for the care of patients with CL and diagnoses approximately 1000 cases of CL managed on an inpatient or outpatient basis each year. Boru Meda serves as the general hospital and specialised referral service for all skin conditions in South Wollo zone. It has two dermatologists, and a 35-bed unit for the management of CL and diagnoses around 700 cases of CL, managed on an inpatient or outpatient basis each year. Study recruitment began in April 2022 and is expected to continue until the sample size is reached or until the cut-off of September 30th, 2023, whichever occurs first. Follow-up is therefore expected to be completed by September 30th, 2024. All adults and children diagnosed with CL and initiated on CL treatment at both study centres are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria: 1. Individuals with parasitologically confirmed CL using microscopy or culture 2. Individuals receiving treatment at the study centres Exclusion criteria: 1. Participants who have previously been treated with systemic anti-leishmania therapy 2. Participants who do not or are unable to give informed consent to participate in the study Participants will be treated using the standard of care for CL at the centre where they are managed and in accordance with the Ethiopian national guideline24 which will be agreed by two clinicians in consultation with the affected individual prior to enrolment. The treatment may be changed if deemed clinically appropriate. In general, if the size of the skin lesion is 4cm in diameter of smaller and not near a mucosal site the first line treatment is most frequently local therapy (cryotherapy and/or intralesional SSG). In general, if the diameter of the skin lesion is greater than 4 cm and/or near or involving a mucosal surface the initial therapy is usually intramuscular SSG for 28 days with or without adjunctive cryotherapy. Participants will be assessed on day 42 and further local or systemic therapy may be offered depending on the degree of clinical response. We will classify CL using the operational definitions shown in Box 1. • CL is diagnosed in a person with skin and/or mucosal lesions with evidence of Leishmania infection in the affected tissues characterised by the presence of Leishmania amastigotes on smear microscopy or growth of Leishmania promastigotes in culture or the detection of Leishmania DNA by polymerase chain reaction. • Localized CL: A confirmed case of leishmaniasis, with no mucosal involvement, characterized by ten or fewer cutaneous papules and/or nodules and/or plaques with or without ulceration involving one body site • Multi-regional localized CL: A confirmed case of leishmaniasis, with no mucosal involvement, characterized by ten or fewer cutaneous papules and/or nodules and/or plaques with or without ulceration involving two or more body sites • Mucocutaneous leishmaniasis: A confirmed case of leishmaniasis characterized by ten or fewer papules and/or nodules and/or plaques with or without ulceration involving skin and an adjacent mucosal surface • Mucosal leishmaniasis: A confirmed case of leishmaniasis characterized by papules and/or nodules and/or plaques with or without ulceration involving exclusively a mucosal surface • Diffuse CL: A confirmed case of leishmaniasis characterized by eleven or more papules and/or nodules and/or plaques with or without mucosal involvement. Body sites are classified as: 1. Head and neck 2. Torso - anterior (including genitalia) 3. Torso - posterior (including buttocks) 4. Right upper limb 5. Left upper limb 6. Right lower limb 7. Left lower limb This prospective observational cohort will integrate different methodologies appropriate to the different “areas” under investigation, using qualitative and quantitative tools: Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient related outcome measure (quantitative) Psychosocial: serial patient related outcome measures (quantitative) and in-depth interviews (qualitative) Economic burden of CL: Serial economic evaluations Immunological investigations on skin and blood samples – quantitative laboratory research Study visits for data collection are scheduled at day 1, 14, 28, 42, 90, 180 and 360 (Table 1). Treatment will be decided by the treating clinicians, who may or may not be one of the study dermatologists. All study specific data collection will be undertaken by a member of the study team. At the baseline visit, we will collect socio-demographic data, perform clinical assessment, and collect data on PROMs and costs. Follow-up data will be collected for all patients at specified time points, as detailed further below. In a subset of patients, additional biological samples will be collected for immunological and genetic studies. A further subset of participants will be selected for in-depth interviews. At the baseline visit, we will record the clinical phenotype of CL including the number, size, morphology25 and location of lesions. Lesion dimensions (defined as the largest diameter of the lesion) will be measured using a disposable tape measure. Individuals with more than one lesion will be asked to indicate the lesion causing greatest concern and this will be designated the “index” lesion. Individuals may indicate two or more lesions of equal concern. In those with mucosal involvement, mucosal signs and symptoms are recorded. Clinicians will then provide a subjective global assessment of CL severity using a 3-point Likert scale (mild, moderate, severe). Slit-skin smears will be collected at baseline for microscopy and species identification using polymerase chain reaction (PCR). At each follow-up visit, we will assess lesions for the degree of re-epithelization or flattening. For those with DCL, the number of resolved lesions and percent of lesions flattened per region is recorded. Clinicians will provide a global assessment of the improvement or worsening of CL using a 5-point Likert scale (worse, no change, minor improvement, major improvement, cure). At each visit, we will record the treatment being administered and any changes since the last visit. The adverse reactions to CL treatments will be assessed including the nature and severity of the reaction, the action taken, the presumed cause and the outcome. The Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be used for describing adverse reactions and grading their severity. Slit-skin smear samples will be collected at day 90 for test of cure if the skin lesion is not healed. At baseline, Amharic or Oromiffa translations of several PROMs will be collected (Table 1): two general measures of HRQoL, the EQ-5D-5L and the SF-36, and two skin-specific measures of HRQoL, the Dermatology Life Quality Index (DLQI) and the Children’s Dermatology Life Quality Index (CDLQI). Participants will complete the Cutaneous Leishmaniasis Impact Questionnaire (CLIQ), a CL-specific measure of HRQoL. At the follow-up visits indicated in Table 1, we will collect PROMs again and ask participants for their global assessment of the improvement of their CL lesion(s) using a 5-point Likert scale (better, somewhat better, same, somewhat worse, worse). The Amharic or Afan Oromo translations of the Patient and Observer Scar Assessment Scale (POSAS) v226 will be completed by both the clinician and participant at the day 42 assessment and each subsequent visit. A selection of participants will be recruited to participate in an in-depth interview to capture data on their lived experience of CL until data saturation has occurred. Initially, we aim to recruit a total of 30 adults and 10 children from both study centres. We will ensure representation of participants by key demographics including age, gender, and CL phenotype. Interviews will be conducted by members of the research team using a topic-guide covering key themes including the impact of the illness, the care-seeking pathways they have undertaken and their experience of traditional and allopathic medicine. Ethnographic observation will be used to enrich the data on treatment experiences in the health facilities. At baseline data will be collected on costs incurred by the individuals and their household that can be attributed to a member having CL and/or seeking treatment for CL including costs incurred since onset of CL symptoms. We will also collect data on household income, expenditure, and asset possession to establish a month of income and expenditure data. At each follow up visit, cost related to CL since the last visit/admission will be collected. These will include costs of medicines, hospitalisation, consultation, transportation, and opportunity costs related to having and treating CL (i.e., missed work or school). We will also collect information on how households cope with the economic burden of CL. Data from the provider perspective will be collected. This will include costs of training health workers, providing services, medicines donated, and other service-related costs. These costs will be collected from the project’s activity and financial records; the value of any donated items will be estimated based on market prices. Where necessary, government and donor sources will be consulted for relevant cost data. To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort. The primary outcome is the proportion of individuals with a healed index lesion by study day 90. In line with the consensus document on clinical trial outcomes cure will be defined as 100% re-epithelialization or flattening of the index lesion. Pre-specified additional outcomes are the proportion healed at day 42 and 180. Secondary outcomes include the frequency of adverse events and measures of HRQoL life. There are no pre-existing high-quality data on cure rates for CL caused by L. aethiopica. We calculated the sample size required to measure a range of cure-rates at 90 days with varying degrees of precision (Table 2). Based on this we aimed to recruit up to 250 participants with each CL phenotype (LCL, MCL, and DCL) across the two sites. We anticipate that mucosal leishmaniasis will be rare and will recruit as many individuals with this type of disease as possible.

1: To assess clinical outcomes in LCL, MCL and DCL in a prospective cohort. & Objective 2: To assess clinical parameters in LCL, MCL and DCL in a prospective cohort. We will use descriptive statistics (means, medians, and proportions) to describe demographics and baseline characteristics of the cohort. In exploratory analysis, we will use univariable logistic regression to examine clinical and demographic variables associated with resolution of CL at Day 42, 90, and 180. Whilst not formally powered for these analyses, the findings will be viewed as hypothesis-generating and potentially form the basis for future studies.

3: To assess the change in HRQoL associated with CL before and after treatment. Changes in general, skin and CL-specific HRQoL will be assessed using the following PROMs: EQ-5D-5L, SF-36, DLQI/ CDLQI and CLIQ applied at the timepoints shown in Table 1, and compared with clinical outcomes to determine validity and responsiveness. Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment).

4: To develop a severity scale for CL. A round table CL expert discussion forum, informed by preliminary data, will be held, and consensus reached about potential items and scoring system for a severity scale. The prototype will be tested in individuals with CL at diagnosis.

5: To understand the lived experiences of individuals with CL. Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data.

6: To assess household costs of CL, and health care provider, and societal costs of treating CL. We will assess costs from a disaggregated societal perspective, examining costs to providers (MOH, donors), households, and society as a whole (i.e., costs to providers and household combined). Incremental costs to providers will be estimated relative to a hypothetical counterfactual in which no treatment for CL is provided. Incremental costs to households will be estimated relative to a hypothetical counterfactual in which the CL infection has not occurred. We will estimate total costs of diagnostic and treatment strategies and average costs, including cost per case diagnosed and cost per case resolved, disaggregated by site, type of CL, and patient characteristics (where possible), with appropriate statistical measures of variation. We will explore the degree of impoverishing and catastrophic expenditure for families of individuals with CL. Descriptive statistics frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis

7: To characterise how cell-mediated immune responses associate with treatment responses in CL due to L. aethiopica and (HLA) variation correlate with different clinical phenotypes. Biopsies of affected skin and blood samples will be analysed to (i) evaluate secretion and quantification of selected host cytokines via the LuminexⓇ platform, (ii) evaluate the expression of innate/adaptive, and pro-and anti-inflammatory biomarkers and by evaluating the change in mRNA expression level of TLRs and pro- and anti-inflammatory cytokines before treatment, during treatment, and after completion of treatment. Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Each participant will be assigned a unique study identification number and all data will be recorded and analysed with this unique identification number. Data will be electronically entered into a GCP-compliant, trial-specific study database [Research Electronic Data Capture (RedCap), Nashville, TN, USA]. Access to the database will be restricted to relevant researchers within the study team. Relevant documentation will be kept for a minimum of 10 years. Moreover, MAXQDA plus 2022 will be used to manage the in-depth interview data. Ethical approval was obtained from AHRI/ALERT Ethics Review Committee (Ref: PO/23/21 dated 15.07.2021), the National Research Ethics Review Committee (Ref: 7/2-506/m259/35 dated 17.12.2021), and the London School of Hygiene and Tropical Medicine (Ref: 26421 dated 11.10.2021). Written individual informed consent will be obtained from all participants in the study (see Extended data27). For study participants under 18 years of age, consent from a responsible adult (parent, relative, guardian, legal representative) will be obtained. In addition, assent will be obtained from study participants aged 12 to 17 years. CL represents a major public health, psychosocial and economic problem both in Ethiopia and in the tropical – subtropical regions. A key target of the 2021–2030 WHO Neglected Tropical Diseases roadmap is a significant increase in the proportion of individuals with CL who are diagnosed and receive appropriate treatment. The 2021–25 Ethiopian strategic plan for Neglected Tropical Diseases, aims to meet the 2030 global target by increasing CL treatment centres from 14 to 30, diagnostic centres from 25 to 170 and to be able to detect 85% of cases and treat 95% of these. A better understanding of strategies to improve accessibility and treatment outcomes is a priority action to achieve this goal28. There are currently few prospective data of well-defined treatment outcomes for individuals with CL caused by L. aethiopica, and no data on PROMs or costs. A large cohort of participants with CL will generate robust data on how outcomes are associated with key clinical characteristics such as lesion size, site and number. This will allow us to gain insights into expected clinical outcomes up to one year following treatment and accurately quantify the burden of adverse reactions associated with current treatments. There are new agents and combination therapies proposed for the treatment of leishmaniasis and our data will be valuable in helping design future trials of these approaches in individuals with L. aethiopica. This prospective cohort study will generate the most comprehensive and holistic dataset of CL, combining clinician determined and patient reported outcomes alongside health economic and laboratory data to better capture the impact of CL for affected individuals, their families, and communities. These data will inform the design of randomised trials to evaluate new treatment strategies, help improve evidence-based guidelines and support evidence-led policy decisions. LSHTM Data Compass: Prospective observational cohort study of cutaneous leishmaniasis in Ethiopia - Informed consent form. https://doi.org/10.17037/DATA.0000361827. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). We wish to thank the individuals and communities for their participation in the work of the Skin Health Africa Research Programme. SPONSOR: London School of Hygiene & Tropical Medicine FUNDERS: UK National Institute of Health Research Grant Ref: NIHR200125 Additional members of the SHARP Collaboration SHARP Steering Committee: Yaw Amoako - Kumasi Centre for Collaborative Research Richard Phillips - Kumasi Centre for Collaborative Research Rachel Pullan - LSHTM Dorothy Yeboah-Manu - Noguchi Memorial Institute for Medical Research Named SHARP Applicants: Sinead Langan -LSHTM David Mabey -LSHTM Jennifer Palmer -LSHTM Prof Asrat Hailu- Addis Ababa University SHARP collaboration members from LSHTM: ETHIOPIA study staff involved in study running: Data team: Fikregabrail Aberra Kassa, Samuel Ayele, Mikiyas Gebremichael; Tedros Nigusse Nurses and runners: Almaz Bedye; Fire Nigusse; Atsede Tesfaye; Aberash Alena; Jawar Mohammed; Aziza Temesigen; Zelalem Tefera New medical member Dr Mulualem Degefa Economics team: Debisa Eshatu; Nebiyu Sherefa; Mosisa Bekele Degefa; Derese Bekele Study co-ordination: Aklilu Adane; Eyerusalem Tesfaye; Yematawork Kebede; Sagni Challi; Demeketch Damte Lab staff: Zemed Biyazen; Eyob Fekadu; Befekadu Debebe Social sciences contribution: Abay Woday; Kibur Engedawork Faculty Opinions recommendedReferences - 1. de Vries HJC, Reedijk SH, Schallig HDFH: Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol. 2015; 16(2): 99–109. PubMed Abstract | Publisher Full Text | Free Full Text - 2. Yanik M, Gurel MS, Simsek Z, et al.: The psychological impact of Cutaneous Leishmaniasis. Clin Exp Dermatol. 2004; 29(5): 464–7. PubMed Abstract | Publisher Full Text - 3. Galvão EL, Pedras MJ, Cota GF, et al.: Development and initial validation of a Cutaneous Leishmaniasis impact questionnaire. Verdonck K, editor. PLoS One. 2018; 13(8): e0203378. PubMed Abstract | Publisher Full Text | Free Full Text - 4. Salimi M, Saghafipour A, Parsa HH, et al.: Economic burden evaluation of cutaneous leishmaniasis in Iran. Shiraz E-Medical J. 2019; 20(6): e82810. Publisher Full Text - 5. Wijerathna T, Gunathilaka N, Gunawardena K: The economic impact of cutaneous leishmaniasis in Sri Lanka. Biomed Res Int. 2018; 2018: 3025185. PubMed Abstract | Publisher Full Text | Free Full Text - 6. Walayat Hussain TS, Motley RJ, Wang TS: Cryosurgery. In: Griffiths CEM, Barker J, Bleiker TO, Chalmers R, Creamer D, editors. Rook’s Textbook of Dermatology. John Wiley & Sons, 2016; 2016. - 7. Aronson N, Herwaldt BL, Libman M, et al.: Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH). Am J Trop Med Hyg. 2017; 96(1): 24–45. PubMed Abstract | Publisher Full Text | Free Full Text - 8. De Vries HJC, Schallig HD: Cutaneous leishmaniasis: a 2022 updated narrative review into diagnosis and management developments. Am J Clin Dermatol. 2022; 23(6): 823–840. PubMed Abstract | Publisher Full Text | Free Full Text - 9. van Henten S, Adriaensen W, Fikre H, et al.: Cutaneous leishmaniasis due to Leishmania aethiopica. EClinicalMedicine. 2018; 6: 69–81. PubMed Abstract | Publisher Full Text | Free Full Text - 10. Fikre H, Mohammed R, Atinafu S, et al.: Clinical features and treatment response of Cutaneous Leishmaniasis in North-West Ethiopia. Trop Med Int Health. 2017; 22(10): 1293–1301. PubMed Abstract | Publisher Full Text - 11. van Griensven J, Gadisa E, Aseffa A, et al.: Treatment of cutaneous leishmaniasis caused by Leishmania aethiopica: a systematic review. PLoS Negl Trop Dis. 2016; 10(3): e0004495. PubMed Abstract | Publisher Full Text | Free Full Text - 12. Akuffo H, Dietz M, Teklemariam S, et al.: The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica. Trans R Soc Trop Med Hyg. 1990; 84(4): 532–4. PubMed Abstract | Publisher Full Text - 13. Van Der Meulen J, Mock B, Fekete E, et al.: Limited therapeutic action of rifampicin/isoniazid against Leishmania aethiopica. Lancet. 1981; 2(8239): 197–8. PubMed Abstract | Publisher Full Text - 14. Negera E, Gadisa E, Hussein J, et al.: Treatment response of Cutaneous Leishmaniasis due to Leishmania aethiopica to cryotherapy and generic sodium stibogluconate from patients in Silti, Ethiopia. Trans R Soc Trop Med Hyg. 2012; 106(8): 496–503. PubMed Abstract | Publisher Full Text - 15. Padovese V, Terranova M, Toma L, et al.: Cutaneous and Mucocutaneous Leishmaniasis in Tigray, northern Ethiopia: clinical aspects and therapeutic concerns. Trans R Soc Trop Med Hyg. 2009; 103(7): 707–11. PubMed Abstract | Publisher Full Text - 16. Heras-Mosteiro J, Monge-Maillo B, Pinart M, et al.: Interventions for old world Cutaneous Leishmaniasis. Cochrane Database Syst Rev. John Wiley and Sons Ltd; 2017; 2017(11): CD005067. PubMed Abstract | Publisher Full Text | Free Full Text - 17. Olliaro P, Grogl M, Boni M, et al.: Harmonized clinical trial methodologies for localized Cutaneous Leishmaniasis and potential for extensive network with capacities for clinical evaluation. Satoskar AR, editor. PLoS Negl Trop Dis. 2018; 12(1): e0006141. PubMed Abstract | Publisher Full Text | Free Full Text - 18. Zewdu FT, Tessema AM, Zerga AA, et al.: Effectiveness of intralesional sodium stibogluconate for the treatment of localized Cutaneous Leishmaniasis at Boru Meda general hospital, Amhara, Ethiopia: pragmatic trial. PLoS Negl Trop Dis. 2022; 16(9): e0010578. PubMed Abstract | Publisher Full Text | Free Full Text - 19. van Henten S, Tesfaye AB, Abdela SG, et al.: Miltefosine for the treatment of Cutaneous Leishmaniasis— A pilot study from Ethiopia. PLoS Negl Trop Dis. 2021; 15(5): e0009460. PubMed Abstract | Publisher Full Text | Free Full Text - 20. Reithinger R, Coleman PG: Treating Cutaneous Leishmaniasis patients in Kabul, Afghanistan: cost-effectiveness of an operational program in a complex emergency setting. BMC Infect Dis. 2007; 7: 3. PubMed Abstract | Publisher Full Text | Free Full Text - 21. Stahl HC, Ahmadi F, Nahzat SM, et al.: Health economic evaluation of moist wound care in chronic Cutaneous Leishmaniasis ulcers in Afghanistan. Infect Dis Poverty. 2018; 7(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text - 22. Refai WF, Madarasingha NP, Sumanasena B, et al.: Efficacy, safety and cost-effectiveness of thermotherapy in the treatment of Leishmania donovani - Induced Cutaneous Leishmaniasis: a randomized controlled clinical trial. Am J Trop Med Hyg. 2017; 97(4): 1120–1126. PubMed Abstract | Publisher Full Text | Free Full Text - 23. Malik F, Hanif MM, Mustafa G: Comparing the efficacy of oral chloroquine versus oral tetracycline in the treatment of Cutaneous Leishmaniasis. J Coll Physicians Surg Pak. 2019; 29(5): 403–405. PubMed Abstract | Publisher Full Text - 24. Federal Ministry of Health- Ethiopia: Guideline for the diagnosis, treatment and prevention of leishmaniasis in Ethiopia. Addis Abeba; 2013. Reference Source - 25. Nast A, Griffiths CEM, Hay R, et al.: The 2016 International League of Dermatological Societies’ revised glossary for the description of cutaneous lesions. Br J Dermatol. 2016; 174(6): 1351–8. PubMed Abstract | Publisher Full Text - 26. POSAS - The Patient and Observer Scar Assessment Scale. Reference Source - 27. Lambert S: Prospective observational cohort study of Cutaneous Leishmaniasis in Ethiopia - Informed consent form. [Data Collection]. London School of Hygiene & Tropical Medicine, London, United Kingdom, 2023. http://www.doi.org/10.17037/DATA.00003618 - 28. Ending the neglect to attain the sustainable development goals: a road map for neglected tropical diseases 2021–2030. [cited 2023 Mar 12]. Reference Source Version 2 VERSION 2 PUBLISHED 03 Dec 2024 Revised Version 1 VERSION 1 PUBLISHED 05 Oct 2023

is closed on this version, please comment on the latest version above. - Author Response 26 Oct 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia26 Oct 2024Author ResponseSTUDY PROTOCOL Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review ... Continue reading STUDY PROTOCOL Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Reviewer comment: The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. Author response: Indeed, this has been designed as a prospective observational cohort into which are integrated different methodologies appropriate to the different “areas” under investigation, using qualitative and quantitative tools: - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations Reviewer comment: Introduction is too long if it is supposed to be quantitative research. Author response: We employ a variety of methods, and the length of the introduction reflects the broad scope of this cohort study. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Reviewer comment: Inclusion and exclusion criteria need to be elaborated more. Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - Reviewer comment: In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Explain in detail how sample size was calculated. Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: Give details of software to be used for data analysis, statistical tests and significant values that will be used. Author response: The following have been expanded in each section Clinical: Data will be analyzed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. Reviewer comment: Some of the abbreviations have also not been explained. Author response: Thank you, this has been amended. Reviewer comment: Repetition of content in discussion. Author response: We feel the brief discussion summarizes the important points presented previously. Reviewer comment: 19 of the 28 ref used are more than 5 years old. Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Ref 26, not in Vancouver style. Author response: We have checked referencing style. Thank you. Reviewer comment: No PMID or DOI for ref provided. Author response: Apologies, this was provided but does not seem to have uploaded correctly. Will amend with editorial team

Reviewer comment: Shorten the background/introduction to be more concise for a quantitative study Author response: It is mixed method research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.

Reviewer comment: Expand on the inclusion/exclusion criteria, especially related to prior treatments Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Elaborate on the sample size calculations and precision expected Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: State which statistical tests and analysis software will be used Author response: This has now been under in each section of the Data Analysis paragraph Analysis Plan Reviewer comment: Specify proposed statistical tests and significance levels for objectives Author response: This has now been under in each section of the Data Analysis paragraph

Reviewer comment: Reduce repetition of content covered elsewhere Author response: We feel the brief discussion summarizes the important points presented previously.

Reviewer comment: Update older references, over 5 years old unless seminal works Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Author response: We believe our paper contains detailed methodological description.STUDY PROTOCOLCompeting Interests: No competing interests were disclosed. Close Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Reviewer comment: The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. Author response: Indeed, this has been designed as a prospective observational cohort into which are integrated different methodologies appropriate to the different “areas” under investigation, using qualitative and quantitative tools: - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations Reviewer comment: Introduction is too long if it is supposed to be quantitative research. Author response: We employ a variety of methods, and the length of the introduction reflects the broad scope of this cohort study. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Reviewer comment: Inclusion and exclusion criteria need to be elaborated more. Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - Reviewer comment: In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Explain in detail how sample size was calculated. Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: Give details of software to be used for data analysis, statistical tests and significant values that will be used. Author response: The following have been expanded in each section Clinical: Data will be analyzed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. Reviewer comment: Some of the abbreviations have also not been explained. Author response: Thank you, this has been amended. Reviewer comment: Repetition of content in discussion. Author response: We feel the brief discussion summarizes the important points presented previously. Reviewer comment: 19 of the 28 ref used are more than 5 years old. Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Ref 26, not in Vancouver style. Author response: We have checked referencing style. Thank you. Reviewer comment: No PMID or DOI for ref provided. Author response: Apologies, this was provided but does not seem to have uploaded correctly. Will amend with editorial team

Reviewer comment: Shorten the background/introduction to be more concise for a quantitative study Author response: It is mixed method research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.

Reviewer comment: Expand on the inclusion/exclusion criteria, especially related to prior treatments Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Elaborate on the sample size calculations and precision expected Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: State which statistical tests and analysis software will be used Author response: This has now been under in each section of the Data Analysis paragraph Analysis Plan Reviewer comment: Specify proposed statistical tests and significance levels for objectives Author response: This has now been under in each section of the Data Analysis paragraph

Reviewer comment: Reduce repetition of content covered elsewhere Author response: We feel the brief discussion summarizes the important points presented previously.

Reviewer comment: Update older references, over 5 years old unless seminal works Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Author response: We believe our paper contains detailed methodological description. - Discussion is closed on this version, please comment on the latest version above. Author details Author details 1 ALERT Hospital, Addis Ababa, Ethiopia 2 Department of Dermatovenereology, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia 3 Boru Meda Hospital, Boru Meda, Ethiopia 4 Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia 5 Department of Malaria and Neglected Tropical Disease Research, Armauer Hansen Research Institute, Addis Ababa, Ethiopia 6 Department of Paediatrics and Neonatal Health Nursing, College of Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia 7 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 8 Department of Global Health and Development, London School of Hygiene & Tropical Medicine, London, UK 9 School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia 10 Department of Medical Statistics, Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK 11 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 12 University College London, London, UK 13 Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, London, UK 2 Department of Dermatovenereology, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia 3 Boru Meda Hospital, Boru Meda, Ethiopia 4 Department of Epidemiology and Biostatistics, Institute of Public Health, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia 5 Department of Malaria and Neglected Tropical Disease Research, Armauer Hansen Research Institute, Addis Ababa, Ethiopia 6 Department of Paediatrics and Neonatal Health Nursing, College of Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia 7 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 8 Department of Global Health and Development, London School of Hygiene & Tropical Medicine, London, UK 9 School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia 10 Department of Medical Statistics, Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London, UK 11 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 12 University College London, London, UK 13 Hospital for Tropical Diseases, University College London Hospitals NHS Foundation Trust, London, UK Amel Beshir Mohammed Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Fewzia Shikur Mohammed Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Feleke Tilahun Zewdu Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Shimelis Doni Nigusse Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation Yohannes Hailemichael Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation Teklu Cherkose Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Abebaw Yeshambel Alemu Roles: Investigation, Project Administration, Writing – Original Draft Preparation Roles: Investigation, Project Administration, Writing – Original Draft Preparation Eshetu Molla Roles: Investigation, Writing – Original Draft Preparation Roles: Investigation, Writing – Original Draft Preparation Kidist Bobosha Roles: Conceptualization, Writing – Original Draft Preparation Roles: Conceptualization, Writing – Original Draft Preparation Vanessa Yardley Roles: Conceptualization, Writing – Original Draft Preparation Roles: Conceptualization, Writing – Original Draft Preparation Iris Mosweu Roles: Investigation, Methodology, Writing – Review & Editing Roles: Investigation, Methodology, Writing – Review & Editing Mirgissa Kaba Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Catherine Pitt Roles: Conceptualization, Methodology, Writing – Review & Editing Roles: Conceptualization, Methodology, Writing – Review & Editing Elizabeth Allen Roles: Conceptualization, Formal Analysis, Writing – Review & Editing Roles: Conceptualization, Formal Analysis, Writing – Review & Editing Saba Maria Lambert Roles: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Data Curation, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Michael Marks Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Stephen L. Walker Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Endalamaw Gadisa Roles: Conceptualization, Data Curation, Project Administration, Writing – Review & Editing Roles: Conceptualization, Data Curation, Project Administration, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This project is funded by the National Institute for Health Research (NIHR) under its Research and Innovation for Global Health Transformation (RIGHT) Programme (Grant Reference Number NIHR200125). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) Copyright © Crown copyright, 2024 Mohammed AB et al.. This open access work is licensed under the Open Government Licence v3.0 metrics VIEWS $counts.viewCount downloads Citations CITE how to cite this article Mohammed AB, Mohammed FS, Zewdu FT et al. Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.13432.2) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. track receive updates on this article Track an article to receive email alerts on any updates to this article. Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 03 Dec 2024 Revised Views 0 How to cite this report: Robledo SM. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.15037.r34052) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-34052 https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-34052 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 17 Jan 2025 Approved VIEWS 0 Thank you for addressing all the previous comments comprehensively. After reviewing the protocol, I found the document complete and well-structured. It includes all the essential components required for such a study, and the methodology is clearly outlined. The authors have ... Continue reading I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close Thank you for addressing all the previous comments comprehensively. After reviewing the protocol, I found the document complete and well-structured. It includes all the essential components required for such a study, and the methodology is clearly outlined. The authors have adequately incorporated feedback from earlier reviews, ensuring clarity and rigor throughout. I have no further suggestions for major revisions at this time. However, as a final refinement, I recommend a careful proofreading to ensure consistency in language, grammar, and formatting. This will enhance the overall readability and presentation of the protocol. I have no further suggestions for major revisions at this time. However, as a final refinement, I recommend a careful proofreading to ensure consistency in language, grammar, and formatting. This will enhance the overall readability and presentation of the protocol. - Is the rationale for, and objectives of, the study clearly described? Yes - Is the study design appropriate for the research question? Yes - Are sufficient details of the methods provided to allow replication by others? Yes - Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Clinical trials, drug development CITE HOW TO CITE THIS REPORT Robledo SM. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.15037.r34052) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-34052 https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-34052 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. Views 0 How to cite this report: Ahmed N. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.15037.r33926) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-33926 https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-33926 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 11 Jan 2025 Najia Ahmed, Bahria University of Health Sciences, Karachi, Pakistan; Dermatology, Pakistan Naval Station Shifa Hospital, Karachi, Sindh, Pakistan Approved VIEWS 0 Thanks for re- review. Most of the observations have been explained except why using ... Continue reading I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close Thanks for re- review. Most of the observations have been explained except why using SPSS version 23 when even 30 is available. This version may be approved. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Section of data analysis, specially the tests of significance to be used, should also be assessed by a statistician. CITE HOW TO CITE THIS REPORT Ahmed N. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.15037.r33926) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-33926 https://openresearch.nihr.ac.uk/articles/3-49/v2#referee-response-33926 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. Version 1 VERSION 1 PUBLISHED 05 Oct 2023 Views 0 How to cite this report: Alharazi T. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.14569.r30757) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30757 https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30757 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 23 Nov 2023 Approved with Reservations VIEWS 0

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close - Shorten the background/introduction to be more concise for a quantitative study - Expand on the inclusion/exclusion criteria, especially related to prior treatments

- Shorten the background/introduction to be more concise for a quantitative study - Expand on the inclusion/exclusion criteria, especially related to prior treatments - Specify which cytokines/biomarkers will be analyzed in the immunology studies - Elaborate on the sample size calculations and precision expected - State which statistical tests and analysis software will be used - Specify proposed statistical tests and significance levels for objectives - Reduce repetition of content covered elsewhere - Update older references, over 5 years old unless seminal works - Is the rationale for, and objectives of, the study clearly described? Yes - Is the study design appropriate for the research question? Partly - Are sufficient details of the methods provided to allow replication by others? Partly - Are the datasets clearly presented in a useable and accessible format? Not applicable Competing Interests: No competing interests were disclosed. Reviewer Expertise: My work primarily focuses on the epidemiology and control of malaria and neglected tropical diseases, including schistosomiasis, leishmaniasis, and other protozoan and helminth parasites .Also conducts research on the epidemiology of microorganisms and multidrug-resistant bacteria. CITE HOW TO CITE THIS REPORT Alharazi T. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.14569.r30757) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30757 https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30757 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. - Author Response 12 Dec 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia12 Dec 2024Author ResponseDear Dr Alharazi, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these ... Continue reading Dear Dr Alharazi, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Introduction- Shorten the background/introduction to be more concise for a quantitative study This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Methods- Expand on the inclusion/exclusion criteria, especially related to prior treatments “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” - Specify which cytokines/biomarkers will be analyzed in the immunology studies “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Elaborate on the sample size calculations and precision expected Table 2 details the sample sizes required for different cure rates. - State which statistical tests and analysis software will be used Analysis Plan- Specify proposed statistical tests and significance levels for objectives Discussion- Reduce repetition of content covered elsewhere References- Update older references, over 5 years old unless seminal works - Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Thank you for helping us improve our paperDear Dr Alharazi,Competing Interests: none Close We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Introduction- Shorten the background/introduction to be more concise for a quantitative study This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Methods- Expand on the inclusion/exclusion criteria, especially related to prior treatments “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” - Specify which cytokines/biomarkers will be analyzed in the immunology studies “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Elaborate on the sample size calculations and precision expected Table 2 details the sample sizes required for different cure rates. - State which statistical tests and analysis software will be used Analysis Plan- Specify proposed statistical tests and significance levels for objectives Discussion- Reduce repetition of content covered elsewhere References- Update older references, over 5 years old unless seminal works - Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Thank you for helping us improve our paper COMMENTS ON THIS REPORT - Author Response 12 Dec 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia12 Dec 2024Author ResponseDear Dr Alharazi, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these ... Continue reading Dear Dr Alharazi, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Introduction- Shorten the background/introduction to be more concise for a quantitative study This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Methods- Expand on the inclusion/exclusion criteria, especially related to prior treatments “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” - Specify which cytokines/biomarkers will be analyzed in the immunology studies “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Elaborate on the sample size calculations and precision expected Table 2 details the sample sizes required for different cure rates. - State which statistical tests and analysis software will be used Analysis Plan- Specify proposed statistical tests and significance levels for objectives Discussion- Reduce repetition of content covered elsewhere References- Update older references, over 5 years old unless seminal works - Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Thank you for helping us improve our paperDear Dr Alharazi,Competing Interests: none Close We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Introduction- Shorten the background/introduction to be more concise for a quantitative study This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Methods- Expand on the inclusion/exclusion criteria, especially related to prior treatments “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” - Specify which cytokines/biomarkers will be analyzed in the immunology studies “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Elaborate on the sample size calculations and precision expected Table 2 details the sample sizes required for different cure rates. - State which statistical tests and analysis software will be used Analysis Plan- Specify proposed statistical tests and significance levels for objectives Discussion- Reduce repetition of content covered elsewhere References- Update older references, over 5 years old unless seminal works - Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Thank you for helping us improve our paper Views 0 How to cite this report: Ahmed N. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.14569.r30538) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30538 https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30538 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Reviewer Report 01 Nov 2023 Najia Ahmed, Bahria University of Health Sciences, Karachi, Pakistan; Dermatology, Pakistan Naval Station Shifa Hospital, Karachi, Sindh, Pakistan Approved with Reservations VIEWS 0 - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Introduction is too long - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Introduction is too long if it is supposed to be quantitative research. - Inclusion and exclusion criteria need to be elaborated more. - In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. - Explain in detail how sample size was calculated. - Give details of software to be used for data analysis, statistical tests and significant values that will be used. - Some of the abbreviations have also not been explained. - Repetition of content in discussion. - 19 of the 28 ref used are more than 5 years old. - Ref 26, not in Vancouver style. - No PMID or DOI for ref provided. - Is the rationale for, and objectives of, the study clearly described? Yes - Is the study design appropriate for the research question? Partly - Are sufficient details of the methods provided to allow replication by others? Partly - Are the datasets clearly presented in a useable and accessible format? Not applicable Competing Interests: No competing interests were disclosed. Reviewer Expertise: Section of data analysis, specially the tests of significance to be used, should also be assessed by a statistician. CITE HOW TO CITE THIS REPORT Ahmed N. Reviewer Report For: Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia [version 2; peer review: 2 approved, 1 approved with reservations]. NIHR Open Res 2024, 3:49 (https://doi.org/10.3310/nihropenres.14569.r30538) The direct URL for this report is: https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30538 https://openresearch.nihr.ac.uk/articles/3-49/v1#referee-response-30538 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. - Author Response 03 Dec 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia03 Dec 2024Author ResponseRESPONSE TO COMMENTS: Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. ... Continue reading RESPONSE TO COMMENTS: Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations - Introduction is too long if it is supposed to be quantitative research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.- Inclusion and exclusion criteria need to be elaborated more. Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Explain in detail how sample size was calculated. - Give details of software to be used for data analysis, statistical tests and significant values that will be used. Clinical: Data will be analysed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. - Some of the abbreviations have also not been explained. - Repetition of content in discussion. - 19 of the 28 ref used are more than 5 years old. - Ref 26, not in Vancouver style. We have checked referencing style. Thank you. - No PMID or DOI for ref provided. RESPONSE TO COMMENTS:Competing Interests: none Close Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations - Introduction is too long if it is supposed to be quantitative research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.- Inclusion and exclusion criteria need to be elaborated more. Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Explain in detail how sample size was calculated. - Give details of software to be used for data analysis, statistical tests and significant values that will be used. Clinical: Data will be analysed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. - Some of the abbreviations have also not been explained. - Repetition of content in discussion. - 19 of the 28 ref used are more than 5 years old. - Ref 26, not in Vancouver style. We have checked referencing style. Thank you. - No PMID or DOI for ref provided. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. COMMENTS ON THIS REPORT - Author Response 03 Dec 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia03 Dec 2024Author ResponseRESPONSE TO COMMENTS: Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. ... Continue reading RESPONSE TO COMMENTS: Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations - Introduction is too long if it is supposed to be quantitative research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.- Inclusion and exclusion criteria need to be elaborated more. Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Explain in detail how sample size was calculated. - Give details of software to be used for data analysis, statistical tests and significant values that will be used. Clinical: Data will be analysed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. - Some of the abbreviations have also not been explained. - Repetition of content in discussion. - 19 of the 28 ref used are more than 5 years old. - Ref 26, not in Vancouver style. We have checked referencing style. Thank you. - No PMID or DOI for ref provided. RESPONSE TO COMMENTS:Competing Interests: none Close Dear Dr Ahmed We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations - Introduction is too long if it is supposed to be quantitative research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.- Inclusion and exclusion criteria need to be elaborated more. Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” - Explain in detail how sample size was calculated. - Give details of software to be used for data analysis, statistical tests and significant values that will be used. Clinical: Data will be analysed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. - Some of the abbreviations have also not been explained. - Repetition of content in discussion. - 19 of the 28 ref used are more than 5 years old. - Ref 26, not in Vancouver style. We have checked referencing style. Thank you. - No PMID or DOI for ref provided. - The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. Version 2 VERSION 2 PUBLISHED 03 Dec 2024 Revised Version 1 VERSION 1 PUBLISHED 05 Oct 2023

is closed on this version, please comment on the latest version above. - Author Response 26 Oct 2024Saba Lambert, ALERT Hospital, Addis Ababa, Ethiopia26 Oct 2024Author ResponseSTUDY PROTOCOL Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review ... Continue reading STUDY PROTOCOL Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Reviewer comment: The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. Author response: Indeed, this has been designed as a prospective observational cohort into which are integrated different methodologies appropriate to the different “areas” under investigation, using qualitative and quantitative tools: - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations Reviewer comment: Introduction is too long if it is supposed to be quantitative research. Author response: We employ a variety of methods, and the length of the introduction reflects the broad scope of this cohort study. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Reviewer comment: Inclusion and exclusion criteria need to be elaborated more. Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - Reviewer comment: In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Explain in detail how sample size was calculated. Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: Give details of software to be used for data analysis, statistical tests and significant values that will be used. Author response: The following have been expanded in each section Clinical: Data will be analyzed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. Reviewer comment: Some of the abbreviations have also not been explained. Author response: Thank you, this has been amended. Reviewer comment: Repetition of content in discussion. Author response: We feel the brief discussion summarizes the important points presented previously. Reviewer comment: 19 of the 28 ref used are more than 5 years old. Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Ref 26, not in Vancouver style. Author response: We have checked referencing style. Thank you. Reviewer comment: No PMID or DOI for ref provided. Author response: Apologies, this was provided but does not seem to have uploaded correctly. Will amend with editorial team

Reviewer comment: Shorten the background/introduction to be more concise for a quantitative study Author response: It is mixed method research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.

Reviewer comment: Expand on the inclusion/exclusion criteria, especially related to prior treatments Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Elaborate on the sample size calculations and precision expected Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: State which statistical tests and analysis software will be used Author response: This has now been under in each section of the Data Analysis paragraph Analysis Plan Reviewer comment: Specify proposed statistical tests and significance levels for objectives Author response: This has now been under in each section of the Data Analysis paragraph

Reviewer comment: Reduce repetition of content covered elsewhere Author response: We feel the brief discussion summarizes the important points presented previously.

Reviewer comment: Update older references, over 5 years old unless seminal works Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Author response: We believe our paper contains detailed methodological description.STUDY PROTOCOLCompeting Interests: No competing interests were disclosed. Close Protocol for a prospective observational cohort study of cutaneous leishmaniasis in Ethiopia RESPONSE TO COMMENTS: Dear Dr Ahmed, We are grateful for your careful review of our protocol paper and your comments are important in helping us to improve it. We are addressing these one by one here. Reviewer comment: The study design has been stated as prospective observational cohort but it appears to be a mixed method study as it also involves patient interviews and their experiences. Author response: Indeed, this has been designed as a prospective observational cohort into which are integrated different methodologies appropriate to the different “areas” under investigation, using qualitative and quantitative tools: - Clinical: Observation of clinical features, treatments given, clinical outcomes as well as patient reported outcome measure (quantitative) - Psychosocial: patient’s treatment experience using ethnographic methods; qualitative (IDI) - Immunological investigations on skin and blood samples – quantitative laboratory research - Economic burden of CL: Serial economic evaluations Reviewer comment: Introduction is too long if it is supposed to be quantitative research. Author response: We employ a variety of methods, and the length of the introduction reflects the broad scope of this cohort study. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible. Reviewer comment: Inclusion and exclusion criteria need to be elaborated more. Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study - Reviewer comment: In immunology samples, expressions of innate and adaptive immunity and pro and anti inflammatory cytokines to be measured, have not been specified. Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Explain in detail how sample size was calculated. Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: Give details of software to be used for data analysis, statistical tests and significant values that will be used. Author response: The following have been expanded in each section Clinical: Data will be analyzed in STATA. Descriptive statistics will be used to describe the cohort. Odds ratio and logistic regression models will be used to identify clinical and demographic characteristics associated with healing. PROMS: Validity and responsiveness of each PROM will be analysed using SPSS version 23.0 (IBM Corporation, Armonk, NY, USA). Descriptive statistics will be presented using frequencies, percentages, tables, and graphs. Mean rank sum scores of each of sub-domains will be recorded using Kruskal Wallis rank sum test among as well as the different clinical (LCL, MCL and DCL) types of lesions to check known-group construct validity. Wilcoxon-signed rank test will be used to compare mean difference of scores before initiation of treatment (Day 0) and at Day 90 (date of initial cure assessment). Laboratory: Statistical analyses of laboratory data will be performed using R (version 4.4.1, the R Foundation for Statistical Computing). Nonparametric Mann-Whitney statistics will be used to compare different groups. Differences in cytokine and chemokine concentrations and mRNA expressions among CL types, treatment outcomes, time points, and other variables will be compared by the Kruskal-Wallis and t-tests. Fisher's exact test will be employed to determine the association between HLA types and CL types. Economics: Descriptive statistics: frequencies, percentages, means and medians will be used as appropriate to estimate costs. Further analysis will be conducted to investigate catastrophic out-of-pocket health expenditure and poverty impact using a linear probability model. STATA version 17 will be used for data analysis In-depth interviews: Recordings will be transcribed and translated verbatim, and interviewer’s notes included for each interview. A codebook will be developed, and thematic analysis will be used. MAXQDA plus 2022 will be used to manage the in-depth interview data. Reviewer comment: Some of the abbreviations have also not been explained. Author response: Thank you, this has been amended. Reviewer comment: Repetition of content in discussion. Author response: We feel the brief discussion summarizes the important points presented previously. Reviewer comment: 19 of the 28 ref used are more than 5 years old. Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Ref 26, not in Vancouver style. Author response: We have checked referencing style. Thank you. Reviewer comment: No PMID or DOI for ref provided. Author response: Apologies, this was provided but does not seem to have uploaded correctly. Will amend with editorial team

Reviewer comment: Shorten the background/introduction to be more concise for a quantitative study Author response: It is mixed method research. This introduction reflects what is known about CL in Ethiopia and what data is available so far, bringing to the fore questions that need to be addressed. In view of the complex multi-disciplinary issues being addressed, and to address CL In Ethiopia holistically, we believe the introduction is as concise and inclusive as possible.

Reviewer comment: Expand on the inclusion/exclusion criteria, especially related to prior treatments Author response: Thank you, we have elaborated these criteria as follows: “All adults and children diagnosed with CL and initiated on CL treatment at both study centers are eligible to be recruited in this study. Each participant will be followed for 360 days. Inclusion criteria:- Individuals with parasitologically confirmed CL using microscopy or culture - Individuals receiving treatment at the study centers Exclusion criteria:- Participants who have previously been treated with systemic anti-leishmania therapy - Participants who do not or are unable to give informed consent to participate in the study” Author response: We have amended the text to: “To interrogate the dynamics of immune responses in different clinical phenotypes of CL, blood and skin lesion samples will be collected at three time points. Participants will have blood samples and a skin biopsy (stored in RNAlater™) taken to evaluate subsets of the innate and adaptive immunity; pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-6, IL-27, IL-10, IL-4, IL-13, TGF-β, and hsp18) cytokines and interleukins (IL)-12, IL-2, IL-1, IL-18, IL-15, IL-8, IL-17A, and IL-22) using Luminex multiplex assay. (ii) The change in mRNA (Ribonucleic acid) expression level of Toll-like receptors (TLRs) and pro-inflammatory (IFN-γ, IL-12, IL-1β, IL-17A, and IL-12p70) and anti-inflammatory (IL-5, IL-6, IL-10, IL-4, TGF-β, and hsp18) cytokines will also be assessed before treatment (at day 0), during treatment (day 28) and after completion of treatment (day 90) using quantitative PCR (Real-time Polymerase Chain Reaction). In addition, the levels of chemokines (such as CXCL8, CXCL10, CCL5, CCL4, CCL3, and CCL2) will be determined in supernatants from the whole blood stimulation assays for the Luminex multiplex assay. HLA typing will be performed to assess if there is an association between HLA types (targeting probes for HLA-A, B, C, DR, DQ, and DP loci) and the clinical phenotypes of CL seen in our cohort.” Reviewer comment: Elaborate on the sample size calculations and precision expected Author response: Table 2 details the sample sizes required for different cure rates. Reviewer comment: State which statistical tests and analysis software will be used Author response: This has now been under in each section of the Data Analysis paragraph Analysis Plan Reviewer comment: Specify proposed statistical tests and significance levels for objectives Author response: This has now been under in each section of the Data Analysis paragraph

Reviewer comment: Reduce repetition of content covered elsewhere Author response: We feel the brief discussion summarizes the important points presented previously.

Reviewer comment: Update older references, over 5 years old unless seminal works Author response: CL is a NTD and research in Ethiopia is limited. We believe we have provided a thorough literature review. There are a few more recent articles, but these were not available at the date of our protocol development. These will be reviewed when results are presented. Reviewer comment: Overall, add more methodological specifics in line with quantitative reporting guidelines to facilitate reproducibility and interpretation. Author response: We believe our paper contains detailed methodological description. - Discussion is closed on this version, please comment on the latest version above. Alongside their report, reviewers assign a status to the article: - Approved - Approved with reservations - Not approved | Invited Reviewers | ||| |---|---|---|---| | 1 | 2 | 3 | | | Version 2 (revision) 03 Dec 24 | read | read | | | Version 1 05 Oct 23 | read | read | Sign up for content alerts You are now signed up to receive this alert Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' - Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. - You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. - You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). - You work at the same institute as any of the authors. - You hope/expect to benefit (e.g. favour or employment) as a result of your submission. - You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' - You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. - You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. - You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Sign up for content alerts and receive a weekly or monthly email with all newly published articles Register with NIHR Open Research Already registered? Sign in close Error If you are a previous or current NIHR award holder, sign up for information about developments, publishing and publications from NIHR Open Research. We'll keep you updated on any major new updates to NIHR Open Research Sign In If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password. Email us for further assistance. The email address should be the one you originally registered with F1000. Email address not valid, please try again You registered with F1000 via Google, so we cannot reset your password. To sign in, please click here. If you still need help with your Google account password, please click here. You registered with F1000 via Facebook, so we cannot reset your password. To sign in, please click here. If you still need help with your Facebook account password, please click here. Code not correct, please try again Server error, please try again. If your email address is registered with us, we will email you instructions to reset your password. If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance. Please wait...