A Study to Assess Immune Response Status in Patients Before and After Treatment for Visceral Leishmaniasis

ImmStat@Cure will be undertaken at 4 treatment centres for leishmaniasis in East Africa; the Institute for Endemic Diseases, University of Khartoum, Khartoum, Sudan, the University of Gondar, Gondar, Ethiopia, Amudat Hospital, Amudat, Uganda and the Kimalel and Chemolingot Health Centres, Baringo County, Kenya.

The primary objective of the study is to assess systemic and skin immune responses and parasite load in patients before, and at end of treatment for visceral leishmaniasis.

Secondary objectives are:

• To identify systemic and local immune correlates of treatment outcome
• To evaluate parasite genotype in relation to immune response characteristics
• To compare immunological and parasitological data across each site and determine possible correlates of progression to PKDL

The outcomes include data on immune cell phenotypes and function in peripheral blood and tissue, as determined using standard methods in immunology (e.g. flow cytometry, transcriptomics and histopathology) and parasite quantitation (e.g. PCR, histopathology). There will be qualitative and quantitative analysis of data across patient cohorts and between patients and controls.

Forty patients diagnosed with VL from each site will be included in the study. Patients must be willing and able to adhere to the study procedures and to give written informed consent. All patients will have been clinically and parasitologically confirmed as having visceral leishmaniasis (VL) and will receive one round of sodium stibgluconate/paramomycin (SSG/PM) treatment as per normal clinical practice and regional guidelines. Thirty healthy endemic controls will also be studied from each country.

For patients, confirmation of VL will be made by visual identification of parasites in stained tissue smears according to recommended diagnostic practice (using bone marrow, spleen or lymph node aspirates as clinically indicated and as per local procedures). Parasites will be isolated from residual tissue aspirate and genotyped. Where tissue aspirate is not a recommended or currently performed diagnostic procedure, parasites will be isolated where possible from blood and diagnosis confirmed by PCR.

The study requires two additional blood draws, one taken before treatment and the second taken at the end of treatment.

The study also requires two 3mm skin biopsies (taken from the back of the neck/shoulder), one taken prior to the beginning of treatment and the second taken at the end of treatment. Biopsies will be taken under local anaesthesia. Patients refusing skin biopsies can still be recruited into the study.

For healthy controls, 23mL of blood will be taken on a single occasion, for diagnostic purposes and to provide blood for immunological analyses.

Inclusion criteria

• Aged 12 to 50 years on the day of diagnosis
• Have confirmed diagnosis of VL and be judged suitable for treatment using a standard regimen of SSG/PM
• Willing and able to give written informed consent
• For adolescents aged 12 to 17 years on the day of screening, written informed consent from a parent must be obtained in addition to assent from the patient
• Without any other significant health problems as determined by medical history, physical examination, results of screening tests and the clinical judgment of a medically qualified Clinical Investigator
• Negative for malaria on blood smear
• Judged, in the opinion of a medically qualified Clinical Investigator, to be able and likely to comply with all study requirements as set out in the protocol

Exclusion criteria

The patient may not enter the study if any of the following apply:

• Has HIV, HBV or HBC
• Has previously had any form of leishmaniasis
• Pregnancy or lactating mothers
• Any confirmed or suspected immunosuppressive or immunodeficient state, including; asplenia; recurrent, severe infections and chronic (more than 14 days) immunosuppressant medication within the past 6 months
• Tuberculosis, leprosy, or severe malnutrition (severe malnutrition in adults defined as a BMI <18.5, and in adolescents (12-17yrs) as a Z score cut-off value of <-2 SD)
• Any other significant disease, disorder or finding, which, in the opinion of a medically qualified Clinical Investigator, may influence the result of the study, or the volunteer's ability to participate in the study
• Unlikely to comply with the study protocol