Community Research and Development Information Service - CORDIS

Final Report Summary - AFRICOLEISH (Care Package for Treatment and Control of Visceral Leishmaniasis in East Africa)

Executive Summary:
Visceral leishmaniasis (VL) is fatal if left untreated in over 95% of cases. It is characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver, and anaemia. The disease affects some of the poorest people on earth with about 200 000 to 400 000 new cases and 20 000 to 30 000 deaths of VL worldwide each year. East Africa is currently the region with the highest VL burden worldwide and the disease affects specifically children (1, 2). New field-adapted VL treatments are badly needed. VL-HIV co-infection has been an additional emerging problem. Treatment efficacy is poor, toxicity is higher and there is a high risk of multiple relapses in this population.
The AfriCoLeish project aimed towards the development and delivery of a package of care for VL patients and co-infected patients with HIV in East Africa through safe and cost-effective treatments, therefore improving current case management and contributing to disease control in the region. It included four clinical studies, which have provided critical information for the development of new improved treatments for primary VL patients and a package of care for HIV-VL co-infected patients - thus contributing to potential new tools for the control of VL ultimately improving public health in East Africa. Also, the Consortium’s research contributed to more attention to the most vulnerable patients in East Africa.
The studies involved in the various work packages successfully demonstrate that carrying out rigorously controlled studies in remote areas with limited resources is feasible, if such projects include adequate capacity building both in health personnel and infrastructure.
In addition to creating impact by conducting the above-mentioned studies, the AfriCoLeish consortium generated impact on disease management and control through briefing health workers in Ethiopia, Uganda, Sudan and Kenya; Information on the disease and treatment to communities living in high endemic areas surrounding the leishmaniasis treatment centres were disseminated, thus improving health seeking behaviour and access to diagnosis and treatment.
This model of development of capacities through the implementation of research activities could very well be an example for others, both in the developed countries and in countries with limited resources, and may strengthen the momentum for subsequent efforts for a comprehensive approach to control.
The experience gained in East Africa will contribute to a deeper understanding of VL control and management, that may also be applicable to other areas. The findings of Work Package 1 paved the way for use of miltefosine in VL paediatric patients (key for future combination studies), whereas the findings of WP2 and WP3 trials provided evidence that will have a direct impact on the management of HIV-VL patients.
The Consortium will continue to disseminate the results, which are expected to influence policy makers´ decision on revising guidelines for VL/HIV management in Ethiopia. By providing evidence based data to policy makers also at European and global level (such as WHO) they are able to make informed choices to what extent results may be extrapolated to other areas.

References: Please refer to endnotes for S&T results section.
Project Context and Objectives:
Visceral leishmaniasis and its burden in East Africa
Visceral leishmaniasis (VL), also known as kala-azar, is fatal if left untreated in over 95% of cases. It is characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver, and anaemia. Untreated, patients become malnourished, immunosuppressed and die within months. It is caused by the parasite Leishmania donovani in Asia and Africa and L.infantum in the Mediterranean region and Latin America, and is transmitted by the sandfly.
The disease affects some of the poorest people on earth, and is associated with malnutrition, a weak immune system, population displacement, poor housing, and lack of financial resources. An estimated 200 000 to 400 000 new cases and 20 000 to 30 000 deaths of VL occur worldwide each year. In 2014, more than 90% of new cases reported to WHO occurred in 6 countries: Brazil, Ethiopia, India, Somalia, South Sudan and Sudan. The disease is reported in rural areas, villages in mountainous regions, and some peri urban areas where Leishmania infantum parasites live mainly on dogs (3). In Europe, cases are found in all European Mediterranean countries such as Spain, France, Portugal or Croatia.
Despite recent scientific breakthroughs and an increased control effort, in March 2010 the WHO stated that “mortality and morbidity from leishmaniasis worldwide show a worrying increasing trend (4).”

East Africa is currently the region with the highest VL burden worldwide with frequent outbreaks in the northern acacia–balanite savanna and the southern savanna and forest areas where sandflies live around termite mounds. Humans are considered the main reservoir of the Leishmania parasites causing VL in East Africa. Reliable statistics do not exist due to poor surveillance systems and frequency of misdiagnosis, but estimates suggest 30 000 new cases per year in Africa, with numbers rising sharply during an epidemic (5). As recently as 2010, South Sudan faced an epidemic which recorded 10 000 cases (6). In East Africa, the disease affects specifically children (i.e.in Southern Sudan 25.9% under 5 years and 58.5% under 15years (7)).

VL-HIV co-infection has been an additional emerging problem and has appeared in one third of the endemic countries. For example, in some areas of Ethiopia, 35% of all leishmaniasis patients are co-infected with HIV. VL-HIV co-infected patients are risky reservoirs of the parasite (8), specifically in East Africa, where the problem is compounded by ineffective treatment, higher mortality and frequent relapses.

The challenging situation of treating VL patients
Existing monotherapies for VL often have significant limitations: They are requiring painful intramuscular injections that generate potential for infections, are toxic, costly and require long treatment, thus affecting patients´ compliance. They often must be dispensed in rural, isolated clinics, where there are limited resources to manage VL patients needing medication with potential toxic effects. Of the four commonly used drugs – sodium stibogluconate (SSG), AmBisome®, miltefosine and paromomycin (PM), only SSG, a one-month painful and toxic treatment, is the only one both effective as monotherapy and widely available in East Africa.
In addition, drug resistance to monotherapies is an increasing problem. For example, SSG is no longer efficacious in most of India (9). Also, the cost of the current VL treatments represents a significant financial burden on patients and the affected families, which may deter them from seeking treatment and contribute to a cycle of poverty (10, 7, 11).

Sodium stibogluconate and paromomycin (SSG&PM) developed by DNDi and its partners is the first combination treatment for VL in East Africa with an efficacy of 91% (12). It is currently recommended as first line therapy. Although an improvement over the previously recommended 30-day SSG monotherapy, it still requires 17 days of two painful injections and hospitalisation throughout treatment. SSG exhibits life-threatening toxicities including; cardiotoxicity, hepatotoxicity and pancreatitis. More than 3 000 VL patients were treated with SSG&PM in a pharmacovigilance program implemented in four countries of East Africa; its efficacy was confirmed and no new safety findings identified. However, the HIV-VL coinfected patients and those with age > 50 years had poorer outcome and a higher mortality rate (13). These patients clearly need a different management and safer treatment.
To sum up, alternative treatments for VL patients that are efficacious, safe, ideally short duration, affordable and suitable for use in remote areas need to be developed. Despite the high disease burden in East Africa, the limited amount of clinical trial data available is a key challenge. Therefore, evidence to deliver an improved package of care is urgently needed.

The challenging situation of treating VL-HIV co-infected patients
While individuals infected with the Leishmania parasite could remain asymptomatic for decades, some may rapidly develop severe VL once their immune system is compromised through HIV; conversely, leishmaniasis accelerates the onset of AIDS in HIV positive individuals and significantly diminishes the positive impact of anti-retroviral (ARV) treatment: multiple VL relapses occur, relapses become more frequent over time until patients become unresponsive to VL treatment (14). Co-infected patients are highly infective to sand flies contributing to the cycle but inversely depending on the CD4 count (15), which is an additional reason to provide them with early treatment (7).

The management of VL-HIV co-infected patients is extremely problematic: treatment failures with currently used monotherapy regimens are common due to drug toxicity and higher relapse rates (5, 14, 16). During treatment, mortality, particularly with SSG, is higher than in non-HIV co-infected patients and may be caused by increased drug toxicity or co-morbidity (17). The latter is important as most HIV infected patients have a CD4 count below 200 cells/mm3 when developing clinical VL. The combination regimen of SSG&PM is also associated with poor efficacy and high mortality in this group, probably associated with the SSG component (13).
Secondary Prophylaxis in VL-HIV co-infected patients
Drugs studied for secondary prophylaxis in Europe include antimonials (sodium stibogluconate and meglumine antimoniate), AmBisome® and pentamidine (18, 19). However, using first line antileishmanial drugs (sodium stibogluconate, liposomal amphotericin B, paromomycin, miltefosine) as secondary prophylaxis bears the risk for developing resistance that can easily be transmitted in anthroponotic transmission regions. In East Africa, antimonials, AmBisome® and miltefosine (MF) are all essential for primary VL treatment, and using this drugs for maintenance therapy or prophylaxis in areas with anthroponotic transmission entails a risk of emergence and transmission of resistant parasites, compromising current first line treatment which is the reason why WHO discourages secondary prophylaxis (4). Pentamidine has therefore been identified as the most suitable candidate for secondary prophylaxis. However, at the start of the project no data was available on the feasibility, safety and effectiveness of a pentamidine secondary prophylaxis for VL patients treated the East-African context.

At the time of starting the project national guidelines in Ethiopia did not have a recommendation for the treatment of VL-HIV co-infected patients, while the guidelines in Sudan recommend SSG or liposomal amphotericin B (AmBisome®) but did not specify dosage; Médecins Sans Frontières (MSF)’s clinical experience showed that AmBisome® 30 mg/kg had cure rates of only 74%, decreasing for each relapse episode.

Concluding, a major need was observed for quality data to ensure improved and effective management of primary VL in HIV co-infected patients in East Africa as well as in secondary prophylaxis.

The Goals and Objectives of the AfriCoLeish Project

Goal: The AfriCoLeish project aimed to carry out studies towards the development and delivery of a package of care for VL patients and co-infected patients with HIV in East Africa through safe and cost-effective treatments, therefore improving current case management and contributing to disease control in the region.
The package of care consists of two parts:
• a possible alternative, safe and effective co-administration of two drugs (see below sub-objective 1) to further shorten treatment duration from the current 17 days for primary VL cases and
• an improved safe and effective treatment for VL in HIV co-infected patients (see sub-objective 2) to prevent relapse and to promote immunological recovery after primary treatment through the use of secondary prophylaxis (sub-objective 3) using drugs with known anti-leishmanial effect, complemented by prompt initiation of antiretroviral treatment (18, 20).

In the original project proposal, it was planned to conduct a large Phase III trial on a short course combination treatment for VL with other currently available drugs, depending on results from the LEAP 0208 phase II clinical trial. LEAP 0208 (21) was trial designed to evaluate two potential short course VL treatments including AmBisome® and miltefosine, as an alternative treatment option to be tested later against the recommended treatment of SSG&PM. In the proposal risk assessment of the original project proposal, the risk of not observing sufficient efficacy during the phase II trial was acknowledged. Indeed, the result of the trial indicated that none of the combinations tested reached the required 90% cure rate at 6-months post treatment to justify moving to a Phase III trial. Therefore, the project was adjusted in June 2014.

The focus was shifted on two studies to assess alternative oral treatments that could potentially be combined in a VL treatment: Fexinidazole, being under late development for Human African Trypanosomiasis (HAT), is a nitroimidazole that can be administered orally. It could have potentially been the most cost effective treatment for VL as it had shown potent activity against L. donovani in vitro and in vivo in a VL mouse model, and studies in healthy volunteers found it to be safe when given as a single or repeated dosing after 14 days. At the time of the project revision it was already in phase II for African Trypanosomiasis. The second treatment selected was again miltefosine: Indeed, after results of the PK/PD analysis it was found that miltefosine data from the LEAP 0208 study indicated that children were under-exposed to miltefosine as compared to adults, which lead to lower efficacy rates in this age group, and indicated that dose adjustment was required.

The second part of the activities related to develop a package of care remained unchanged.


In detail, the objectives of the AfriCoLeish project were outlined as follows:

Original Sub-objective 1: Short Course Combination treatment for Primary VL. To deliver a safe and effective combination of two drugs that will further shorten current treatment periods (to around 11 days).
Adjusted Sub-objective 1: Fexinidazole Phase II Proof of Concept (PoC) study (in adults) and miltefosine allometric dose pharmacokinetics and safety study in paediatric VL patients. To provide evidence that will guide further research on combination of oral drugs for the treatment of VL in East Africa, ideally an innovative treatment of short course therapy which is efficacious, safe, cost-effective and adapted to field conditions to guarantee access to treatment.

Sub-objective 2: Secondary Prophylaxis for VL in HIV co-infected patients. To deliver a preventive intervention for VL in HIV co-infected patients that limits the recurrence of VL through use of secondary prophylaxis (pentamidine 4mg/kg once monthly) that is safe and feasible to implement in the East African context.

Sub-objective 3: Treatment for VL in HIV co-infected patients. To identify and deliver a safe and highly effective treatment for VL in HIV co-infected patients as part of a package of management that will improve long-term survival of these patients.

Sub-objective 4: Capacity building. To ensure relevant staff of the AfriCoLeish project have the knowledge and skills to conduct all aspects of clinical trials, including data management, statistical analysis planning and reporting and ensure that good practice guidelines and ethical criteria are applied coherently across all Consortium activities.

Sub-objective 5: Dissemination of results to support translation into policy and implementation of treatments.

Sub-objective 6: Project management. To ensure successful implementation of the AfriCoLeish project by month 48 by meeting milestones and deliverables.

The AfriCoLeish Consortium: Networking to improve the care of VL patients in East Africa:
In a context of remote settings and dispersed difficult to reach populations in remote areas and poor health care structures, it is challenging to develop high-quality scientific evidence base through clinical trials. Six well-respected AfriCoLeish partners brought together research knowledge and capacity to form a unique Consortium with expertise in both VL and HIV:

The Drugs for Neglected Diseases initiative (DNDi), The Institute of Tropical Medicine, Antwerp (ITM-A), The London School of Hygiene and Tropical Medicine (LSHTM), Tropical Epidemiology Group, Médecins Sans Frontières-Holland (MSF), The IED - Institute of Endemic Diseases, University of Khartoum and The College of Medicine and Health Sciences, University of Gondar Hospital (Gondar).
A key multilateral initiative that the project interacted with is the Leishmaniasis East Africa Platform (LEAP), a regional forum that involves and seeks the input and political support of stakeholders in the region, including national control programmes, Ministries of Health (MoH), and international organisations, to facilitate implementation and scale-up of new treatments and policy change in East Africa. The AfriCoLeish participants are members of the LEAP platform; in turn, the LEAP played a major role in allowing AfriCoLeish project team to maintain and strengthen relationships with national control programmes and key research institutions or members of the research community.

References: Please refer to endnotes for S&T results section.
Project Results:
The project was divided into 6 Work Packages (WP) coinciding with the objectives listed above:
• Original WP1 (work conducted in 2013 and 2014): Short course combination for primary VL
• Adjusted WP1 starting in 2014: Fexinidazole Phase II PoC study and miltefosine allometric dose pharmacokinetics and safety study in paediatric VL patients
• WP2: Prospective study to document outcomes of Secondary Prophylaxis for VL in HIV co-infected patients
• WP3: Improved treatment options for VL in HIV co-infected patients
• WP4: Capacity building
• WP5: Dissemination of results to support translation into policy and implementation of treatments
• WP6: Project management

The main S&T results of the work performed:
Work conducted between January 2013 and June 2014 for the Original WP1 “Short course combination for primary VL” – main results: DNDi, IED, KEMRI, LSHTM conducted a Phase II clinical trial (LEAP 0208) to evaluate two potential short course VL treatments as an alternative treatment option to the recommended treatment of SSG&PM. Unfortunately, none of these treatment regimens showed sufficient efficacy to move to a Phase III trial. When analysing efficacy by age groups, it was clear that children had poorer efficacy as compared to adults, especially for the miltefosine 28-days monotherapy arm (59% vs 86%, p= 0.05) (21). PK data from this trial showed that children were underexposed to miltefosine as compared to adults. This is in line with previous published miltefosine PK data from India and Nepal (22, 23). The data available was analysed to assess if an adjusted regimen of AmBisome®+ combined with miltefosine could be a suitable option for the region.
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The conclusion of this analysis was that the doses of AmBisome® would have to be increased and divided in several administrations to achieve desirable efficacy, and the miltefosine treatment would have to be adjusted to an allometric regimen (24) with duration of at least 14 days. This optimized regimen with increased doses and duration of AmBisome®+miltefosine would not meet the Target Product Profile (25) (TPP) for VL and would not provide the needed cost-effective solution for East Africa.

The final decision for next steps was made after a review with the LEAP Platform partners at the LEAP Principle Investigators meeting held in Nairobi in February 2014. The experts concluded that the dose of AmBisome®+miltefosine needed to reach the efficacy target did not meet the minimal requirements of the VL TPP and would not be feasible in the East African context (not suitable for women of child-bearing age, high cost, need for cold chain, etc.). It was consequently decided not to go ahead with a Phase III trial, but to focus efforts on the development of new combinations of orally administered, effective and safe drugs.


Therefore, the original WP1 was revised and the following activities were planned:
• A Phase II PoC trial to assess safety and efficacy of fexinidazole as an oral treatment for primary VL.
• A miltefosine allometric dose pharmacokinetics and safety study in pediatric VL patients assessing if the allometric dose of miltefosine gives drug exposures required for high efficacy levels in children in East Africa.

Main S&T results from the adjusted WP1 “Fexinidazole Phase II PoC study and miltefosine allometric dose pharmacokinetics and safety study in paediatric VL patients”:

Fexinidazole PoC study on primary VL in Sudan
This open label, single arm, Phase II, proof of concept (PoC) clinical trial was to assess the efficacy and safety of fexinidazole in adult patients (> 15 years) with primary VL conducted in Gedaref state, Sudan. Recruitment took place at a single site: The Professor EL-Hassan treatment centre in Dooka. A total of 164 subjects were screened. A total of 14 subjects (8.54%) who met the inclusion/exclusion criteria were enrolled and assigned to fexinidazole treatment. Since VL is a disease affecting primarily children in East Africa, the main reason for exclusion was age, followed by no confirmatory VL diagnosis, abnormal laboratory values and severe malnutrition.

Results
The VL patients treated with fexinidazole have tolerated well the medication, all patients completed the treatment and no Serious Adverse Events (SAEs) have been reported. All patients presented improvement in VL clinical signs, symptoms and lab parameters, and 80% had negative microscopy at the end of treatment. However, the majority of patients presented relapse of the disease 2-4 months after the treatment and had to receive rescue treatment. Due to the higher than expected rate of relapse, the study recruitment was put on hold and the available data for the 1st interim analysis was analysed and presented to the Data and Safety Monitoring Board (DSMB).

The DSMB reviewed study results and recommended that the study should not resume in its current form. Efficacy, safety, Pharmacokinetics (PK) and Pharmacodynamics (PD) data obtained in the clinical trial, together with safety data available from previous fexinidazole studies on other indications were then analyzed and guided the rationale for the next steps of this project.

Conclusions from this trial:
This first Phase II PoC study has shown evidence of some activity of fexinidazole against VL, with clear clinical improvement during treatment, majority of patients presenting negative microscopy at the end of treatment and a reduction of parasitemia (by qPCR). However, the 10-days regimen was not sufficient to sustain the response, and a large proportion of patients relapsed during the 6-months follow-up period. Survival analysis performed with secondary pharmacokinetic parameters using a Cox proportional hazard model, showed that overall exposure of the sulfoxide (M1) and sulfone (M2) metabolites (AUC0-264h, M1+M2) and time above the threshold of 99% effective concentration (EC99) for sulfone metabolite (Time>EC99, M2), among other related variables, had a significant effect on survival and onset of rescue treatment time. This was an indication of an exposure-response relationship between fexinidazole metabolites and recurrence of disease. Therefore, increased exposure to M2 (either by increasing the daily dose or treatment duration) should improve treatment efficacy.
Fexinidazole was not foreseen to be used as monotherapy, but in combination with another oral treatment. The currently available oral treatment for VL is miltefosine, which has been extensively used in Indian sub-continent either as monotherapy or more recently in combination with paromomycin. In order to develop an efficacious oral combination treatment for VL, the fexinidazole regimen would need to be increased, whereas miltefosine will have to be adjusted to allometric dose.

S&T results of the work conducted in regards to the miltefosine PK Study:
In a region where patients have to travel long distances to access treatment, miltefosine, the only currently available oral drug for VL, is a component offering significant advantages for improved treatment acceptance. Although miltefosine is potentially teratogenic, requiring contraceptive coverage in women of child-bearing age, a combination treatment with miltefosine will particularly benefit children (being the biggest patient group, 60-70% of the total) as it would avoid painful injections and long hospitalisations. Pharmacokinetic (PK) results from the previous LEAP 0208 study had indicated that children treated with miltefosine were underexposed to the drug as compared to adults, and this reflected in poorer clinical outcomes. It was then decided to carry out a study to assess the PK, safety and efficacy of miltefosine allometric dose in children with primary VL in Kenya and Uganda. Since most patients in Eastern Africa are children under the age of 5, this pivotal study sought to further optimize miltefosine treatment in children with VL. The hypothesis was that by adjusting the dose based on an allometric scale, children would reach similar exposure as compared to adults, and therefore, higher efficacy of the treatment would be achieved. Results from this study were expected to guide how to best adjust miltefosine dose, as well as on the duration of treatment when used in future combination of oral drugs.

This was a multicentre, non-comparative, Phase II, open-label proof of concept study whose primary objective was to characterize the drug exposure and PK properties as well as safety of miltefosine using allometric dosing in children with primary VL in eastern Africa.

Results
It was shown in this trial that miltefosine remains an attractive option for combination with other drugs. A total of 30 primary VL patients aged 4 to 12 years were included in the trial and treated with miltefosone allometric dose for 28 days. Final efficacy assessment was done at the 6 months follow-up visit.The results showed that efficacy level was increased in children treated with the allometric dose as compared to the conventional dose, reaching similar efficacy observed in adults in the LEAP0208 trial. In general, miltefosine was well tolerated with no major safety concerns identified; compliance in the hospital setting was 100%.

The overall miltefosine exposure was higher compared to conventional dosing in the same paediatric age group. Miltefosine seemed to accumulate faster in this trial (LEAP0714) with the allometric dosing in the first week of treatment, resulting in a higher drug exposure during the first half of the treatment than for the previous trial (LEAP0208), although this difference was not significant due to high variability. While total miltefosine exposure was thus increased, children treated with miltefosine allometric dosing did not achieve the expected exposure i.e. similar to adults treated with conventional therapy of 2.5 mg/kg/d for 28 days.

Conclusion
Based on the results of this trial, Miltefosine is an oral drug that remains an attractive option for combination with other drugs, and for children (< 30Kg), the allometric dosing is a better regimen, both in terms of drug exposure and therapeutic outcome, while maintaining a good safety profile. Results from this study will guide how to best adjust miltefosine dose in VL patients (especially children), as well as duration of treatment when used in future combination of oral drugs.


Main S&T results of WP2 “Prospective study to document outcomes of Secondary Prophylaxis for VL in HIV co-infected patients”
In northwest Ethiopia, the HIV co-infection rate reaches 20 to 30% of VL patients, with up to 56% relapse in a year in patients on ART but without secondary prophylaxis (29). Until cellular immunity returns with ART, the patient is at risk of VL relapses, which can result in death, severe illness, reduced ART efficacy, drug-resistance and possibly transmission of drug-resistant Leishmania donovani. Patients most vulnerable to relapses are those with high levels of immunosuppression, with previous VL episodes, or with opportunistic infections (OIs). The most important factor to prevent relapses seems to be the clearance of visible parasites and effective ART. This open label, single arm study assessed the effectiveness, safety and feasibility of monthly pentamidine (PM) infusion to prevent recurrence of VL in HIV co-infected patients. A placebo group was not included, due to the clear advantages of the intervention to the patient population (30).

This was an open label, single arm trial designed to investigate the effectiveness, safety and feasibility of monthly pentamidine prophylaxis to prevent VL relapse in patients with HIV. The study had three phases, an initial 12 months of monthly pentamidine (main study period), six months extended treatment period (with monthly pentamidine) for those who remained with CD4 count less than 200 cells/μl at the end of 12months follow-up, and a subsequent 12months follow-up after the prophylaxis to assess long term outcomes. All patients received antiretroviral therapy (ART). Time-to-relapse or death was the primary end point.

Results
A total of 74 subjects were included in the study, of whom 41 completed the regimen taking at least 11 of the 12 doses. The probability of relapse-free survival at 6 months and at 12 months was 79% and 71% respectively.
The results of the trial found that pentamidine secondary prophylaxis led to a 29% failure rate within one year, much lower than reported in historical controls (50%-100%). Importantly, the intervention was found to be safe and feasible to organize, including in remote settings. Patients with low CD4 counts, however, remained at increased risk of relapse despite effective initial VL treatment, ART and secondary prophylaxis.

Conclusion
It was found out that longer VL relapse free survival was achieved using pentamidine as secondary prophylaxis in addition to antiretroviral therapy for VL in people with HIV infection. However, patients with profound immune deficiency (CD4 count less than 200/µL) were still at risk of relapse. Thus, there is a need to investigate additional treatment options for this group of patients. Early VL case detection is crucial for effective management and prevention of relapses. This is the first adequately powered trial that has addressed the use of secondary prophylaxis for prevention of VL relapse in HIV co-infected patients.


Main S&T results of WP3: “Improved treatment options for VL in HIV co-infected patients” HIV and VL mutually influence each other as they both affect cellular immunity. The most important features of co-infection include poor outcome, increased drug toxicity and relapse of treatment with the need for maintenance therapy.
Not only can HIV cause a rapid development of severe VL in patients who may have been asymptomatic carriers for many years; but conversely leishmaniasis accelerates the onset of AIDS in HIV positive individuals and significantly diminishes the positive impact of antiretroviral treatment. As a result, multiple VL relapses occur and can become more frequent over time until patients become unresponsive to treatment.
Few studies exist in regards to co-infected patients. Experience from MSF in Ethiopia (25) treating HIV-VL patients with Ambisome 30mg/Kg showed an overall initial cure rate of 60%, with higher risk of failure for patients who were relapse cases as compared to primary VL episode (38% vs 74% initial cure rate). The latest national guidelines in Ethiopia (June 2013) recommend AmBisome® 40mg/kg total dose for VL-HIV co-infected patients as a first-line treatment, following recommendations by the WHO 2010 Expert Committee. The second-line treatment remains pentavalent antimonials, to be administered under careful monitoring of adverse events.

This work package comprised a Phase III multi-centre randomised, non-comparative trial (LEAP 0511) to evaluate the recommended regimen of 40mg/kg dose of AmBisome® (in divided doses of 5mg/kg given at day 1-5, 10, 17, 24) and a combination of AmBisome® (30mg/kg given in divided doses of 5mg/kg at day 1, 3, 5, 7, 9, 11) and miltefosine (100mg/day for 28 days) in VL-HIV co-infected patients.Primary efficacy endpoint was assessed by a test of cure (parasitology) and clinical improvement at the end of first round of treatment (Day 29). Patients who had a positive parasitology at Day 29 had a 2nd cycle of treatment (same regimen as before), and outcome assessed at Day 58. Patients continued a 12-month follow-up period to assess risk of relapse. During follow-up, patients who presented CD4 count less than 200/µL were eligible to start pentamidine prophylaxis treatment. All patients received ART during entire study.
The study design was based on an adaptive triangular design with sequential interim analysis after every 10 patients/arm completed the Day 29 assessment.

Results
The number of patients required for the first interim analysis (20 patients) was reached mid-December 2014. Following the first interim analysis, a stopping rule was met for the AmBisome® monotherapy arm due to lower than expected efficacy, and as per protocol and DSMB recommendation, recruitment was stopped for this study arm.

The second interim analysis included data available from 20 patients in the AmBisome® monotherapy arm and 28 patients in the AmBisome®+miltefosine arm, who had completed Day 29 assessment. The second DSMB meeting was held on 7 July 2015, during which the stopping rule met for the AmBisome® monotherapy arm was confirmed. In this second analysis, the lower boundary of the triangular test was also crossed for the AmBisome®+miltefosine arm, indicating that the stopping rule was met for lower than expected efficacy. Therefore, the recruitment was interrupted.
However, DSMB acknowledged an apparent better efficacy of the combination regimen compared to the AmBisome® monotherapy regimen, although the study was not planned to be comparative and was not powered for this comparison.

A secondary analysis was performed to evaluate the efficacy of the extended treatment strategy (test of cure at Day 58). Success at D58 was defined as test of cure (parasitology) negative at D29 and symptom free at D58; or test of cure (parasitology) negative in tissue aspirate and symptom free at D58 if patient got the extended treatment. The proportion of success at D58 included success following initial and extended treatment. The efficacy of AmBisome® monotherapy arm was below the threshold of 75% and there was not a significant gain in efficacy after the 2nd cycle of treatment; whereas in the combination arm, the efficacy was significantly increased after the 2nd cycle, with cure rate at Day 58 above the 90% threshold.

Conclusion
Although the trial design did not plan for statistical comparison of efficacy between arms, the combination arm seems to be more promising. The extended treatment strategy based on individual patient response provides an interesting guidance to define the best duration of treatment for each individual affected by HIV/VL co-infection.

The final clinical trial report and publication of results are expected in Q2 2017.

After discussion with WHO and investigators, and based in the encouraging results found in a limited number of patients, it was concluded that more patients should be treated with the same combination regimen to confirm the findings and strengthen the evidence. Results of the LEAP 0511 study will be presented to the Ethics and regulatory authorities in Ethiopia, aiming to facilitate the implementation of a cohort observational study where HIV-VL patients will be treated with Ambisome and miltefosine combination, using the same strategy designed for the LEAP 0511 study. The evidence obtained from these studies is expected to guide policy makers (in Ethiopia and international levels) for a revision on the HIV-VL treatment recommendations.

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(8) Molina R, Gradoni L, Alvar J. (2003) HIV and the transmission of Leishmania. Ann Trop Med Parasitol. Oct;97 Suppl 1:29-45. doi: 10.1179/000349803225002516
(9) Lira R, Sundar S, Makharia A, et al. (1999) Evidence that the High Incidence of Treatment Failures in Indian Kala-azar is due to the Emergence of Antimony-resistant Strains of Leishmania donovani. J. Infect. Dis.; 180(2): 564–567. doi: 10.1086/314896
(10) Boelaert M, Meheus F, Sanchez A, et al. (2009) The poorest of the poor: a poverty appraisal of households affected by visceral leishmaniasis in Bihar, India in Trop Med Int Health 14 :639-644 (June). doi:10.1111/j.1365-3156.2009.02279.x
(11) Boelaert M, Meheus F, Robays J and Lutumba P. (2010). Socio-economic aspects of neglected diseases: Sleeping sickness and visceral leishmaniasis. Pathogens and Global Health, October; doi: 10.1179/136485910X12786389891641
(12) Musa A, Khalil E, Hailu A, et al. (2012) Sodium stibogluconate (SSG) & paromomycin combination compared to SSG for visceral leishmaniasis in East Africa: a randomised controlled trial. PLoS Negl Trop Dis. ;6(6):e1674. doi:10.1371/journal.pntd.0001674.
(13) Kimutai R, Musa AM, Njoroge S, et al. (2017). Safety and Effectiveness of Sodium Stibogluconate and Paromomycin Combination for the Treatment of Visceral Leishmaniasis in Eastern Africa: Results from a Pharmacovigilance Programme. Clinical Drug Investigation. ;37(3):259-272. doi:10.1007/s40261-016-0481-0
(14) Cota GF, de Sousa MR, Rabello A. (2011) Predictors of Visceral Leishmaniasis Relapse in HIV-Infected Patients: A Systematic Review. PLoS Negl Trop Dis 5(6): e1153. doi:10.1371/journal.pntd.0001153
(15) The CD4 count literally counts the number of CD4 T-cells in a blood sample and tracks whether a person's immune strength is going up or going down, with higher values indicating a stronger, more robust response. Along with the HIV viral load, a CD4 count is considered an invaluable diagnostic tool in the care and treatment of people living with HIV. (Source: https://www.verywell.com/what-is-a-cd4-count-and-why-is-it-important-49548).
(16) Ter Horst R, Collin SM, Ritmeijer K, et al (2008) Concordant HIV Infection and Visceral Leishmaniasis in Ethiopia: The Influence of Antiretroviral Treatment and Other Factors on Outcome. Clin Infect Dis; 46: 1702.
(17) Ritmeijer K, Dejenie A, Assefa Y, et al. (2006) A Comparison of Miltefosine and Sodium Stibogluconate for Treatment of Visceral Leishmaniasis in an Ethiopian Population with High Prevalence of HIV Infection. Clin Infect Dis; 43: 357-364.
(18) Alvar J, Aparicio P, Aseffa A, et al. (2008) The Relationship between Leishmaniasis and AIDS: the Second 10 Years in Clinical Microbiology Reviews; 21: 334-359. doi: 10.1128/CMR.00061-07
(19) Perez-Molina JA, Lopez-Velez R, Montilla P, and Guerrero A. (1996) Pentamidine isethionate as secondary prophylaxis against visceral leishmaniasis in HIV-positive patients in AIDS 10: 237–238.
(20) Patel TA, Lockwood DN. (2009) Pentamidine as secondary prophylaxis for visceral leishmaniasis in the immunocompromised host: report of four cases. Trop Med Int Health; 14: 1064-1070.
(21) Wasunna M, Njenga S, Balasegaram M, et al. (2016) Efficacy and Safety of AmBisome in Combination with Sodium Stibogluconate or Miltefosine and Miltefosine Monotherapy for African Visceral Leishmaniasis: Phase II Randomized Trial. PLoS Negl Trop Dis 10(9): e0004880. doi:10.1371/journal.pntd.0004880
(22) Dorlo, T, Rijal, S, Ostyn, B., et al. (2014) Failure of miltefosine in visceral leishmaniasis is associated with low drug exposure. Journal of Infectious Diseases, 210(1), 146–153. https://doi.org/10.1093/infdis/jiu039
(23) Ostyn, B, Hasker, E, Dorlo, T, et al. (2014) Failure of miltefosine treatment for visceral leishmaniasis in children and men in South-East Asia. PLoS ONE, 9(6). https://doi.org/10.1371/journal.pone.0100220
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(25) As a prerequisite to building the strategy, the target product (treatment) profile (TPP) has been established. It is based on discussions with various VL experts, consultation with VL national control programmes in endemic countries, and specifically with leading physicians and health workers who deal with this disease on a daily basis. The priority is to develop a safe, effective, oral, short-course (11 days maximum) VL drug to replace current treatments. This will improve and simplify current case management. The aim is to develop combinations of drugs that are effective against VL in all foci of the disease.
(26) Diro E, Ritmeijer K, Boelaert M, et al. (2015) Use of Pentamidine As Secondary Prophylaxis to Prevent Visceral Leishmaniasis Relapse in HIV Infected Patients, the First Twelve Months of a Prospective Cohort Study. PLoS Negl Trop Dis 9(10): e0004087. doi:10.1371/journal.pntd.0004087
(27) For further information, refer to https://www.clinicaltrials.gov/ct2/show/NCT01360762?term=HIV+AND+VL
(28) Ritmeijer K, Ter Horst R, Chane S, et al. (2011) Limited Effectiveness of High-Dose Liposomal Amphotericin B (AmBisome) for Treatment of Visceral Leishmaniasis in an Ethiopian Population with High HIV Prevalence. Clin Infect Dis; 53 (12): e152-e158. doi: 10.1093/cid/cir674
(29) SDG 3: “Ensure healthy lives and contribute to well-being for all at all stages”
(30) “Guideline for diagnosis, treatment, prevention of Leishmaniasis in Ethiopia, 2nd edition, June 2013, page 30-31.
(31) See also www.africoleish.org
(32) Adhikari, S. R., Maskay, N. M. & Sharma, B. P. (2009). Paying for hospital-based care of kala-azar in Nepal: assessing catastrophic, impoverishment and economic consequences. Health Policy and Planning, 24, 129–139.
(33) Bolo S. Omae H, Wasunna M (2012) The Economic Impact of Visceral Leishmaniasis in Baringo, Kenya. https://su-plus.strathmore.edu/handle/11071/2023
(34) Kasili S, Okindo ES, Kutima HL, Mutai JM (2016). Socioeconomic Impacts of Leishmaniases on Households of Marigat Sub County, Baringo County of Kenya. J Trop Dis 4: 226. doi:10.4172/2329-891X.1000226

Potential Impact:
(for references, please refer to endnotes for S&T results section.)

IMPACT of the AfriCoLeish PROJECT

The AfriCoLeish project aimed to contribute to VL control in East Africa by introducing innovative new drug regimens, and providing evidence for the development of new treatments based on combination of oral drugs. The trials conducted in WP2 and WP3 constituted an important evidence base to move towards a standard package of care for VL-HIV co-infected patients including initial VL treatment and secondary prophylaxis to prevent or delay VL relapse.
The project brought together European institutions and partners from disease-endemic countries in East Africa ensuring that their distinct expertise and skills multiplied in a collective effort to combine fragmented knowledge and address knowledge gaps in the management of VL in East Africa. Local partners, IED and Gondar University, together with KEMRI, LEAP and other institutions including Addis Ababa University, played a key role in liaising with the relevant government departments in order to facilitate dissemination of clinical trial results.
The evidence generated is expected to provide important tools to support efforts to improve treating patients with VL-HIV co-infection, to provide data to update public health policies and treatment guidelines in the region and to inform scientists and medical staff in Europe. In addition, the evidence generated on VL treatments will be useful to guide new treatments for primary VL in East Africa.

1. Public health impact in East Africa
Leishmaniasis affects the poorest of the poor in East Africa and though patient numbers are high, the disease and its patients fail to receive adequate attention through the public health system. This is a typical constraint when talking about neglected diseases and may also be due to lack of adapted, efficacious and affordable diagnostics and medicines (see below in socio-economic impact section) as patients and families turn away from seeking potentially expensive or pain-causing treatment. It is obvious, that conducting research activities increases attention to the disease in the health community and an improved package of care leads to better public health. It is also a contribution to the Sustainable Development Goals (26) given the high patient numbers (specifically in children) and the heavy disease burden of VL in East Africa. Appropriate treatment regimens for co-infections, appropriate dosing of treatments, and effective field-adapted treatments reduce the disease burden, increase adherence to treatments and therefore prevent ultimately disabilities and death.

Thus, improved treatment outcomes for children and HIV co-infected patients but also better knowledge about the disease in the field through capacity building has been the desired long-term impact of the AfriCoLeish project. Well informed and trained health personnel help to better manage patients and thus improve control of the disease. This was achieved with measures in work package 4 and 5. Increased knowledge of the disease, improved case management and better compliance to existing treatments delay also the emergence of resistance to available treatments which impacts disease control.

In regards to adapting of VL treatment to the paediatric population it was important to have learnt through the studies how to treat children with miltefosine using allometric dosing, as this allows the only oral treatment currently available is still to be considered as an option for future combinations with new chemical entities.
In addition, to have learnt how to treat HIV-VL patients, and how to prolong the time free of relapse by using pentamidine secondary prophylaxis, gives patients new hope improving not only their life standard but also preventing transmission. The implications are beyond Africa and can be extrapolated to other regions.

As outlined in the summary on scientific results, the AfriCoLeish project included four clinical studies, which have provided critical information for the development of a package of care for VL patients and HIV-VL co-infected patients thus contributing in several ways to prevention and control of VL and therefore to improve public health in East Africa:

1.1 Improved treatment: impact for control and patient management
In the 2012 proposal and in the 2014 amendment, the AfriCoLeish consortium outlined indicators for assessing the impact of the project in terms of improved treatment. They are:
a. By 2016 endemic countries in East Africa (Ethiopia and Sudan) will have improved tools for the treatment and control of VL available and fundamental work for a new oral combination treatment for VL will have been laid out through the Proof of Concept trial.
b. Clinical trials in WP1 will pave the way for innovative treatments based on combination of oral drugs that will be adapted to be used in the settings where VL occurs in East Africa. Ultimately these new treatments should be efficacious, safe, affordable and adapted to the field – offering the potential of home administration of treatment, high cure and survival rates and low relapse rates.
c. Patients with VL-HIV co-infection will benefit from the implementation of safe and effective treatments leading to improved survival in East Africa (target of initial cure rates of 90%, improvement on the current cure rates of 70%).
d. Prolonged life of current treatments due to reduced risk of resistance.

Below, an assessment of the project impact against these indicators is provided.

a. By 2016 endemic countries in East Africa (Ethiopia and Sudan) will have improved tools for the treatment and control of VL available and fundamental work for a new oral combination treatment for VL will have been laid out through the PoC trial.

The original trial planned for WP1 was a Phase III multi-centre non-inferiority randomised controlled trial to evaluate short course combinations involving AmBisome® and miltefosine in comparison to the standard treatment (SSG&PM). However, results of the Phase II proof-of-concept trial (LEAP 0208) has shown that the efficacy of the Ambisome® and miltefosine combination from the study would be inferior to the existing WHO recommended treatment (did not reach the expected target of 90% efficacy).

Conclusion
While LEAP 0208 did not provide a better combination treatment, an extensive analysis of miltefosine conventional versus allometric dosing to identify the optimal dose of miltefosine for children resulting in appropriate drug exposure was performed. Simulations were done to assess what would be the expected exposure for miltefosine dosing of 10, 14 & 28 days, based on demographic data obtained from 974 individuals enrolled in previous DNDi trials in East Africa. Also, efficacy and pharmacodynamics results from the LEAP 0106 trial, assessing multiple and single dose AmBisome® regimens, were used to determine the optimal dosing regimen for liposomal amphotericin B (LAmB). The conclusion was that to reach the target efficacy of 90%, the doses of AmBisome® and miltefosine would have to be increased. Miltefosine would have to be used based in allometric dosing for a minimum of 14 days. The AmBisome® regimen would require a higher total dose and split into 4 infusions to achieve faster and more extensive parasite clearance, as assessed by kinetoplast-DNA PCR analysis of patient blood samples. The experts concluded that the increased dose regimen of AmBisome®+miltefosine did not meet the minimal requirements of the VL Target Product Profile and would not be feasible in the East African context (especially the high cost, need for cold chain, etc.).

It was consequently decided not to go ahead with a Phase III trial, but to use this knowledge and focus efforts on the development of new combinations of orally administered, effective and safe drugs.


a. Clinical trials in WP1 will pave the way for innovative treatments based on combination of oral drugs that will be adapted to be used in the settings where VL occurs in East Africa.

To be prepared for future studies on drug combinations, it was decided to evaluate two oral treatments: fexinidazole in a Phase II PoC trial, and to identify the optimal dose of miltefosine for children resulting in appropriate drug exposure.

Information generated by the fexinidazole Phase II PoC trial (WP1):
All patients showed clinical improvement during treatment and the majority had parasite clearance (negative test of cure by microscopy) at the end of treatment. Three patients remained cured until 6-months follow-up, however the response was not sustained in other patients and relapses were observed. The study was interrupted in 2014 as it failed to show conclusive efficacy in the majority of patients.

Conclusion
Efforts were made to assess if a combination of fexinidazole with miltefosine would be an appropriate option for VL treatment. However, this would require an initial drug-drug-interaction study in healthy volunteers before proceeding to VL patients. Due to long timelines for this development and a narrow therapeutic index, it was decided to terminate the development of fexinidazole for VL and focus on the development of new chemical entities, which are innovative promising alternatives and well advanced in pre-clinical development.

Information generated by of the miltefosine PK and safety trial (WP1) that paves the way for better treatment of VL patients:
These results showed that efficacy level was increased in children treated with the allometric dose as compared to the conventional dose, reaching similar efficacy observed in adults in the LEAP0208 trial (i.e. 86.2%). In general, miltefosine was well tolerated with no major safety concerns identified; Compliance in the hospital setting was 100%. The overall miltefosine exposure was higher compared to conventional dosing in the same paediatric age group. Still, children treated with miltefosine allometric dosing did not achieve the expected exposure i.e. similar to adults treated with conventional therapy of 2.5 mg/kg/d for 28 days.

Conclusion:
Based on the results of this trial, miltefosine is an oral drug that remains an attractive option for combination with other drugs, and for children (< 30Kg), the allometric dosing is a better regimen, both in terms of drug exposure and therapeutic outcome, while maintaining a good safety profile. Results from this study will guide how to best adjust miltefosine dose in VL patients in Eastern Africa (especially children), as well as duration of treatment when used in future combination of oral drugs. In fact, the DSMB recommended that the allometric dosing should be considered for any future study involving paediatric patients in Eastern Africa. This is going to be employed in the approved Phase III trial to assess miltefosine/paromomycin combination.

b. Patients with VL-HIV co-infection will benefit from the implementation of safe and effective treatments leading to improved survival in East Africa (target of initial cure rates of 90%, improvement on the current cure rates of 70%.

The AfriCoLeish Consortium conducted two clinical trials to improve treatments for patients co-infected with VL-HIV, in WP2 and WP3.

The information generated and impact on treating patients generated by the VL-HIV pentamidine secondary prophylaxis study (WP2) is as follows:
It was found out that longer VL relapse free survival was achieved (29% as compared to 50-100% in historical controls) using pentamidine as secondary prophylaxis in addition to antiretroviral therapy in people with HIV infection. However, as previously described, it was also found that patients with low CD4+cell counts remained at increased risk of relapse despite effective initial VL treatment, anti-retroviral treatment and secondary prophylaxis.

Conclusion:
It can be concluded that pentamidine prophylaxis associated with anti-retroviral therapy is efficacious in preventing the occurrence of relapse in VL-HIV co-infected patients. In addition, VL should be detected and treated early enough in patients with HIV infection before profound immune deficiency installs.
This is the first adequately powered trial that has addressed the use of secondary prophylaxis for prevention of VL relapse in HIV co-infected patients.

The information and impact generated by treating patients on the VL-HIV treatment study to find improved treatment options (WP3):
This work package evaluated AmBisome® monotherapy and a combination of AmBisome® and miltefosine in VL-HIV co-infected patients in a Phase III trial (LEAP 0511).
During the 1st interim analysis the stopping rule for the AmBisome® monotherapy arm was met; and in a second analysis, the lower boundary was also crossed for the AmBisome®+miltefosine arm, indicating stopping rule was met, for lower than expected efficacy at end of first round of treatment (Day 29).
However, an apparent better efficacy of the combination regimen compared to the AmBisome® monotherapy regimen was acknowledged, even though the study was not planned to be comparative.

A secondary analysis was performed to evaluate the efficacy of the extended treatment strategy. This strategy was defined in the protocol as allowing the patients with clinical improvement but positive parasitology at D29 to undergo a second round of the same treatment, i.e. doubling the dose and duration of treatment for patients showing partial response to the initial treatment.

Conclusion:
Although the trial design did not plan for statistical comparison of efficacy between arms, the combination arm is the more promising treatment option, as it reached the target of > 90% cure rate at the end of the 2nd cycle of treatment. The extended treatment strategy based on individual patient response provides an interesting guidance to define the best duration of treatment for each individual affected by HIV/VL co-infection.
The immunology assessments are still ongoing at the time of writing this report. However, the assessment of pharmacokinetics of AmBisome® and miltefosine, as well as antiretroviral drugs, showed a lower exposure to miltefosine in this population compared to historical reference in non HIV-coinfected population.
The final clinical trial report and publication of results are expected in Q2 2017. Two abstracts were submitted and accepted to the Worldleish 6 conference in Toledo (May 2017), one on the clinical results and one on the pharmacokinetics of AmBisome®, miltefosine and antiretroviral drugs in this population.
In regards to the impact on the treatment of patients and control of disease, the discussions with WHO experts have already started. Current guidelines for treatment were based on a limited number of European patients treated and with low level of evidence.
Specifically, data from this trial are responding to doubts stated in the treatment guidelines of Ethiopia (27) on the appropriate combination regimen to be recommended for relapse cases occurring with HIV-VL patients because of lack of clinical trial data. Therefore, the data generated will be presented to Ethics Committee and regulatory authorities in Ethiopia to promote the implementation of a cohort/compassionate study where a larger number of patients will be treated with the AmBisome®/miltefosine combination, using the same strategy of 1 or 2 cycles, depending on the individual response at the end of treatment. ART and pentamidine prophylaxis would also be part of the patient management care. The objective is to build more solid data and to generate additional evidence that is expected to support new recommendations for HIV/VL co-infection for the management of VL treatment (primary VL or relapses) as well as for secondary prophylaxis to avoid occurrence of relapses.
The results of the LEAP 0511 trial and additional data to be gathered from a cohort observational study are likely impacting the policy at world and country level. DNDi is thus planning to organize a stakeholder meeting including WHO representatives in the region, to provide evidence to support policy change for the HIV/VL co-infected patients based on patient-adapted combined therapy of AmBisome® and miltefosine, and post-treatment prophylaxis with pentamidine to reduce the rate of occurrence of relapses.

c. Prolonged life of current treatments due to reduced risk of resistance.

The higher efficacy achieved with the use of miltefosine allometric regimen in children (the great majority of VL patients in East Africa) will decrease the risk of relapses and therefore prevent the selection of resistant clones that in the case of anthroponotic transmission has a huge importance, opposite of that occurring in zoonotic VL where the dog is the main reservoir implying a diluting factor in the resistance transmission to humans. This applies to both VL in immunocompetent and immunosuppressed patients.
The strategy of combining drugs has shown satisfactory results in VL-HIV co-infected patients, and this should also contribute to the reduced risk of long-term resistance.


To sum up, the project was partially readjusted according to data and results from previous studies saving resources and time. However, the study results from the fexinidazole and mitelfosine studies were very important to guide the next steps in research for finding better treatments for VL patiewnts. The adjustment of the original proposal responded also to the still unmet medical public health gap-filling strategy. Certainly, the research conducted through the AfriCoLeish consortium contributed to more attention to the most vulnerable patients in East Africa, improved knowledge about the use of current treatments and generated solid data that paved the way for future studies for better treatment and increased disease control of VL and HIV/VL co-infected patients.

In addition to creating impact by conducting the above-mentioned studies, the AfriCoLeish consortium generated impact on disease management and control through briefing health workers in Ethiopia, Sudan and Kenya on the diagnosis and disease management, current treatment guidelines, trial results and potential new treatment options. This was done in close collaboration with Médecins sans Frontières (MSF) but also respective Ministries of Health, IED, University of Gondar and KEMRI. MSF outreach teams organized VL health promotion activities in communities in 24 target sites. As a direct result, awareness among the target population was increased which was indicated by the shorter duration between onset of symptoms and reporting in a treatment centre and increased referral numbers.

Also, research capacity was greatly improved through extensive trainings and workshops on Good Clinical Laboratory Practices (GCLP) and Good Clinical Practices (GCP).
In VL clinical trials, monitoring of laboratory parameters (chemistry and haematology) are key for the safety assessment, whereas parasitology by microscopy or molecular biology techniques remains the key efficacy parameter to assess cure. Therefore, good laboratory practice is essential for the implementation of any VL clinical trial. AfriCoLeish worked to strengthen and harmonize the GCLP component of the laboratories across all LEAP sites with the support of an expert and during two workshops followed by on the ground assessments and corrective plan of actions.
Overall, there was a significant improvement on the general organization of the labs in regards to personnel management (organigram, definition of tasks and responsibilities), equipment maintenance, files, records, in-house trainings on equipment and Standard Operating Procedures (SOPs), supply management and an improvement of the lab’s safety. SOPs were developed and are now up to date, quality systems were improved and an External Quality Assessment was implemented at all labs.
Also, all clinical trials implemented under AfriCoLeish have followed international standards of ICH GCP and local regulations. Before initiation of any clinical activity, the study teams at the sites were trained on GCP, the study protocol, study specific procedures, etc. to ensure conduct of trial in compliance with protocol and ICH GCP; to ensure protection of rights and safety of participants; and to confirm that the reported data are accurate, complete and verifiable against source documents.
Clinical teams from participating institutions have provided appropriate training throughout the implementation of the clinical trials.
Finally, in order to enhance knowledge and improve patient management with this neglected disease, online clinical case studies were developed by ITM-A and the University of Gondar to train physicians in disease-endemic countries and internationally. The cases address a wide range of scenarios in the management of the disease. They can be used by different health worker groups and students (i.e. from ITM-A) and are made easily and freely available at the website of AfriCoLeish. Also, a CD-Rom is produced that will be distributed to the national program, to regional health bureaus, medical colleges in VL endemic countries (focus in East Africa) in 2017.
2. Impact on policy

The objective of the AfriCoLeish consortium was to generate quality data resulting from the clinical trials and to disseminate them widely in order to help implement new treatments in East Africa but also in other affected regions and to contribute to a policy change in regards to treating VL and VL co-infected patients.

Various dissemination measures were conducted to support the intended policy change: (Interim-) results were and will be published in peer-reviewed and widely accessible journals such as PLOS NTDs and presented during international scientific conferences to enhance and share knowledge about VL among the scientific community in North and South.
Information on the project and trial results was also disseminated through other media channels such as East African press (28) (i.e. The East African, The Monitor).
AfriCoLeish consortium members and Leishmaniasis East Africa Platform (LEAP) members briefed decision-makers in Africa on the trial designs, the results and potential new treatment modalities to facilitate recommendation and implementation of new treatments. LEAP played a key role in coordinating a regional clinical research network. Currently composed of academic and government research institutions from the region, Ministries of Health, and non-governmental organizations from four countries, LEAP supports policy changes such as the revision of the Ethiopian treatment guidelines for VL.
The Consortium has also started to share results on HIV-VL management with WHO that hopefully will result in new guidelines not only for East Africa but also beyond.
The largest opportunities for LEAP and Consortium members to brief decision-makers in East Africa occurred during the annual LEAP meetings, held in 2014 (LEAP conference in Ethiopia with 133 participants from 14 countries), 2015 (in Sudan with 68 participants) and 2016 (in Kenya with 87 participants from 10 countries). It gave the AfriCoLeish Consortium the opportunity to sensitise others in the region, particularly the MOHs, to the programme and its progress to facilitate any future implementation activities.
In May 2017, the results of the AfricCoLeish projects will be presented during the Worldleish 6 congress.

Summary of the impact created on policy: The AfriCoLeish findings from WP2 & WP3 are expected to influence an informed decision making by national control programs and to improve current treatment guidelines such as the Ethiopian guidelines of treating VL-HIV co-infection. Findings of the work conducted by AfriCoLeish will be discussed during the next East African stakeholders meeting organized by WHO in 2017. If validated in a larger cohort observational study, results will be of enormous value to provide solid evidence for recommendations by the next WHO Expert meeting that may take place in 2019.
This will be facilitated through the fact that the Project Advisory Committee of the AfriCoLeish consortium includes representatives of WHO regional offices and MoHs.
For East Africa specifically, the findings have been communicated regularly within LEAP and LEAP meetings (attended by policy makers) and to the scientific community at multiple occasions via scientific conferences. Consortium members have also organized symposia on VL/HIV coinfection (ECTMIH, Worldleish 5) which also covered AfriCoLeish studies, and a special session on VL/HIV coinfection (covering also AfriCoLeish findings) is foreseen for Worldleish 6 congress in May 2017. Consortium members also organized a special issue on VL/HIV coinfection (PLOS NTD). The pentamidinetrial has received a prestigious price from the Ethiopian authorities, which was widely covered by media in Ethiopia, providing additional communication on the study.

3. Socio-economic impact on patients

It was anticipated by the Consortium that shorter and cost-effective treatments will have a direct impact on the socio-economic situation of VL patients and that of their immediate families by reducing the financial burden of the treatment.

As mentioned above, leishmaniasis affects some of the poorest people on earth in countries with limited resources and weak health systems. Every member of a household where a VL case occurs may lose many days of productive life. This loss, when added to the costs of treatment, may push an already poor house-hold further into poverty (32), reduce food security and keep children from school. Also, high expected costs associated with a patient infected by VL may deter the search for treatment (13). For example, a study measuring the economic impact of VL on households in Baringo (Kenya) indicated that a single VL treatment episode costs about triple the average household monthly income which is beyond the reach of majority poor households. It was shown that they were forced to employ a combination of coping strategies including asset sales, reliance on community support, spending family savings or borrowing. In addition, indirect costs may be much higher than direct costs especially as the patient is incapacitated and is not able to work again. It was concluded that the economic burden of caring for VL and the subsequent stripping and compromise of coping mechanisms can institute a vicious cycle of poverty in a household and may undermine sustainable development of endemic communities (33). Another study proved similar affects indicating that “the cost of treating a patient with VL was way above the monthly expenditure of residents resulting in sinking the affected families in poverty. (...) And “the mean number of economic days lost was 178 days (34).”

Consequently, direct and indirect costs of treatments are a key factor when developing a new treatment. The AfriCoLeish project sought to deliver a package of care that provides cost-effectiveness at various levels:

Health systems and health providers would potentially benefit from reduced treatment costs through fewer treatment days, a decreased hospital occupancy, potential home administration of treatments, high cure and survival rates, and low relapse rates. It was expected to further reduce treatment duration – depending on the results of trials, it was hoped that the duration could potentially be 10 days for fexinidazole and 14 days in a combination with miltefosine. The fact that this combination didn’t progress further aborted the possibility of shortening the treatment duration, time of hospitalization and reduction of costs. However, the progress made in learning how to use miltefosine in children, always in combination with a second drug, is an important achievement that sooner or later will benefit the health systems at all levels.

In the future, a combination of drugs against VL which remains the long-term objective of DNDi beyond the AfriColeish consortium, will potentially reduce the costs of a treatment. Preliminary data from a DNDi/ITM-A cost-effectiveness analysis completed in Sudan suggests that combination treatments are more cost-effective than monotherapies. The current preferential price for the miltefosine kit containing 56 capsules (28-days treatment) is €54. Considering that the duration of miltefosine treatment when used in combination should be around 14 days, it is likely that total amount of miltefosine required for a combination treatment will be half of the miltefosine kit.

In parallel, a shorter treatment duration would reduce also indirect costs (i.e. working days in the field due to a family member being hospitalized) for VL patients and their families; VL-HIV co-infected patients would see a reduction in the cost associated with repeated treatments/relapses and an improvement in overall health and thus and improved ability to work and care for themselves and their families.


4. Organisational impact

Within the AfriCoLeish project, European and African partners have strengthened links and contacts in numerous meetings and trainings between state-run hospitals, universities, African researchers in the field and an NGO, and between European academic institutions, the LEAP platform members, and WHO field offices, thus reinforcing North-South collaboration.

Next to many daily contacts made, an improvement of work and management of clinical trials was discussed and implemented during meetings along conferences, trainings, seminars. For example, as part of the trainings of medical assistants in Sudan (WP5), the attendees were enrolled in a referral network. A network for medical consultation was formed between the participants of the training and the medical team at EL-Hassan Center for Tropical Medicine (Dooka/Sudan). This network has since helped provide medical advice on various tropical diseases.

Second, the AfriCoLeish Consortium met annually over the course of the grant, in Nairobi (2013), Geneva (2014), and Antwerp (2015). Additionally, the consortium took advantage of their attendance at LEAP meetings to meet, in October 2014, October 2015, and October 2016. In addition, the AfriCoLeish Project Advisory Committee (PAC) met on the annual LEAP meeting in Sudan (2015) and in Kenya (2016).

Additionally, the AfriCoLeish consortium supported two important structures that were critical to the undertaking of the AfriCoLeish clinical trials: LEAP and the DNDi LEAP Data Centre.

Leishmaniasis East Africa Platform (LEAP)
In 2016, the LEAP partners launched LEAP 2.0, a restructuring of the platform to enable it to adjust to expanding clinical trial needs in the region. During the period 2003-2016, LEAP made impressive progress in fulfilling its mandate of facilitating clinical testing and registration of new treatments for VL in the region. Despite process improvements, LEAP nevertheless continues to face certain limitations, for example: slow recruitment for specific patient population (ex. adult VL patients), slow transition from clinical trial results to patient access, and some problems within the governance structure. Some of these limitations also have been highlighted in the discussion of clinical trial delays and limiting factors in WP1-3. LEAP 2.0 will focus on sustained advocacy and communication, proactive engagement with stakeholders, and a focus on building stakeholder capacity to ensure access.

DNDi LEAP Data Centre
Data management (DM) is a critical activity in clinical trials. For DNDi clinical trials (WP1 and WP3), data management for the trials was undertaken by the DNDi LEAP Data Centre based at DNDi’s Regional Office in Nairobi. The DNDi LEAP Data Centre recognise that continuous improvement in data management is a prerequisite for high quality data and efficient delivery of clinical trial results.
Thus, the Data Management team participated in a number of trainings and workshops that were conducted in collaboration with LSHTM or at the Association of data management in the Tropics (ADMIT) at ITM-Antwerp gathering DM colleagues from ITM’s partner institutions from the North and South. Since creation, ADMIT has been organizing data management workshops, every two years, in order to bring on board data managers to share their experience and learn from one another.
Over the course of the grant the above mentioned trainings and workshops resulted in improved time to database lock (3 months compared to 6 months before) and improved processes for generating queries for statisticians.
The future objective is to improve local capacity in data management for clinical trials to such an extent that the Centre is engaged by external partners, thus optimizing its operations, and serving as a local centre of excellence for data management.


5. Impact on African research capacity

One of the proposed impacts of the AfriCoLeish project was to strengthen the capacity of African partners through access to networks, strengthened relationships with European researchers, improved project management and reporting skills, and training of young researchers and health personnel. The European members of the Consortium saw it as part of their mission to transfer their experience and expertise, especially in relation to new technology and knowledge gained in other regions (e.g. VL in Asia) to African research colleagues and partner organisations and invested time and resources as needed.

In the study sites in Sudan, Kenya and Ethiopia, young researchers have pursued PhD or MSc study related to the AfriCoLeish project through academic institutions involved in the Consortium. Two PhDs and two Master thesis programmes were supported.
Dr. Ermias Diro’s research was supported through 2010-2014. Dr. Diro successfully defended his thesis in May 2015. The title of his PhD research was: “Approach for Better Visceral Leishmaniasis Control in HIV Patients: Secondary Prophylaxis and HAART Optimization”. The host institution for his PhD was ITM, and his degree was awarded by the University of Antwerp.
The second PhD, based at the University of Gondar, submitted his proposed research, with the title, “Identification of risk factors and prediction of adverse treatment outcomes in visceral leishmaniasis in Ethiopia”, which was approved by the ITM PhD committee and the University of Antwerp. His progress report has been approved in December 2016 and at the time of writing this final report, he was about to submit the thesis.
In addition, in 2014 the AfriCoLeish team proposed in their amended proposal that VL research could be carried out by more mature and experienced Master´s students. For this reason, the Consortium supported two Masters students in their thesis projects. MSc Student 1 has proposed to study factors associated with nutritional status among children with Visceral Leishmaniasis at Kacheliba Subcounty Hospital, West Pokot County, Kenya (WP1). MSc Student 2 has proposed the evaluation of a latex agglutination test (KATEX) in VL-HIV co-infected patients for diagnosis, monitoring or treatment response and prediction of relapse (WP2).

The AfriCoLeish consortium activities within WP4 fostered exchange between institutions and the training and learning of individual researchers in each organisation by conducting two GCLP workshops (in Ethiopia and Kenya) for laboratory heads and technicians from all LEAP sites in 4 African countries and one GCP training in Kenya focusing on Clinical Monitoring. This training was organized for Clinical Monitors to refresh their knowledge and skills to better apply the concepts and principles of GCP and international ethical standards in their routine work. Also, the team of the DNDi LEAP Data Centre participated in numerous trainings and shared knowledge with other professionals and experts. Trained and skilled data managers are a key success factor for conducting research in remote settings and create quality data that can be disseminated to create impact. The impact of these trainings can be measured by a shorter turnaround time in the clinical data cleaning process to lock the clinical trial database and come up with results (example being the LEAP 0714 Miltefosine PK study, 3 months compared to 6 months before).

The studies involved in WP1, WP2 and WP3 successfully demonstrate that carrying out these rigorously controlled studies in remote areas with limited resources is feasible, if such projects include adequate capacity building both in health personnel and infrastructure. Specifically, the immunological data collected within WP3 provides a unique dataset that will be used within the scientific capacity building collaboration between ITM-A and the University of Gondar.

This model of development of capacities through the implementation of research activities could very well be an example for others, both in the developed countries and in countries with limited resources, and may strengthen the momentum for subsequent efforts for a comprehensive approach to control and disease elimination.

The evidence base gathered also strengthens the position of the European academic participants (ITM-A and LSHTM) as leading research institutions in field contexts and contributed to academic material (i.e. online tutorials with case studies developed in WP5) for their students, both European and third country nationals. European Partners also benefit through access to field-specific knowledge being facilitated by African research partners. African research partners have been key in facilitating access to stakeholders, particularly at government and community level.

6. Impact on the wider scientific and public health community, including in Europe

The experience gained in East Africa will contribute to a deeper understanding of VL-HIV control and management and may also be applicable to other areas. By providing evidence based data to policy makers they are able to make informed choices to what extent results may be extrapolated to other areas. As mentioned in the section on impact on policy, the findings are likely to be discussed during the next regional WHO meeting planned for 2017 and eventually in the next WHO Expert Committee at the end of 2019 if organized by WHO. In addition, findings from WP2 & WP3 are very likely to influence the guidelines for VL/HIV management in Ethiopia.


Main dissemination activities and exploitation of results
As mentioned previously, the consortium has disseminated interim and final project results to improve treatment options for patients with VL and to contribute to an improvement of the control of VL in the region and beyond.

The dissemination of results focused on several target audiences in the South and in the North:
• Scientific communities through peer-reviewed journals, and conferences;
• Decision-makers and public authorities in East Africa through conferences and meetings to change the guidelines;
• Members of the public, especially in Africa through media work;
• Health workers at the trial sites in Ethiopia, Kenya, and Sudan through trainings;
• Physicians in disease-endemic countries and internationally through interactive online clinical case studies.

Summary (please also refer to Table A2 for further details):

Two press releases were published.

Three scientific articles were published:
• Innovative approaches to clinical data management in resource limited settings using open-source technologies by Omollo R, Ochieng M, Mutinda B, Omollo T, Owiti R, Okeyo S, Wasunna M, Edwards T. PLOS NTDs 2014, doi:10.1371/journal.pntd.0003134.
• Use of pentamidine as secondary prophylaxis to prevent visceral leishmaniasis relapse in HIV-infected patients, the first twelve months of a prospective cohort study by Diro E, Ritmeijer K, Boelaert M, Alves F, Mohammed R, Abongomera C, Ravinetto R, De Crop M, Fikre H, Adera C, Colebunders R, van Loen H, Menten J, Lynen L, Hailu A, van Griensven J. PLoS Neglected Tropical Diseases 2015, doi:10.1371/journal.pntd.0004087.
• The Leishmaniasis East Africa Platform (LEAP): strengthening clinical trial capacity in resource-limited countries to deliver new treatments for visceral leishmaniasis by Wasunna M, Musa, A, Hailu A, Khalil EAG, Olobo J, Juma R, Wells S, Alvar J, Balasegaram M. Transactions of the Royal Society of Tropical Medicine and Hygiene 2016, doi:10.1093/trstmh/trw031.

Two more scientific articles are in preparation at the time of submitting this report
• Pharmacokinetics, safety and efficacy of miltefosine allometric regimen for the treatment of primary visceral leishmaniasis in Eastern African children: open-label, phase II clinical trial
• Efficacy and safety of AmBisome® monotherapy and AmBisome® plus Miltefosine for patients coinfected with HIV and Visceral Leishmaniasis in Ethiopia

18 oral presentations and 2 posters and 1 exhibition were developed for scientific events and conferences. In addition, four conferences were organized.

Case studies for further training on VL diagnostic and its management: http://www.africoleish.org/casestudies.html

For a list of the 5 articles in the (African) media and 4 TV clips, please see www.africoleish.org

A video was produced: AfriCoLeish: Collaborating for VL and HIV/VL in East Africa Interview with Dr Ermias Diro: https://youtu.be/fX_CWfi86E0?list=PLAZhFyV67xKxDggGGyJw3sl-5PgMpbGYf

List of Websites:
www.africoleish.org

Related information

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DRUGS FOR NEGLECTED DISEASES INITIATIVE FONDATION*DNDI
Switzerland
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