The 17 day SSG & PM combination treatment had a good safety profile and was similar in efficacy to the standard 30 day SSG treatment, suggesting suitability for VL treatment in East Africa.

The PM versus SSG comparison enrolled 205 patients per arm with primary efficacy data available for 198 and 200 patients respectively. The SSG & PM versus SSG comparison enrolled 381 and 386 patients per arm respectively, with primary efficacy data available for 359 patients per arm. In Intention-to-Treat complete-case analyses, the efficacy of PM was significantly lower than SSG (84.3% versus 94.1%, difference = 9.7%, 95% confidence interval, CI: 3.6 to 15.7%, p = 0.002). The efficacy of SSG & PM was comparable to SSG (91.4% versus 93.9%, difference = 2.5%, 95% CI: −1.3 to 6.3%, p = 0.198). End of treatment efficacy results were very similar. There were no apparent differences in the safety profile of the three treatment regimens.

A multi-centre randomized-controlled trial (RCT) to compare efficacy and safety of PM (20 mg/kg/day for 21 days) and PM plus sodium stibogluconate (SSG) combination (PM, 15 mg/kg/day and SSG, 20 mg/kg/day for 17 days) with SSG (20 mg/kg/day for 30 days) for treatment of VL in East Africa. Patients aged 4–60 years with parasitologically confirmed VL were enrolled, excluding patients with contraindications. Primary and secondary efficacy outcomes were parasite clearance at 6-months follow-up and end of treatment, respectively. Safety was assessed mainly using adverse event (AE) data.

Alternative treatments for visceral leishmaniasis (VL) are required in East Africa. Paromomycin sulphate (PM) has been shown to be efficacious for VL treatment in India.

Visceral leishmaniasis (VL) is a parasitic disease with about 500,000 new cases each year and is fatal if untreated. The current standard therapy involves long courses, has toxicity and there is evidence of increasing resistance. New and better treatment options are urgently needed. Recently, the antibiotic paromomycin (PM) was tested and registered in India to treat this disease, but the same dose of PM monotherapy evaluated and registered in India was not efficacious in Sudan. This article reports the results of a clinical trial to test the effectiveness of injectable PM either alone (in a higher dose) or in combination with sodium stibogluconate (SSG) against the standard SSG monotherapy treatment in four East African countries—Sudan, Kenya, Ethiopia and Uganda. The study showed that the combination of SSG &PM was as efficacious and safe as the standard SSG treatment, with the advantages of being cheaper and requiring only 17 days rather than 30 days of treatment. In March 2010, a WHO Expert Committee recommended the use of the SSG & PM combination as a first line treatment for VL in East Africa.

Competing interests: The authors have read the journal's policy and have the following conflicts: Manica Balasegaram is employed by DNDi as Head of VL Clinical Program. Sally Ellis is employed by DNDi as Clinical Manager (VL). Robert Kimutai is employed by DNDi as a Clinical Trial Manager. Raymond Omollo works for DNDi. Marius Mueller works for MSF.

Funding: The study was funded by Medecins Sans Frontieres (MSF) and the Drugs for Neglected Diseases initiative (DNDi). DNDi would like to thank the following donors for their support: Department for International Development (DFID), UK; Médecins Sans Frontières/Doctors without Borders, International; Ministry of Foreign and European Affairs (MAEE), France; Region of Tuscany, Italy; République and Canton de Geneva, Switzerland; Medicor Foundation, Liechtenstein; Fondation Pro Victimis, Switzerland; Fondation André & Cyprien, Switzerland; Spanish Agency for International Development Cooperation (AECID), Spain; Swiss Agency for Development and Cooperation (SDC), Switzerland; and private foundations and individual donors. The funders had no role in study design, data collection and analysis, decision to publish, and preparation of the manuscript.

Copyright: © 2012 Musa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The objectives remained the same; to compare the efficacy and safety of PM monotherapy and SSG & PM combination therapy to SSG. The results of this trial are reported here.

The aim was to compare safety and efficacy of PM monotherapy and SSG & PM combination therapy with standard SSG treatment. An interim analysis showed that the PM monotherapy had an efficacy of <50% parasite clearance 6 months after the end of treatment in Sudan [13] . This arm was discontinued while a separate dose-finding trial of alternative PM regimens was conducted in Sudan [14] . The original Phase III trial was then restarted with a higher PM monotherapy dose (20 mg/kg/day for 21 days), while the other two arms remained unchanged.

For registration of PM and evaluation of the SSG & PM combination therapy throughout East Africa, efficacy and safety data were required from a Phase III trial. A multi-centre phase III trial has been conducted in six clinical trials sites in 4 East Africa countries (Ethiopia, Kenya, Sudan and Uganda). The trial started in 2004 with three arms; SSG monotherapy (20 mg/kg/day for 30 days: reference arm), PM monotherapy (15 mg/kg/day for 21 days) and SSG & PM combination (SSG: 20 mg/kg/day, PM: 15 mg/kg/day both given for 17 days).

The efficacy of paromomycin sulphate (PM) monotherapy for the treatment of VL has been demonstrated in India, where it is now registered for the treatment of VL [9] and the safety and efficacy of the combination of SSG and PM has been demonstrated in trials in India and a small Kenyan study [10] , [11] . A large case series of 4,263 VL patients carried out by Médecins sans Frontières – Holland (MSF) in South Sudan showed that treating patients with a combination of SSG & PM for 17 days yielded better results than treating them with SSG alone: the initial cure rate was 97% for SSG & PM for 17 days versus 92% for SSG alone for 30 days [12] .

VL treatment options in East Africa are primarily limited to the antimonial sodium stibogluconate (SSG), which is efficacious, but requires 4 weeks of hospitalization for daily intra-muscular injections and has been associated with serious adverse events such as cardiotoxicity; a concern in areas of HIV co-infection [3] , [4] , [5] . In India, leishmania parasites have developed resistance to SSG, with up to 65% of the population in the hyper endemic region of Bihar being unresponsive [6] , [7] . SSG unresponsiveness is emerging in East Africa and treatment with a combination of SSG & PM may limit the spread of the emerging resistant strains of leishmania parasites [8] .

The parasitic disease visceral leishmaniasis (VL) has an incidence of 500,000 new cases annually occurring primarily in India, Bangladesh, Nepal, Sudan, and Brazil and is fatal if untreated [1] . However, it is also an important disease in several other East African countries, with an incidence rate of 30,000 cases per year and a mortality rate of 4,000 deaths per year [2] , [3] .

Methods

Ethics statement The trial was conducted in accordance with the Declaration of Helsinki (2002 version) relating to the conduct of research on human subjects and followed the International Committee on Harmonization guidelines for the conduct of clinical trials. All trial site personnel received training in Good Clinical Practice (GCP). The relevant ethics committees from each country approved the study and the details are listed in the attached supporting text document. Patients and their legal guardians (if they were minors) provided signed informed consent prior to being randomized to the different treatment arms. GCP-trained monitors recruited from all four participating countries regularly monitored the trial at all sites.

Design An open label, parallel-arm multi-centre individually randomized controlled trial.

Interventions The three arms were SSG monotherapy (20 mg/kg/day for 30 days: reference arm), PM monotherapy (20 mg/kg/day for 21 days) and SSG & PM combination (SSG: 20 mg/kg/day, PM: 15 mg/kg/day both administered for 17 days). Administration of PM (Gland Pharma, India) was intramuscular (IM). SSG (Albert David, India) was administered IM, or intravenous (IV) in Kenya. Patients requiring rescue medication were given liposomal amphotericin B, (manufactured as Ambisome®, Gilead, USA) according to national dosage guidelines of the participating countries. Patients were hospitalized for treatment and weekly monitoring of clinical and biological parameters. Follow-up visits were conducted 3 months and 6 months post end of treatment (Figures 1 and 2).

Outcome Measures The primary efficacy endpoint was definitive cure, defined as parasite clearance from splenic, bone marrow or lymph node tissue aspirates 6 months after the end of treatment. Any patient who died from VL, received rescue medication during the trial, or had parasites detected at the 6-month assessment was considered a treatment failure. The secondary efficacy endpoint was parasite clearance from tissue aspirates at the end of treatment (SSG: day 31, PM: day 22, SSG & PM: day18). Treatment failure at the end of treatment was defined as death or receipt of rescue medication during initial hospitalization or presence of parasites at end of treatment necessitating rescue treatment. The presence of parasites at the end of the treatment, subsequently cleared without need for rescue treatment was considered a treatment success for primary outcome (definitive cure at 6 months follow-up), but a treatment failure for secondary outcome (cure at end of treatment). Slow responders were defined as patients with detectable parasites at end of treatment and parasite clearance at 6 months follow-up, without need for rescue treatment at any time. Parasitology was performed and reported according to an approved World Health Organization (WHO) method [1]. The numbers of parasites in slide fields were counted under oil emersion at 100× magnification and counts recorded.

Other Data Collection Safety was evaluated based on the occurrence of adverse events (AE), laboratory parameters (haematology and biochemistry), electrocardiogram (ECG) readings, and audiometry. AEs were classified according to the Medical Dictionary for Regulatory Activities (MedDRA) version 10 [15]. A treatment emergent AE (TEAE) was defined as an AE with onset between the first day of treatment and 30 days after end of treatment. ECGs were performed at all sites using a portable self-reporting ECG machine (Cardiofax, Model ECG 9620, Nihon Kohden) with patients resting supine on their beds. Trial physicians reviewed tracings and reported any abnormality. Post-kala-azar dermal Leishmaniasis (PKDL) was recorded actively as an adverse event during patient follow-up or reported directly by the patients in between follow-up dates. Audiometric testing was performed at all trial sites except Um el Kher using Voyager 522 Portable Diagnostic Audiometer (Madsen, Taastrup, Denmark). In recruitment period 1, investigators reported audiometric data as normal, clinically insignificant or clinically significant [13]. In period 2, hearing levels were recorded in detail for each ear at six frequencies. The following definitions were used to measure abnormalities; 1) disabling hearing impairment (DHI): an average hearing level, over frequencies 500, 1000, 2000, 4000 Hz, of ≥31 dB in both ears for those <15 years and ≥41 dB for those aged ≥15 years; 2) audiometric shift: a change in hearing level from baseline of ≥25 dB at ≥1 threshold frequency or ≥20 dB at ≥2 adjacent threshold frequencies. All patients were offered counselling and HIV testing in accordance to national guidelines at screening.

Sample Size Determination The trial was designed to have 90% power (β = 0.1) to detect, at the 5% significance level (α = 0.05), an absolute difference in efficacy of 15% between PM and SSG and 10% between SSG & PM and SSG regimens [16]. An 85% efficacy was assumed in the reference arm and adjusting for 10% HIV co-infection and 10% loss to follow-up at 6 months post end of treatment, it was estimated that 404 and 195 patients per arm were required for the respective comparisons. Being HIV-positive was not an exclusion criteria but the original protocol stated that there was to be a sufficient number of patients for a subgroup analysis excluding HIV patients (if deemed necessary).

Randomization As described at the end of the Introduction, recruitment and randomisation was carried out during two periods. In the first period, patients were randomised to SSG or SSG & PM combination arms, as part of a randomisation into three arms. Data from the third arm, a lower dosage regimen of PM found to be ineffective are not included here. In the second period, randomisation continued into one of three arms; SSG, SSG & PM arms as per period 1 and a PM monotherapy arm at a higher dosage regimen than previously (see Introduction and Interventions sections.) In recruitment period 2 (using the higher 20 mg/kg dose of PM), randomization into 3 arms was continued until the desired sample size was reached for the PM versus SSG comparison. Randomization was then continued into one of two arms (SSG or SSG & PM) until reaching the sample size for the SSG versus SSG & PM comparison. Um el Kher site participated in period 1 only and Amudat site in period 2 only (during the two-arm randomization). A computer-generated randomization list was produced with stratification by centre and block sizes of 15 until recruitment in the PM arm was completed, and block sizes of 10 thereafter. Allocation was concealed using opaque, sequentially numbered sealed envelopes. The randomization list and envelopes were prepared and stored securely at the LEAP Data Centre, based at the trial co-ordination centre in Nairobi. Blinding of patients and investigators was not possible due to the different treatment durations and additional placebo injections were considered inappropriate.

Statistical Methods Data were double-entered and validated in Epi-Info. Bespoke query generation programs were developed using Stata software, version 11 [17]. All statistical analyses were performed using Stata. Baseline data were summarized using mean and standard deviation (SD) or proportions where appropriate. Nutritional status was classified as normal, underweight, or severely underweight according to WHO Child Growth Standards in those <19 years and body mass index (BMI) in those ≥20 years [18].