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Pntd 0002613
  Safety and Efficacy of Single Dose versus Multiple Dosesof AmBisome H  for Treatment of Visceral Leishmaniasis inEastern Africa: A Randomised Trial Eltahir A. G. Khalil 1 , Teklu Weldegebreal 2 , Brima M. Younis 1 , Raymond Omollo 3 , Ahmed M. Musa 1 ,Workagegnehu Hailu 4 , Abuzaid A. Abuzaid 1 , Thomas P. C. Dorlo 5,6 , Zewdu Hurissa 4 , Sisay Yifru 4 ,William Haleke 2 , Peter G. Smith 7 , Sally Ellis 8 , Manica Balasegaram 8 , Ahmed M. EL-Hassan 1 ,Gerard J. Schoone 9 , Monique Wasunna 3,10 , Robert Kimutai 3,10 , Tansy Edwards 7 , Asrat Hailu 11 * 1 Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan,  2 Arba Minch Hospital, Regional Health Bureau of SNNP State, Arba Minch, Ethiopia, 3 Drugs forNeglected Diseases initiative (DND i  ) Africa Regional Office, Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya,  4 University of Gondar, College of Medicine & Health Sciences, Gondar, Ethiopia,  5 Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands,  6 Slotervaart Hospital/The NetherlandsCancer Institute, Amsterdam, The Netherlands,  7 MRC Tropical Epidemiology Group, London School of Hygiene and Tropical Medicine, London, United Kingdom,  8 Drugsfor Neglected Diseases initiative (DND i  ), Geneva, Switzerland,  9 Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands,  10 Centre for ClinicalResearch, Kenya Medical Research Institute, Nairobi, Kenya,  11 School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia Abstract Background:   Anti-leishmanial drug regimens that include a single dose AmBisome H  could be suitable for eastern Africanpatients with symptomatic visceral leishmaniasis (VL) but the appropriate single dose is unknown. Methodology:   A multi-centre, open-label, non-inferiority, randomized controlled trial with an adaptive design, wasconducted to compare the efficacy and safety of a single dose and multiple doses of AmBisome H  for the treatment of VL ineastern Africa. The primary efficacy endpoint was definitive cure (DC) at 6 months. Symptomatic patients withparasitologically-confirmed, non-severe VL, received a single dose of AmBisome H  7.5 mg/kg body weight or multiple doses,7 times 3 mg/kg on days 1–5, 14, and 21. If interim analyses, evaluated 30 days after the start of treatment following 40 or80 patients, showed the single dose gave significantly poorer parasite clearance than multiple doses at the 5% significancelevel, the single dose was increased by 2 ? 5 mg/kg. In a sub-set of patients, parasite clearance was measured by quantitativereverse transcriptase (qRT) PCR. Principal Findings:   The trial was terminated after the third interim analysis because of low efficacy of both regimens. Basedon the intention-to-treat population, DC was 85% (95%CI 73–93%), 40% (95%CI 19–64%), and 58% (95%CI 41–73%) inpatients treated with multiple doses (n=63), and single doses of 7 ? 5 (n=21) or 10 mg/kg (n=40), respectively. qRT-PCRsuggested superior parasite clearance with multiple doses as early as day 3. Safety data accorded with the drug label. Conclusions:   The tested AmBisome H  regimens would not be suitable for VL treatment across eastern Africa. An optimalsingle dose regimen was not identified. Trials Registration: NCT00832208 Citation:  Khalil EAG, Weldegebreal T, Younis BM, Omollo R, Musa AM, et al. (2014) Safety and Efficacy of Single Dose versus Multiple Doses of AmBisome H  forTreatment of Visceral Leishmaniasis in Eastern Africa: A Randomised Trial. PLoS Negl Trop Dis 8(1): e2613. doi:10.1371/journal.pntd.0002613 Editor:  Elodie Ghedin, University of Pittsburgh, United States of America Received  April 14, 2013;  Accepted  November 18, 2013;  Published  January 16, 2014 Copyright:    2014 Khalil et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  DNDi, the sponsor of the study, was involved in the final stages of study protocol development and data analysis; and also supported the preparationof the draft manuscript by a medical writer. The decision to publish was made by investigators as well as the sponsors. DNDi acquired funds for the conduct of thetrial from: Department for International Development (DFID), UK; Me´decins Sans Frontie`res/Doctors without Borders; International; Medicor Foundation,Liechtenstein; Ministry of Foreign and European Affairs (MAEE), France; Spanish Agency for International Development Cooperation (AECID), Spain; Swiss Agencyfor Development and Cooperation (SDC), Switzerland; private foundations and individual donors. Competing Interests:  The authors have declared that no competing interests exist. Gilead Sciences, USA, provided the investigational product, AmBisome H .Gilead Sciences and the sponsors, DNDi, have professional collaborations. This collaboration does not alter our adherence to all PLOS NTDs policies on sharingdata and materials.* E-mail:, Introduction Visceral leishmaniasis (VL) is a life-threatening disease and amajor health burden in developing countries [1,2,3]. WHOestimates there are approximately 0.2–0.4 million cases of VLannually; and more than 90% of global VL cases occur in sixcountries: India, Bangladesh, Sudan, South Sudan, Ethiopia andBrazil [1]. In eastern Africa approximately 30,000 people developsymptomatic VL and 4,000 die every year [3,4].For decades, the mainstay of VL treatment in eastern Africa hasbeen antimonials such as sodium stibogluconate (SSG), but thistreatment is cardiotoxic [5] and requires a 4-week hospitalisation PLOS Neglected Tropical Diseases | 1 January 2014 | Volume 8 | Issue 1 | e2613  imposing a huge economic burden on families [6]. Monotherapywith intramuscular paromomycin (PM) for 3 weeks was shown tobe less efficacious in eastern Africa [7] than in Asia [8], but a 17-day treatment with a combination of SSG and PM showed goodefficacy and is now recommended as first-line treatment by WHO.However, this treatment also requires a relatively long treatmentcourse and twice daily injections [8]. Currently, the safest anti-leishmanial drug is AmBisome H , a liposomal amphotericin Bformulation with significantly diminished renal toxicity [9]. Intrials in India, cure rates of around 90% were obtained with single AmBisome H  doses of 5 mg/kg [9]. In addition, 95% efficacy wasachieved with higher single doses (10 mg/kg) or when used incombination with miltefosine or paromomycin [10]. Althoughlicensed and recommended for first-line treatment of VL inimmunocompetent patients [11], Ambisome H  use in eastern Africahas been mostly limited to second line treatment in a few centresmainly due to its high cost and cold storage requirements [12]. Asmall study with AmBisome H  conducted in Kenya indicatedhigher doses were required than had been used in studies in India.Doses of 2 mg/kg given 3, 5, or 7 times to groups of 10 patientsresulted in cure rates of 20%, 90%, and 100% respectively [13].The aim of this study was to determine the minimum efficaciousand safe single dose for the likely future use of the drug as part of ashorter course of treatment regimen for eastern African VLpatients. The trial was undertaken with the goal of ultimatelyidentifying a short and simplified treatment regimen that includes AmBisome H . Such a regimen will improve patient compliance andwill have the advantage of a reduced cost. Methods The study design and protocol have been published, incompliance with CONSORT requirements [14]. The protocolwas approved in Ethiopia, Sudan and UK by the authors’Institutional and National Ethics Committees. These committeeswere: the Research Ethics Institutional Review Board of theFaculty of Medicine – Addis Ababa University; The InstitutionalReview Board of the University of Gondar; the National EthicsReview Committee (NERC) at the Ethiopian Science & Technol-ogy Commission (ESTC); the Health Research Ethics Committeeof the Institute of Endemic Diseases (University of Khartoum) andthe Directorate of Health Research in the Federal Ministry of Health (Sudan); and the Ethics Committee of the London Schoolof Hygiene and Tropical Medicine. The study was conducted inaccordance with the declaration of Helsinki, ICH GCP guidelines,and all applicable legal requirements. All study subjects partici-pated in the study voluntarily, and signed a written ‘InformedConsent Form’ (ICF). The parents or guardians of studyparticipants under the age of 18 years provided written informedconsent on their behalf. In addition, minors (age 12–17 years)signed a written assent form. The study was registered at (Clinical Trials Registration numberNCT00832208) prior to trial initiation and patient recruitment. Study design The study was designed as a multi-centre, open-label, non-inferiority, randomized controlled trial, using a sequential-stepdesign to evaluate the efficacy and safety of a single dose treatmentregimen of intravenous AmBisome H  (either 7.5, 10.0, 12.5 or15.0 mg/kg body weight) compared to the reference multiple doseregimen currently approved in the USA: 3 mg/kg body weight ondays 1 to 5, 14, and 21. The single dose tested in the first cohortwas 7 ? 5 mg/kg body weight (figure 1, the consort flowchart). Twointerim analyses were planned, after enrolment of 20 and 40patients per arm, for early detection of inefficacious single doses,based on parasite clearance at day 30 (figure 2) and/or worsening clinical conditions. If the stopping rule was met, the single dosewas increased by 2 ? 5 mg/kg and recruitment into the two armsrestarted. The multiple dose treatment remained the samethroughout. Non-responders to treatment were considered treat-ment failures and received rescue medication (multiple dose AmBisome H  regimen for single-dose failures and SSG for multipledose failures). Study population Patients with age of at least 4 years, confirmed HIV-negative,parasitologically-confirmed non-severe VL, were enrolled in threecentres: (1)  Gondar   University Hospital, Amhara Regional State,Northern Ethiopia; (2)  Arba Minch   Hospital, Gamo Gofa Zone,Southern Nations, Nationalities and Peoples Regional State,Southern Ethiopia; and per protocol amendment (3) Ministry of Health Hospital,  Kassab , Gedaref State, Eastern Sudan from May2009 to September 2010. Exclusion criteria were signs/symptomsof severe VL (patients who were very weak, unable to walk,bleeding, jaundiced, suffering from sepsis and other concomitantinfections/illnesses); anti-leishmanial or unlicensed investigationaltreatments within six months; underlying chronic disease such assevere cardiac, pulmonary, renal, or hepatic impairment; serumcreatinine outside the normal range; liver function tests more than3 times the normal range; platelet count less than 40,000/mm3;known alcohol abuse; pregnancy or lactation; concomitant acutedrug usage for malaria and bacterial infection; pneumonia withinlast 7 days; known hypersensitivity to AmBisome H  or amphotericinB; any other condition which may invalidate the trial. Interventions On pre-specified treatment days, AmBisome H  dosage wascalculated according to body weight. Preparation includedreconstitution with sterile water for injection and filtrationaccording to the manufacturer’s instructions. Administration wasby slow intravenous infusion in 5% dextrose solution. During infusion, patients were closely observed with regular monitoring of  vital signs (blood pressure and pulse). A test dose was administeredin the first 10 minutes of infusion, and the patients carefully Author Summary Visceral leishmaniasis is a potentially fatal disease whichaffects 0.2–0.4 million people every year, principally inSouth-East Asia, Latin America or Eastern Africa. Currentlythe safest drug in use is AmBisome H , which cures 90% of patients in India at 5 mg/kg, and is even more effective athigher doses (10 mg/kg) or in combination with miltefo-sine or paromomycin. These regimens have been shown tobe equally cost-effective in India. However, the drugrequires a cold chain for storage and reconstitution priorto injection. Although it is licensed for use in easternAfrica, in practice it is mainly used as a second-linetreatment. A small study carried out in Kenya indicatedthat a higher dose is necessary in eastern Africa in contrastto Asia. This study aimed to determine the minimum singledose that is safe and effective for treatment of easternAfrican VL patients so as to be used in simplified treatmentregimens. However, the tested regimens were found to beineffective, and an optimal single dose that couldpotentially be used in simplified treatment regimens wasnot identified. AmBisome H  for East Africa VL: Clinical Study ReportPLOS Neglected Tropical Diseases | 2 January 2014 | Volume 8 | Issue 1 | e2613  observed for 30 minutes. The time of treatment and dosage wasrecorded. Randomisation and blinding Patients were randomized to receive either treatment using acomputer-generated randomisation list, stratified by site. Individ-ual treatment allocations were placed in sealed, opaque envelopeswhich were opened after a patient had been entered into the trial.It was not possible to blind patients or treating physicians due tothe nature of the intervention. Endpoints Primary (definitive/final cure) and secondary (initial cure)efficacy endpoints were determined by parasitological assessment,as the most objective and consistent method across treatmentcentres at six months follow-up and at Day 30, respectively. Day30 was considered as the end of treatment time point for initialcure assessment. Definitive cure was defined as absence of parasites in tissue aspirates (bone marrow, lymph node or spleen)with no relapse of signs and symptoms of VL during six monthsfollow-up. Initial cure was defined on day 30 by absence of parasites in tissue aspirates. For patients with detectable parasitesat initial cure assessment, clinical and biological assessments wereused, in addition to parasitological results, to ascertain the need forrescue treatment at the end of study treatment at the discretion of the treating clinician, according to the protocol. Patients with apresence of parasites at initial assessment of cure but who wereclinically well were invited to return after one month to furtherassess their status and need for rescue treatment. Patients who didnot clear parasites at end of treatment but did by six monthsfollow-up, in the absence of a requirement for additional rescuetreatment, were classified as treatment successes at six months aspatients who were slow responders, in line with previous trials inthe region [7]. Assessment of safety involved monitoring vital signs,documentation of patients’ complaints about the treatments, andhaematological and biochemical measurements evaluated on days2 to 5, 7, 14, 21 and 30, and at 3 and 6 months. Treatmentemergent adverse events (TEAE) were classified according to theMedical Dictionary for Regulatory Activities (MedDRA). Treat-ment emergent events were those with onset between day 1 of treatment and day 60 inclusive.Peripheral Blood Parasitaemia: In Kassab, per protocolamendment, peripheral blood samples were analysed for parasiteload in a subset of 5 consenting patients in each of the 10 mg/kg single dose and multiple dose arms. For this, a validatedquantitative reverse-transcriptase polymerase-chain-reaction(qRT-PCR) method targeting   Leishmania   18S ribosomal RNAwas used [15]. Genetic material was extracted using a modifiedBoom-method [15,16,17]. qRT-PCR analysis using a Bio-RadCFX-96 real-time machine (Bio-Rad, Veenendaal, the Nether-lands) was performed at Koninklijk Instituut voor de Tropen(KIT). Figure1.Patientflowchart. Consort Patient flow chart: AmBisome H  multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost toFollow-up.doi:10.1371/journal.pntd.0002613.g001AmBisome H  for East Africa VL: Clinical Study ReportPLOS Neglected Tropical Diseases | 3 January 2014 | Volume 8 | Issue 1 | e2613  Sample size For the primary endpoint comparison, 120 patients perarm would provide 80% power to detect non-inferiority withina margin of 10%, assuming 95% cure in the reference arm, aone-sided alpha of 0 ? 05 and 15% loss-to-follow-up. In interimanalyses, 20 patients per arm would provide 90% power to detecta difference of at least 35% in parasite clearance rate at day30, assuming 95% cure in the reference arm and a two-sidedalpha of 0.05. With 40 patients per arm, there would be 90%power to detect a difference of at least 25% under the sameassumptions. Statistical analysis Interim analyses were based on day 30 cure in the Intention-to-Treat (ITT) population. Decision-making at each interim analysiswas based on a test of difference between the parasite clearancerates in the single dose arm and multiple dose arms. If the singledose arm showed significantly poorer efficacy (P , 0.05), the single-dose was increased prior to re-starting recruitment. Patientsallocated to the multiple-dose arm for discontinued single-dosecomparisons were not included in comparisons of higher singledoses.Key assumptions for the planned final analysis were not met dueto low efficacy in the multi-dose arm and the trial was terminatedprematurely (see Results). Cumulative data for each treatmentregimen were used to calculate the percentage of patients cured,with exact binomial 95% confidence intervals (CI), at day 30 and 6months follow-up in ITT and per-protocol (PP) analysis popula-tions. Patients with missing outcome data were excluded fromanalyses. For safety, the number and percentage of patients perarm experiencing adverse events (AEs) were summarised, for AEswith cumulative incidence higher than 10%. For parasiteclearance from peripheral blood, a linear mixed effects regressionmodel using the natural log-transformed parasite loads was appliedto estimate the time to clear 50% and 90% of parasites for eachindividual. Model performance and significance were assessed byanalysis of variance (ANOVA). Results Baseline characteristics The most commonly presenting VL symptoms were fever andweight loss, followed by loss of appetite, abdominal swelling, andcough; less commonly observed were epistaxis, diarrhoea, and skin Figure2.Summaryofstudydesign. Interim analyses were based on day 30 cure in the Intention-to-Treat (ITT) population. iv intravenous, mg/kg/day milligram/kilogram body weight/day.doi:10.1371/journal.pntd.0002613.g002AmBisome H  for East Africa VL: Clinical Study ReportPLOS Neglected Tropical Diseases | 4 January 2014 | Volume 8 | Issue 1 | e2613  lesions. Other characteristics of patients at entry to the trial aresummarizedinTable1.Overall,82%ofpatientsweremaleandabouthalf were children. About two thirds of patients were underweightor severely underweight. Mean haemoglobin concentrations were , 8 ? 0 g/dl, and anaemia was common, but neither baselinelaboratory parameters nor vital signs suggested any major differenceamong dose groups. Baseline characteristics were generally compa-rableinthemultipleand the10 mg/kgsingledosegroup,whereasthesmaller 7 ? 5 mg/kg dose group showed some imbalances: on average,patients in this group were older and accordingly had higher bodyweight and larger spleen size, but also had the highest baselineparasitaemia. Patients were younger in Kassab (10 ? 5 6 5 ? 0 years) thanin Gondar (20 ? 9 6 6 ? 3) or Arba Minch (17 ? 4 6 10 ? 2 in) and more oftenfemale in Kassab (33 ? 3%) than in Gondar (10 ? 5%) or Arba Minch(12%). In Arba Minch, they were more often normal weight (42 ? 0%)than in Gondar (21 ? 1%) or Kassab (28 ? 6%). Efficacy In the first section of Table 2, parasite clearance rates at day 30are shown for the three interim analyses. Summary data for theparasite clearance rate at day 30 and the cure rate at 6 months areshown for all patients and for those treated at each of the 3 centres.The IC and DC rates with the standard multiple dose treatmentwere both 85%. IC rates with single doses of 7 ? 5 and 10 mg/kg were 50% and 73%, respectively; and DC rates were lower, at40% and 58%, respectively. However, there were variations intreatment response between treatment centres, with poor efficacyin Kassab and Gondar, particularly with single doses. By contrast,at Arba Minch, the multiple doses as well as the single dose of 10 mg/kg resulted in complete cure, and treatment failures wereobserved with the 7 ? 5 mg/kg dose only. All non-responders werecured after receiving rescue medication. Early termination For the first interim analysis, comparing the 7 ? 5 mg/kg singledose to the multiple dose regimen with 20 and 18 patients per arm,respectively, in the two Ethiopian sites, the stopping rule was met(Table 2: Fisher’s exact test, p=0 ? 015). The single dose wasincreased to 10 mg/kg, and recruitment restarted at bothEthiopian sites and in an additional site, Kassab, Sudan. Therewas no significant difference in efficacy found at the next interimanalysis comparing 10 mg/kg to the multiple dose arm(p=0 ? 748), but when 44 patients had been recruited into themultiple dose and 40 patients into the 10 mg/kg single-dose arm,the third interim analysis indicated unexpectedly low initial curerates in both arms; 84% in the multiple dose and 73% in thesingle-dose arm. The stopping rule was not met (chi-squared test,p=0.196), but based on the observed poor efficacy overall, andfollowing discussions with the Data Safety and Monitoring Board(DSMB) and investigators, the sponsor terminated the trial. Table 1.  Baseline data on patient demographics, clinical characteristics and laboratory values. Multiple dose 21 mg/kgN=63Single dose 7.5 mg/kgN=21Single dose 10 mg/kgN=40 Age in years Mean (SD) 16 (9.0) 21 (9.2) 14 (7.4)Children (4–17 y), n (%) 37 (59) 4 (19) 25 (63)Adults ( $ 18 y), n (%) 26 (41) 17 (81) 15 (37)Sex Female, n (%) 10 (16) 2 (11) 10 (25)Male, n (%) 53 (84) 19 (91) 30 (75)Spleen size in cm Mean (SD) 9.5 (5.3) 12.0 (6.0) 9.1 (5.3)Nutritional status {  Weight [kg], mean (SD) 36 (14.5) 44 (12.2) 33 (14.4)Severe underweight, n (%) 18 (29) 9 (43) 11 (28)Underweight, n (%) 22 (35) 7 (33) 17 (43)Normal weight, n (%) 22 (35) 5 (24) 12 (30)Hemoglobin (g/dl) Mean (SD) 7.9 (1.7) 7.7 (1.6) 7.7 (1.4)AST Mean (SD) 52 (27.1) 48 (28.5) 54 (29.0)ALT Mean (SD) 32 (20.7) 35 (21.3) 33 (20.0)Parasite Count (log scale) 6 + , n (%) 2 (3) 2 (10) 05 + , n (%) 7 (11) 5 (23) 7 (18)4 + , n (%) 14 (22) 6 (29) 4 (10)3 + , n (%) 15 (23) 4 (19) 9 (22)2 + , n (%) 11 (17) 4 (19) 9 (22)1 + , n (%) 12 (20) 0 10 (25)0 or missing, n (%) 2* (3) 0 1** (3)Data are n (%) or means (SD). AST aspartate aminotransferase, ALT alanine aminotransferase. { classified using weight for height and BMI for age in those aged # 19 years and BMI in those aged . 19: normal if  2 2SD # weight for height or BMI for age , + 1SD or18.5 # BMI , 25.0; underweight if  2 3SD # weight for height or BMI for age ,2 2SD or 16.0 # BMI , 18.5; severely underweight if weight for height or BMI for age ,2 3SDor BMI , 16.0.*1 case of unconfirmed VL with no parasites detected (major protocol deviation, excluded from analysis) and one case of no parasite count recorded on a log scale inwhich VL was confirmed by lymph node aspirate.**no parasite count recorded on a log scale in which VL was confirmed by lymph node aspirate.doi:10.1371/journal.pntd.0002613.t001 AmBisome H  for East Africa VL: Clinical Study ReportPLOS Neglected Tropical Diseases | 5 January 2014 | Volume 8 | Issue 1 | e2613
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