Randomized trial of artesunate+Amodiaquine, sulfadoxine-pyrimethamine+amodiaquine, chlorproguanal-dapsone and SP for malaria in pregnancy in Tanzania

Background: Malaria in pregnancy is serious, and drug resistance in Africa is spreading. Drugs have greater risks in pregnancy and determining the safety and efficacy of drugs in pregnancy is therefore a priority. This study set out to determine the efficacy and safety of several antimalarial drugs and combinations in pregnant women with uncomplicated malaria. Methods: Pregnant women with non-severe, slide proven, falciparum malaria were randomised to one of 4 regimes: sulfadoxine-pyrimethamine [SP]; chlorproguanil-dapsone [CD]; SP+amodiaquine [SP+AQ] or amodiaquine+artesunate [AQ+AS]. Randomisation was on a 1:2:2:2 ratio. Women were admitted for treatment, and followed at days 7, 14, 21, 28 after the start of treatment, at delivery and 6 weeks after delivery to determine adverse events, clinical and parasitological outcomes. Primary outcome was parasitological failure by day 28. Results: 1433 pregnant women were screened, of whom 272 met entry criteria and were randomised; 28 to SP, 81 to CD, 80 to SP+AQ and 83 to AQ+AS. Follow-up to day 28 post treatment was 251/272 (92%), and to 6 weeks following delivery 91%. By day 28 parasitological failure rates were 4/26 (15%, 95%CI 4-35) in the SP, 18/77 (23%, 95%CI 14-34) in the CD, 1/73 (1% 95%CI 7-0.001) in the SP+AQ and 7/75 (9% 95%CI 4-18) in the AQ+AS arms respectively. After correction by molecular markers for reinfection the parasitological failure rates at day 28 were 18% for CD, 1% for SP+AQ and 4.5% for AQ+AS. There were two maternal deaths during the trial. There was no apparent excess of stillbirths or adverse birth outcomes in any arm. Parasitological responses were strikingly better in pregnant women than in children treated with the same drugs at this site. Conclusions: Failure rates with monotherapy were unacceptably high. The two combinations tested were efficacious and appeared safe. It should not be assumed that efficacy in pregnancy is the same as in children © 2009 Mutabingwa et al. This open-label study was conducted among pregnant women who attended Muheza Designated District Hospital (Muheza DDH). The lowlands of Muheza district experience hyperendemic to holoendemic malaria. The day 28 parasitological failure rates to AQ monotherapy in a recent effectiveness trial in children under 5 years was 76%, with the comparative rates of 61% and 40% for AQ+SP and AQ+AS21. Throughout the study period SP (defined as monotherapy for the purposes of this paper) was national first-line treatment for malaria, and this was taken as the comparator arm for this study. Pregnant women with mild-moderate, slide proven, falciparum malaria were recruited from the Antenatal wing (ANC) of the Reproductive and Child Health (RCH) clinic at Muheza Designated District Hospital. Pregnant women from Muheza Township and surrounding villages attend this clinic for their medical care. Nurses at the RCH identified all febrile pregnant women with a fever or recent history of fever (within 48 hours), symptoms compatible with anaemia or malaria and referred them to the study team. All referrals were re-interviewed and examined by a medical officer from the study team to exclude concomitant infection(s). Duplicate thick and thin blood smears were Giemsa stained and examined microscopically for malaria parasites. Inclusion criteria were pregnancy with either a positive blood smear for P.falciparum with at least 800 asexual parasites/µL in an asymptomatic woman or any of the following symptoms within 2 days prior to consultation; history of fever; headache, vomiting, chills/rigors, and/or any of the following signs: temperature ≥37.5°C and <39.5°C, Hb≥7 and <9 g/dl) together with P.falciparum parasitaemia at any density. Additionally, all cases had to be 14–34 gestation weeks pregnant on the day of attending the clinic, have a viable foetus defined by the presence of foetal heartbeat by sonicaid or pinnard, able to take drugs orally, able to attend follow up clinic, and gave written informed consent to participate or a finger-print witnessed consent for women unable to read. The main exclusion criteria were; severe and complicated forms of malaria [30], pregnancy in the first trimester or >34 gestation weeks (because they had a high chance of delivering during the 28 day follow-up period), mixed plasmodial infection, complicated pregnancy e.g. signs/symptoms of toxaemia, 2 or more abortions or stillbirths, presence of concomitant disease masking assessment of the response to treatment, intake of drugs contraindicated in pregnancy or drugs with effective antimalarial activity within the last 2 weeks, multiple gestation pregnancies, mother aged 38 years or above. Withdrawal criteria were withdrawal of consent, appearance of other species of Plasmodium or major protocol violation. Women who met the inclusion criteria were randomised to one of 4 regimes: three tablets of sulfadoxine-pyrimethamine (500 mg sulfadoxine/25 mg pyrimethamine per tablet) orally at once [SP] in line with the national policy; chlorproguanil-dapsone (1.2 mg/kg and 2.4 mg/kg respectively for 3 days) [CD]; SP 3 tablets once+amodiaquine (10 mg/kg for 3 days) [SP+AQ]; amodiaquine (10 mg/kg for 3 days)+artesunate (4 mg/kg for 3 days) [AQ+AS]. Randomisation was on a 1∶2∶2∶2 ratio for SP, CD, SP+AQ and AQ+AS to maximise information about the drugs whose use in pregnancy is less known; it was assumed the difference in outcome would be greatest for SP compared to other arms so the size of this arm could be smaller. Randomisation was in blocks of random sizes, and conducted in London using Stata 7. Treatment allocations were placed in a sealed opaque envelope, with pregnant women picking their own envelope. Patients were allocated a study number sequentially, and after consenting to participate, participants picked an envelope in front of the attending clinician. Opening the envelope constituted entry to the trial and analysis was conducted on that basis (defined as analysis of all cases in which there was an outcome, irrespective of actual treatment given). All women were admitted to a ward dedicated to research for the first three days to facilitate supervised drug administration and to monitor clinical response and adverse events. Drugs were administered by study nurse-midwives employed by the project. After each administration, the patient was observed for 45–60 minutes. The dose was repeated if vomiting occurred within the observation period. Vomiting the second dose was registered as an adverse event and led to withdrawal from the study. Such cases were treated with parenteral quinine. Patients were treated for symptoms with standard medications e.g. paracetamol for fever. Women continued to receive routine antenatal medicaments of iron supplements, folic acid (5 mg), and tetanus toxoid (TT) given by the RCH. In addition to daily clinical observations and laboratory tests, foetal viability (presence of foetal heartbeat) was monitored daily during admission using a Doppler machine, and at each follow-up visit. Adverse events were classified by severity and potential causal relationship to study drugs. All serious adverse events (SAEs) were independently investigated by a local safety monitor. Subjects with an AE were followed up until the condition had disappeared or stabilized. Parasite counts on Giemsa-stained blood films were performed daily during admission, repeated on days 7, 14, 21 and 28, and on any other day(s) of complaints. Counts were made against 200 white blood cells (WBC) on a thick blood smear. All slides had a second reading done in an independent research laboratory. Discordant results were read by a third reader, with the majority taken as the definitive outcome. All microscopists were blind to treatment allocation. A separate read for gametocytes was undertaken counting against 500 white blood cells. To quantify the effect of treatment on gametocyte carriage, we determined the area under the curve (AUC) of gametocyte density over time which incorporates both the magnitude and the duration of gametocyte carriage [31]. Blood samples for haematology and clinical chemistry, and in anaemic women stool microscopy for intestinal helminths were obtained on admission. Blood and urine samples were repeated at day 3 and where indicated at day 7. Haemoglobin, total and differential white blood cell count, platelet count, creatinine, total bilirubin, alanine aminotransferase (ALT) and albumin were measured (using CBC machine for haemogram and Reflectron for biochemistry) on days 0, 3 and 7, and whenever else indicated. Pre-test counselling for HIV-testing was undertaken, and where consent was given an HIV test was performed. HIV-positive mothers were referred to the HIV care unit of Muheza DDH for counselling and for consideration of antiretroviral drugs. On discharge from the ward, patients were followed up on days 7, 14, 21 and 28 post initiation of treatment and at any time they felt unwell before day 28. At the end of each clinic, members of the study team followed all non-attendees to their homes to establish and record reasons for non-attendance and to collect a blood smear. Patients with either early or late treatment failure following treatment with any of the 4 study regimens were treated with quinine 10 mg/kg 8 hourly for 7 days as rescue therapy. Birth outcome and Dubowitz assessment were recorded for all deliveries taking place at the hospital. Mothers and their newborns attended follow up clinics 6 weeks after delivery. All non-attendees (whether delivered at Muheza DDH or not) were followed up at home. Assessment at this time included a further check of the newborn by the paediatrician for any abnormality that may have been missed at birth or for any serious problem that may occur after birth such as kernicterus. Whenever the mother had moved from the study area, all possible efforts were made to ascertain birth outcome verbally from close relatives. A DSMB reviewed all SAEs, which were notified as they occurred. Blood for PCR was collected on glass-fibre membranes from all patients at enrolment and at each follow-up. The polymorphic repetitive regions were amplified by nested-PCR for block 3 of msp2 [32]. Using the template of the first PCR reaction, allele-specific primer pairs was used to test for the presence of the allelic variants from FC27 and IC of the families of the msp2 region. Amplification patterns of the various allelic families in DNA samples from day 0 were compared to other samples from the same patient when parasitaemic. If the allelic family(ies) amplified on day 0 included those which were identical in size to those amplified during a subsequent episode, then the patient was classified as carrying a recrudescent infection. The primary end-point of the trial was parasitological failure by day 28. This was defined as any of: a need for rescue treatment due to clinical deterioration defined by altered sensorium, seizures, persistent vomiting, renal impairment, respiratory distress, a fall in Hb below 7 g/dl, or in cases where the initial haemoglobin dropped 20% or more from baseline Hb, at any time during admission; persistence of fever with parasitaemia on day 3; increased parasite density on day 2 or 3 compared with baseline density; failure to clear parasites on day 7; rescue medication for recurrent malaria before day 28; slide parasite positivity at day 14, 21 or 28. Major secondary endpoints were: clinical failure by day 28 (parasitological in the presence of symptoms compatible with malaria), parasitological or clinical failure by day 14, incidence of foetal death during treatment, defined as absence of foetal heartbeat assessed by Doppler; change in haemoglobin from baseline on day 14; incidence of perinatal and neonatal mortality, assessed 4–6 weeks after due date of delivery; clinically apparent neonatal abnormality 4–6 weeks after due date of delivery; preterm delivery and other adverse events during treatment. Initially the study was powered to detect a 4 fold difference in treatment failure between SP+amodiaquine and amodiaquine+artesuante groups (8% vs 2%) with 95% precision and 80% power, which would require a samples size of 80 women in the SP+placebo group and 240 women in each of the other three groups. Vigorous measures to protect pregnant women in the district from malaria on a general background of reduced transmission of malaria in this area fortunately led to substantial reductions in the number attending the antenatal clinic with clinical malaria. The data from this and other sites was reviewed on October 2004 and it was decided that given the absence of other data, the question was important enough and of sufficient public health priority that a trial able to detect a larger difference would still be of public health importance. A revised sample size was calculated to detect a difference from 1% (the best likely failure rate in any arm) and 15% (above which no drug could be deployed). This gave a sample size of 72 in each arm when α was .05 and β 0.8. The unbalanced sample size (1∶2∶2∶2) was by this time established and could not be revised retrospectively although the statistical rationale for it was not present with the revised design. Data were double entered into Microsoft Access, and analysed using Stata 8. The analytical plan was finalised before the analysis was undertaken. For primary and major secondary outcomes, proportions with confidence intervals were calculated. Odds ratios were calculated for the difference between all arm and the best and worst arms for parasitological failure unadjusted, and adjusted for the predefined risk factors age, parity, HIV serostatus, initial parasitaemia and initial haemoglobin. Ethical permission was granted by the ethics committees of the National Institute for Medical Research, Tanzania, and the London School of Hygiene & Tropical Medicine and conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent (or witnessed where whey could not read). The trial was monitored by an independent external clinical monitor and was prospectively registered on ClinicalTrials.gov No. {“type”:”clinical-trial”,”attrs”:{“text”:”NCT00146731″,”term_id”:”NCT00146731″}}NCT00146731. The protocol for this trial and supporting CONSORT checklist are available as supporting information: see CONSORT S1 and Protocol S1.