This was a prospective, parallel arm, randomized, placebo-controlled, blinded trial of iNO versus placebo (1:1 ratio), among children with severe malaria, all of whom were treated with artesunate. The trial protocol has been described in detail previously .
Ethics, consent and permissions
The study was reviewed and approved by the Makerere University School of Medicine Research Ethics Committee (REC Protocol # 2010-107), the Uganda National Council on Science and Technology (Ref: HS 857), the National Drug Authority of Uganda (Ref: 297/ESR/NDA/DID-01/2011), and the University Health Network Research Ethics Committee, Toronto, Canada (UHN REB Number 10-0607-B). A data and safety monitoring board (DSMB) was convened and met periodically to review trial quality and adverse events. An interim analysis at the trial midpoint was conducted to review trial quality and safety, at which time the DSMB recommended that the trial proceed without modifications. The trial is registered (ClinicalTrials.gov Identifier: NCT01255215).
Setting and participants
The trial was conducted at a single centre, the Jinja Regional Referral Hospital, in Uganda. Malaria transmission is moderate and seasonal in Jinja and the surrounding Busoga catchment area . The hospital operates under severe resource constraints, and over 30 % of all admissions are due to malaria.
Children (age 1–10 years) were included if they had a positive rapid diagnostic test for both P. falciparum histidine rich protein 2 (HRP2) and lactate dehydrogenase (pLDH)(First Response Malaria Ag. (pLDH/HRP2) Combo Rapid Diagnostic Test, Premier Medical Corporation Limited, India) , as well as selected criteria for severe malaria: repeated seizures (two or more generalized seizures in 24 h), prostration, impaired consciousness (Blantyre Coma Score <5), respiratory distress (age-related tachypnea with sustained nasal flaring, deep breathing or sub-costal retractions). Patients were not included if they had methaemoglobin (metHb) >2 % at baseline, known chronic illness (renal, cardiac or hepatic disease, diabetes, epilepsy, cerebral palsy, or AIDS), severe malnutrition (weight-for length or height below −3 standard deviations based on WHO reference charts, or symmetrical oedema involving at least the feet). Modifications to the exclusion criteria were made with regulatory committee approval after experience with the first 20 enrolled participants. The following exclusion criteria were added: haemoglobinopathy, clinical suspicion of acute bacterial meningitis, unlikely to tolerate mask for study gas delivery, and prior quinine in the emergency department. Trial nurses or clinicians from the emergency department screened patients for eligibility using a uniform checklist and clinicians made final decisions about inclusion in the study.
Randomization and blinding
In order to blind clinicians, nurses, parents, and participants to treatment while titrating and monitoring concentrations of iNO and dose-related levels of metHb and NO2, a dedicated unblinded team was used, the members of which were not involved in clinical care decisions or outcome assessments.
Eligible patients were randomly assigned to treatment with either iNO or room air placebo (both arms received intravenous artesunate). Simple randomization was employed, using a computer-generated list created by unblinded team leader (AC) prior to trial commencement. Treatment assignment was recorded on paper and kept in sequentially numbered, sealed, opaque envelopes in a locked cabinet accessible only to the unblinded study team. After patient stabilization and informed consent, the next envelope was drawn by an unblinded investigator.
iNO was indistinguishable from room air in colour and delivery apparatus (mask, tubing, a stream of vehicle air). An unblinded team member initiated the study gas while treating nurses and clinicians were out of the room. Flowmeters and monitoring devices were in locked opaque boxes accessible only to the unblinded study team. MetHb measurements were performed using non-invasive pulse CO-oximetry (Masimo Rad-57™, Masimo Corporation, Irvine, CA, USA) by unblinded study team members. All laboratory assays and statistical analyses were performed blinded to treatment allocation.
iNO was delivered continuously at a target concentration of 80 ppm by non-rebreather mask for up to 72 h. An air compressor was used to deliver continuous flow of vehicle air, and NO from compressed cylinders was titrated into the air stream to a concentration of 80 ppm, measured continuously at the bedside using a NO-NO2 analyser (Pulmonox Sensor; Pulmonox Research and Development Corporation, Tofield, Alberta, Canada). Methaemoglobinaemia and inspired NO2 were monitored at least every 4 h. The concentration of iNO administered was adjusted downward if the metHb level in peripheral blood rose above 7 %, and was temporarily discontinued for metHb >10 %. Participants in the control group received room air by non-rebreather mask. Both groups received intravenous artesunate, the recommended first-line treatment for severe malaria, at recommended dose and frequency . Follow-on oral therapy was with artemether-lumefantrine tablets or suspension for 3 days.
Bloodwork for clinical and study purposes was drawn at admission and daily during the first 72 h of hospital admission. Admission venous blood samples were analysed at the bedside for haematocrit, creatinine, lactate, and glucose  and at a central laboratory for parasite density, as previously described . Lumbar puncture was performed at the clinician’s discretion and was analysed for cell count and differential, total protein, Gram stain and bacterial culture.
The analysis was undertaken according to a pre-specified analytical plan . There were no changes to any trial outcomes after the trial commenced. The primary endpoint was the longitudinal serum Ang-2 concentration over the first 72 h of hospital admission. Ang-2 was measured from serum samples using commercially available enzyme-linked immunosorbent assay (ELISA) kits (DuoSets, R&D Systems, Minneapolis, MN, USA).
Secondary trial outcomes included: mortality, recovery times, parasite clearance kinetics, and safety. Adverse events were monitored daily using paediatric toxicity tables modified from the US National Institute of Allergy and Infectious Diseases .
Inclusion of 180 children with severe malaria was needed to show, with 80 % power and 95 % confidence, a 50 % difference in the rate of change of Ang-2. This calculation was supported by a simulation study under various assumptions of variance and treatment effect .
The primary outcome, longitudinal course of Ang-2, was compared between study arms using linear mixed-effects (LME) models. All available data was used for the primary analysis. Because of (non-random) missing longitudinal data due to death, withdrawal and lost samples, sensitivity analyses were performed with different methods of adjusting for missing data (‘intention-to-treat’ analysis), as outlined in Additional file 1. Model fit was assessed by visual inspection of residuals.
For secondary binary outcomes, Chi squared or Fisher exact test were used, as appropriate. Time to event outcomes were compared with the log-rank test, and hazard ratios (HRs) together with 95 % CIs were estimated by a Cox proportional hazard model.
Statistical analyses were done with SPSS (version 16.0) and R (version 3.0.1).
Role of the funding source
The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.