Inhaled nitric oxide as adjunctive therapy for severe malaria: a randomized controlled trial

Severe malaria due to Plasmodium falciparum claims 0.6–1.2 million lives annually, 86 % of whom are children in sub-Saharan Africa [1, 2]. Despite the use of highly effective anti-malarial medications, 10–30 % patients with severe malaria will die [3], highlighting the need for new adjunctive therapy. Nitric oxide (NO), with its modulating effects on host endothelial activation, is a promising agent based on pre-clinical findings and an established safety profile in clinical practice [4, 5].

NO is a gaseous, lipid-soluble, free radical, endogenously produced from l-arginine and molecular oxygen by members of the nitric oxide synthase family [6]. NO regulates a broad range of physiologic and pathologic processes, including vasodilation, platelet aggregation, apoptosis, inflammation, chemotaxis, neurotransmission, antimicrobial defence, and endothelial activation [6].

The vascular endothelium plays a central role in the pathogenesis of severe malaria. Parasitized erythrocytes (PEs) adhere to the endothelial cells via constitutive and cytokine-inducible receptors. NO decreases endothelial cell adhesion molecule expression, and has been shown to reduce the adherence of PEs to endothelial cells [7]. Upon activation, endothelial cells release angiopoietin-2 (Ang-2) from intracellular Weibel-Palade bodies (WPB) storage granules [8]. Ang-2 functions as an autocrine regulator by sensitizing the endothelium to the effects of tumour necrosis factor, resulting in increased adhesion receptor expression [9]. Ang-2 is associated with malarial retinopathy and is an independent and quantitative marker of disease severity and clinical outcome in malaria [10‐12]. Ang-2 has also been used to follow disease progression and recovery in previous studies of malaria [13]. NO inhibits the exocytosis of WPB contents through S-nitrosylation of critical regulatory proteins [14].

Reduced bioavailability of NO contributes to the pathogenesis of severe malaria. African children with severe malaria have impaired production of NO [15] and low plasma arginine levels [16], the substrate for NO synthesis. Treatment of Indonesian adults with severe malaria with intravenous l-arginine increased levels of exhaled NO, and reversed malaria-associated endothelial dysfunction [17]. Pathways of endothelial activation and dysfunction are reflected in experimental models of severe malaria, where inhaled NO enhances endothelial integrity, reduces parasite accumulation in the brain vasculature, and improves survival [5, 18].

Administration of exogenous NO at 5–80 ppm is approved for use by the US FDA for neonates with hypoxic respiratory failure [19].Inhaled nitric oxide (iNO) has been safely used in clinical practice and in large clinical trials involving critically ill neonates [19], as well as in children and adults with refractory hypoxia [20]. Adverse effects attributable to iNO in these studies are rare and include methaemoglobinaemia, renal insufficiency [21] and hypotension.

To test the hypothesis that iNO would improve outcomes in children with severe malaria, a randomized, blinded, controlled trial was conducted. The primary objective was to determine if iNO at 80 ppm, relative to placebo room air, would improve endothelial function as determined by an accelerated rate of decline of Ang-2 in peripheral blood among African children with severe malaria receiving artesunate.

Figure 1 shows the trial profile. Recruitment occurred between 12 July, 2011 and 14 June, 2013, with last follow-up visit on 28 June, 2013. The trial ended when the pre-specified sample size was reached.

No significant differences in baseline characteristics between the two treatment groups were observed (Table 1). Ten patients were positive for both HRP2 and pLDH bands on screening rapid diagnostic test but negative on microscopy of the admission sample. One additional patient had a microscopist diagnosis of Plasmodium ovale. All these samples tested positive for P. falciparum by PCR. These cases were equally distributed between groups (5/88 (5.7 %) iNO and 6/92 (6.5 %) placebo, p = 1.0). No alternative diagnosis was apparent (blood culture negative in all cases) and two patients with negative microscopy died.

The median (IQR) linear rate of change of Ang-2 over the first 72 h of hospitalization was −2.1 (−3.1 to −1.2) ng/mL/day in patients receiving iNO vs −1.9 (−3.6 to −0.57) ng/mL/day in patients receiving placebo (p = 0.68). A LME model did not show a statistically significant effect of iNO on the rate of change of Ang-2 over time (p = 0.72). Figure 2a, b shows the Ang-2 levels over time for patients in the iNO and placebo groups, together with best fit curve from the LME model.

Mortality outcomes (Table 2) and survival curves (Fig. 2c) show that most deaths occurred within 48 h of admission and occurred at similar frequency in both groups. Incidence of complications during hospitalization (development of coma, deterioration of coma score, new or persistent seizures, hypoglycaemia, development of severe anaemia, and haemoglobinuria) was also similar between groups (Table 2). Ang-2 levels were higher at admission among patients who subsequently died compared to survivors (median (IQR) 20 (8.8–29) ng/mL among fatal cases vs 8.9 (4.9–14) ng/mL among survivors; p = 0.0068). Co-treatments, administered by physicians blinded to randomization arm, were similar between groups, with the exception of anticonvulsants (Table 2). In particular, the use of antibiotics was not statistically significantly different between groups (82/87 (94 %) iNO and 80/91 (88 %) placebo, p = 0.19).

Among survivors, the recovery times (time to eat, sit unsupported, localize pain, fever resolution, recovery of consciousness, and discharge) were similar between groups (Table 3). Parasite clearance kinetics with artesunate were unaffected by iNO (Table 3). No parasite recrudescence or re-infection was detected at day 14 follow-up in either group. At the time of discharge, 5/88 (5.7 %) iNO recipients and 8/92 (8.7 %) placebo recipients had neurologic deficits, including inability to sit, spastic or flaccid paresis of one or more limbs, seizures, unilateral weakness, vision loss, gaze palsy, and poor head control.

Adverse events were systematically recorded using standardized tables on a daily basis in all patients (Table 4). The study gas was temporarily or permanently discontinued in 19/88 (22 %) iNO patients vs 12/92 (13 %) placebo (p = 0.13) for reasons shown in Table 5. In 5/88 (5.7 %) patients receiving iNO, study gas was temporarily discontinued because of elevated metHb levels. In all cases, iNO was resumed after metHb levels declined and iNO therapy did not need to be permanently discontinued in any patient due to recurrent or refractory methaemoglobinaemia. The study gas did not need to be titrated downward or discontinued in any patient for elevated inhalational levels of NO₂.

Evaluation of the quality of blinding and indices of the quality of clinical care provided in the trial are provided in Additional file 1.

iNO at 80 ppm (iNO₈₀) by non-rebreather mask was safe but did not accelerate the decline in circulating Ang-2 levels during the first 72 h of hospitalization in this study of African children with severe malaria. No differences in clinical outcomes (e.g., mortality, recovery times) were observed, likely because a measurable biological effect on the endothelium was not achieved with this dose and route of administration of NO. Of note, parasite clearance under artesunate treatment was not affected by the addition of iNO.

Among survivors of severe malaria in this study, the rate of change of Ang-2 over the first 72 h of hospitalization was −2.2 ng/mL/day (iNO group) and −1.9 ng/mL/day (placebo group), very similar to that observed in a study involving Indonesian adults, at −2.7 ng/mL/day [13]. Among four patients who died but had repeated measurements of Ang-2, the level increased on average by +15 ng/mL/day, compared to +9.5 ng/mL/day in the study from Indonesia [13]. Moreover, elevated Ang-2 levels at admission were strongly predictive of mortality in this study and in several previous reports from several populations [12, 26, 28, 29]. These findings lend further validity to Ang-2 as a clinically informative biomarker for prognosis in children with severe malaria that can be used to follow the course of disease progression and as a surrogate endpoint for severe malaria studies.

In-hospital mortality was similar in the present trial to the largest published clinical trial of paediatric severe malaria to date, AQUAMAT [25]. In the placebo arm of the present trial (patients receiving artesunate alone) the mortality was 8.7 %, compared to 8.5 % in the artesunate arm of AQUAMAT. Mortality was similar despite evidence that patients in the present trial had more severe disease at presentation: 57 vs 32 % coma; 82 vs 30 % convulsions; 55 vs 30 % severe anaemia; 50 vs 16 % respiratory distress; 93 vs 62 % severe prostration, in the placebo arm of the present trial vs artesunate arm of AQUAMAT, respectively [25]. The clinical care in the present trial (see Additional file 1) was superior to that provided at Uganda’s national referral hospital by several indices: (1) 77 vs 23 % were seen by a clinician within one hour of presentation; (2) 68 vs 12 % received the first dose of anti-malarial drug within 2 h of presentation; (3) no missing supplies and two instances of lack of blood in the hospital compared to 50 % lacking an essential drug or supply needed for resuscitation; (4) 92 vs 29 % highest parental satisfaction rating with medical care [30].

Safety was objectively and blindly assessed using standardized toxicity tables. The only clinical sign that differed between patients receiving iNO₈₀ and placebo was peri-orbital oedema, which resolved without discontinuation of study gas. Increased interstitial fluid may be most evident in the loose areolar tissue around the eye in a recumbent patient, and may indicate transient alterations in vascular permeability [although there was no difference in Ang-2 levels), renal dysfunction with fluid retention (although was no difference in creatinine and rates of acute kidney injury (AKI)], or other reversible disturbances of capillary hydrostatic or oncotic pressures. As expected, levels of metHb rose in patients receiving iNO₈₀. These levels could be controlled with downward titration of the study gas and required temporary discontinuation of iNO in only 5.7 % of patients. This dose-dependent adverse event provided evidence of biological activity of the administered therapy to alter the redox state of circulating haemoglobin, although an effect on the endothelium was not observed, based on Ang-2 measurements. AKI was identified as a toxicity of iNO in a meta-analysis pooling data from multiple randomized trials involving mostly adult patients, although this effect was not evident in individual studies [21]. The rate of AKI among patients in the present study was 8.0 % in the iNO compared to 3.3 % in the placebo group, which did not represent a statistically significant difference, although the present trial, like former randomized controlled trials of iNO, may have been underpowered to detect small differences in AKI rates. Co-treatment with diazepam for acute seizure management was statistically more frequent among patients receiving iNO. The significance of this finding is unclear since the proportion of patients with new onset or persistent seizures, frequency of neurologic sequelae, and time to recovery of consciousness was similar to placebo recipients.

Efficacy of iNO₈₀ in a murine model of experimental cerebral malaria did not translate to efficacy in humans in this clinical trial; however, pre-clinical data from this model were critical for generating the hypothesis and designing the trial. In mice, unlike humans, iNO altered Ang-2 levels, decreased blood–brain barrier dysfunction, decreased brain accumulation of parasites, and improved survival [5]. Inter-species differences in iNO₈₀ pharmacokinetics may account for lack of efficacy in the present trial. Rapid conversion of iNO to nitrate and other stable adducts may have reduced bioavailable NO at the endothelium. On the other hand, iNO has been shown to exert pharmacological effects beyond the pulmonary vasculature in other studies in humans [31]. Although no evidence of efficacy of iNO₈₀ was observed, other doses, routes of administration or NO donor molecules remain viable options for future investigation. Likewise, the Ang-Tie2 pathway remains a valid target for experimental interventions, and the pre-clinical murine model remains a useful tool to test novel therapy in vivo prior to clinical trials.

Attempts to find host-directed treatments for severe malaria over the past several decades have not yet yielded an effective adjunctive therapy. iNO₈₀ did not affect mortality or alter the rate of change of Ang-2 in this study; however, targeting the endothelium to improve outcomes in severe malaria remains a viable strategy with broader implications for other life-threatening infections such as sepsis characterized by endothelial dysfunction. Alternative methods to increase NO bioavailability at the endothelial barrier (higher dose, donor molecules, route of delivery) deserve further investigation. Given the global burden of childhood malaria and the relatively high rate of morbidity and mortality despite treatment with anti-malarials, continued investigation of adjunctive therapy is warranted.