e250 | www.pidj.com The Pediatric Infectious Disease Journal • Volume 31, Number 12, December 2012
allowing survival in patients with severe and cerebral malaria. The
debate has culminated in the approval of 2 phase II randomized pro-
spective clinical trials in Uganda studying the use of inhaled NO (iNO)
as adjuvant therapy in children with severe malaria. The strategy under-
lying both trials is to use the antiinflammatory properties of iNO in the
lung and systemic circulation to “buy time” for chemical antiparasite
therapy to lower the parasite load. This article reviews the nexus of
malaria and NO biology with a primary focus on CM in humans.
There are approximately 225–600 million new malaria infec-
tions worldwide each year.2 Severe malaria is defined by the World
Health Organization as vital organ dysfunction and/or high parasite
burden in the setting of demonstrated parasitemia.3 P . falciparum
is the organism primarily responsible for severe malaria.4 Common
perturbations in severe malaria include anemia, metabolic acidosis,
hypoglycemia, respiratory failure and CM.4 CM is typically defined
as coma in the setting of parasitemia in the absence of other causes of
reduced consciousness, such as other central nervous system infec-
tions, hypoglycemia, a postictal state or ongoing seizure.4 As is true
with severe malaria, definitions of CM must balance sensitivity and
specificity in clinical and research contexts. In locations such as sub-
Saharan Africa in which incidental parasitemia is common and the
resources necessary to definitively rule out other causes of altered
mental status are often limited, reliable diagnosis of CM is difficult.5
Severe and cerebral malaria tend to occur in young children who
have not yet developed effective immunity or in adult travelers from
nonendemic regions. The overall fatality rate for patients with CM
who seek medical attention is roughly 10–40%4 and remains roughly
9% among children who receive the most effective and rapidly act-
ing therapy, intravenous artesunate.6 Children who survive CM have
an approximately 1 in 10 chance of suffering long-term neurologic
sequelae4 and a 1 in 4 chance of persistent cognitive impairment.7
NO is a free radical with protean effects in the human body.
Initially described in the vasculature as the endothelium-derived
relaxing factor, it is an important signaling molecule in diverse pro-
cesses including neurotransmission, immune system and cytokine
modulation, platelet inhibition, vascular homeostasis and regulation
of hematopoiesis. Endogenous production occurs via 3 NO syn-
thase (NOS) isoform enzymes: neuronal NOS (nNOS or NOS1),
inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or
NOS3).8 Exogenous delivery by inhalation has been used therapeu-
tically since the 1990s for a growing number of indications.9 NO
exists as a gas at physiologic temperature and pressure and has a
half-life estimated to be under 2 ms in whole blood.10
L-arginine is converted into NO and L-citrulline by NOS1,
NOS2 and NOS3. NOS1 and NOS3 are constitutively expressed
and are calcium/calmodulin-dependent,8 whereas NOS2 is only
expressed when its transcription is activated.8
NO has many sites of action. Made in the endothelium, it
diffuses into subjacent smooth muscle cells where it activates
soluble guanylate cyclase, increases intracellular levels of cyclic
Copyright © 2012 by Lippincott Williams & Wilkins
Inhaled Nitric Oxide and Cerebral Malaria: Basis of a
Strategy for Buying Time for Pharmacotherapy
Brian Bergmark,* MD, Regan Bergmark, MD, PhD,† Pierre De Beaudrap, MD, PhD,‡ Yap Boum, PhD,§
Juliet Mwanga-Amumpaire, MD,¶ Ryan Carroll, MD, MPH,|| and Warren Zapol, MD||
Abstract: There are approximately 225–600 million new malaria infec-
tions worldwide annually, with severe and cerebral malaria representing
major causes of death internationally. The role of nitric oxide (NO) in the
host response in cerebral malaria continues to be elucidated, with numer-
ous known functions relating to the cytokine, endovascular and cellular
responses to infection with Plasmodium falciparum. Evidence from diverse
modes of inquiry suggests NO to be critical in modulating the immune
response and promoting survival in patients with cerebral malaria. This line
of investigation has culminated in the approval of 2 phase II randomized
prospective clinical trials in Uganda studying the use of inhaled NO as adju-
vant therapy in children with severe malaria. The strategy underlying both
trials is to use the sytemic antiinflammatory properties of inhaled NO to
“buy time” for chemical antiparasite therapy to lower the parasite load. This
article reviews the nexus of malaria and NO biology with a primary focus
on cerebral malaria in humans.
Key Words: inhaled nitric oxide, cerebral malaria, nitric oxide synthase,
(Pediatr Infect Dis J 2012;31: e250–e254)
originally considered as solely an environmental toxin, was discov-
ered to be a physiologically critical signaling molecule in 1988.
Subsequently, NO was studied, among an enormous amount of
worldwide research, as an inhaled gas with eventual approval in
the United States for the treatment of term neonates with persistent
pulmonary hypertension. Over the past 2 decades, a growing num-
ber of studies have examined the role of endogenous NO produc-
tion in the pathophysiology of malarial disease in humans, particu-
larly severe and cerebral malaria (CM).1
These efforts have generated controversy regarding the role of
NO in malaria. Initial findings were interpreted to suggest that high
NO levels contributed to the development of CM and severe anemia,
but tended to be retrospective chemical pathology correlations. More
recent work has indicated that NO is, in fact, likely to be critical in
lasmodium falciparum has coevolved with Homo sapiens and
its predecessors for at least 70,000 years. Nitric oxide (NO),
Accepted for publication May 01, 2012.
From the *Department of Medicine, †Department of Surgery, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA; ‡Institut de
Recherche pour le Développement, Marseilles, France; §Epicentre Research
Base; ¶Mbarara University, Mbarara, Uganda; and ||Department of Anesthe-
sia, Critical Care and Pain Medicine, Massachusetts General Hospital, Har-
vard Medical School, Boston, MA.
The authors are members of the team leading one of the clinical trials that will
evaluate the use of inhaled nitric oxide as adjunctive therapy in cerebral
malaria in Uganda. The authors have no other funding or conflicts of interest
BB and RB contributed equally to this manuscript.
Address for Correspondence: Brian Bergmark, MD, Brigham and Women’s
Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.
The Pediatric Infectious Disease Journal • Volume 31, Number 12, December 2012 Inhaled Nitric Oxide and Malaria
© 2012 Lippincott Williams & Wilkins www.pidj.com | e251
guanosine monophosphate and, thereby, lowers the intracellular
calcium concentration causing vasorelaxation. In the vascular
lumen, NO inhibits platelet aggregation, also through a cyclic
guanosine monophosphate–dependent mechanism, and has
numerous effects on endothelial adhesion molecule expression and
endothelial homeostasis. Two other mechanisms by which NO exerts
intracellular effects are through activation of adenylate cyclase
and inhibition of phospholipase C, both effected via adenosine
diphosphate ribosylation.11 Soluble guanylate cyclase activation by
NO in bone marrow–derived cells, principally monocytes and other
leukocytes, is also a critical pathway.12 Of less importance, a minority
of the NO intracellular signaling effect is due to posttranslational
S-nitrosylation of cysteine thiols on target proteins.11
Initial use of iNO was directed at pulmonary effects rather
than systemic. More recent work has demonstrated systemic effects
of iNO, initially in animals, such as reducing cardiac ischemia-rep-
erfusion injury in mice.12 In humans, evidence of systemic effects
of iNO has been seen in hepatic ischemia-reperfusion injury.13 As
in CM, low NO bioavailability in hepatic ischemia mediates hepatic
ischemia-reperfusion injury via cytokine disregulation, increased leu-
kocyte adhesion and decreased endothelial function with increased
microcirculatory tone.13 Among patients undergoing orthotopic liver
transplantation, the use of iNO at 80 ppm was associated with more
rapid restoration of liver function posttransplant, decreased hospital
length of stay and a 75% reduction in hepatocellular apoptosis.13
Catabolism of NO takes place through multiple pathways.
NO reacts with oxyhemoglobin to produce methemoglobin and
nitrates, and also reacts with the superoxide anion to create perox-
ynitrite.11 Additionally, NO is degraded enzymatically by peroxi-
dases.11 Nitrates and nitrites, typically denoted NOx, are frequently
measured as the breakdown products of NO.
Numerous challenges exist in the reliable and physiologically
relevant study of NO. Because of its short half-life and propensity
for diffusion, NO tends to exert effects near the site of production.
It is difficult, however, to measure NO levels directly at the site of
action in vivo. Additionally, nitrate and nitrite levels in plasma or
other body fluids, such as cerebrospinal fluid are influenced not only
by NO production, but also by diet, renal function and the array of
catabolic pathways available to NO. Debate continues regarding the
relevance of transport in vivo of NO and its potential for distant
effects, including transport bound to hemoglobin or thiol residues.14
Multiple pathways lead to low NO bioavailability in malaria
infection, including hemolysis with release of NO-scavenging cell-
free oxyhemoglobin and arginase,15 as well as inflammatory pro-
duction of superoxide, which reacts with NO to produce peroxyni-
trite. Anstey et al16 demonstrated an inverse relationship between
disease severity and monocyte NOS2 activity in the presence of
high levels of interleukin (IL)-10. Concurrently, however, tumor
necrosis factor (TNF) and interferon-gamma induce NOS2 mRNA
expression in bone marrow–derived cells.17
PATHOPHYSIOLOGY OF CEREBRAL MALARIA
The mechanisms by which P. falciparum infection leads
to altered mental status and coma are not completely understood,
although both disruption of microcirculatory flow and the host
immune response appear to be central processes. Although initial
attention was drawn to parasite sequestration within the cerebral
vasculature and the resulting microcirculatory flow reduction, this
explanation is no longer considered sufficient. Now under investi-
gation are the consequences of the exuberant cytokine response.
The term sequestration describes the tendency of para-
sitized erythrocytes to adhere to the endothelium as well as to other
parasitized erythrocytes (agglutination), uninfected erythrocytes
(rosetting) or platelets (platelet-mediated clumping), a finding that
is particular to falciparum malaria among the human malaria spe-
cies.1 The coma of CM can resolve, with permanent neurologic
sequelae seen in only 10% of pediatric patients who survive and
persistent measurable cognitive deficits seen in one quarter,4,7,18
which are lower rates than would be expected after ischemic injury
to the brain. Additionally, the extent and location of sequestration
found on autopsy does not correspond with high fidelity to the
severity of clinical malaria.19
Recent study of the pathophysiology of CM has focused
on the cytokine and inflammatory response to the parasite and the
interaction between the immune system and the endothelium. The
balance between proinflammatory signals (TNF, interferon-gamma,
IL-1 and IL-12) and immunomodulatory signals (IL-10, NO) is of
particular importance.20 Disease severity correlates with TNF and
IL-1 concentrations independent of degree of parasitemia.21 Fur-
thermore, patients with a TNF promoter mutation associated with
elevated TNF activity are at increased risk for mortality or serious
neurologic sequelae.22 NO is known to inhibit cytochrome C oxi-
dation via competition with oxygen and could theoretically inhibit
the neuronal respiratory cycle in the setting of low partial pressure
of oxygen.23 Angiopoietin-1 (Ang-1) depletion and Angiopoietin-2
(Ang-2) elevation have been investigated as diagnostic and prognos-
tic markers in severe and cerebral malaria. Ang-1 and Ang-2 regu-
late endothelial activation. Ang-1 is produced by endothelial cells
constitutively and appears to maintain endothelial homeostasis via
activation of the Tie-2 receptor.24 Ang-1 levels fall during inflam-
mation and endothelial activation, as is seen in sepsis or severe
falciparum malaria.24 Ang-2 is stored in endothelial Weibel–Palade
bodies from where it is released, displaces Ang-1 from the Tie-2
receptor and propagates endothelial activation, including through
adhesion molecule expression and sensitization of the endothelium
to TNF.24 Weibel–Palade bodies exocytosis is inhibited by NO, limit-
ing the release of Ang-2.25 Additionally, erythrocyte cytoadherence
is reduced in vitro by NO via inhibition of intercellular adhesion
molecule 1 and vascular cell adhesion molecule 1 expression.26
Some authors have additionally posited a central role of
increased intracranial pressure in the pathophysiology of mental
status changes and death in CM. Possible mechanisms of intracra-
nial hypertension development include increased intracranial blood
volume, decreased adenosine triphosphate availability leading to
adenosine triphosphate–dependent ion pump failure and extracel-
lular fluid shift and blood–brain barrier breakdown.1
Intracranial hypertension is undoubtedly responsible
for some patients’ neurologic deficits and deaths, and in fact
papilledema and other findings of intracranial hypertension are
significantly associated with poor outcome.27 Evidence has not
supported a critical effect of elevated intracranial pressure in all
patients, however. Cerebral edema is commonly seen on autopsy,
although studies have failed to show evidence of herniation
regularly in patients with CM who have died.28 Similarly, opening
pressure on lumbar puncture and low cerebral perfusion pressure
have not been shown to be associated with mortality.29
NITRIC OXIDE IN CEREBRAL MALARIA
Accumulating evidence suggests a protective role for NO
in severe and cerebral malaria attributed to NO’s modulating the
Bergmark et al The Pediatric Infectious Disease Journal • Volume 31, Number 12, December 2012
e252 | www.pidj.com
© 2012 Lippincott Williams & Wilkins
L-arginine concentrations fall in the settings of inflamma-
tion, hemolysis and malaria infection, and L-arginine levels vary
inversely with malaria disease severity. It is hypothesized that
the low bioavailability of arginine for NO synthesis contributes
to pathogenesis. L-arginine levels are low in patients with CM,
moderately low in patients with uncomplicated malaria and nor-
mal in healthy controls.30 Hypoargininemia caused by arginase
release from hemolyzed erythrocytes is significantly associated
with fatal outcome in patients with CM.30 Exogenous L-arginine
rescues reactive hyperemia-peripheral artery tonometry, a nonin-
vasive measurement of vascular NO production, in patients with
severe malaria.31 Asymmetric dimethylarginine levels are higher in
patients with severe malaria than in those with moderately severe
malaria and each micromolar increase of this endogenous inhibi-
tor of NOS conversion of L-arginine to NO is associated with an
18-fold increase in mortality.32
MURINE CEREBRAL MALARIA
Mice provide a reliable, although imperfect, model of CM.4
Mice deficient in NOS2 or NOS3 have equivalent courses of CM
as do controls when infected with Plasmodium berghei ANKA, and
delivery of an exogenous NO donor to NOS-deficient mice pre-
vents CM and reduces the inflammatory response.33 Similarly, CM
has been reported in a NOS2-deficient murine model with pharma-
ceutical NOS antagonism, suggesting that NO is not necessary for
CM.34 Exogenous NO as compared with saline placebo in Plasmo-
dium berghei ANKA–infected mice leads to improved pial blood
flow, fewer hemorrhagic foci and less leukocyte adherence.35 Most
recently, iNO was shown to reduce cerebral erythrocyte sequestra-
tion, endothelial activation and systemic inflammation as well as
improve survival in Plasmodium berghei ANKA–infected mice.36
Taken together, these findings demonstrate that NO is not necessary
for the development of CM and that restoring NO bioavailability
reduces the inflammatory response and improves clinical status.
POPULATION AND GENETIC STUDIES
A number of early studies found a positive correlation
between NOx in the blood, cerebrospinal fluid or urine and dis-
ease severity, yet many of these studies did not control for dietary
intake or renal function or compare results with healthy controls
within the same population.37–39 Later studies have demonstrated
lower levels of NOS2 activity, NO and NOx in children with severe
malaria compared with those with mild malaria or asymptomatic
parasitemia, suggesting that higher NOS2 activity and NO produc-
tion protect children against severe malaria.16,40,41 Although it does
appear that people in malaria-endemic regions have higher NOS2
expression at baseline and that low NOS2 levels may be associated
with severe malaria, the differences in and weaknesses of method-
ology preclude definitive conclusions as to whether NOS2 expres-
sion is correlated with disease severity.
Genetic studies have investigated whether haplotypes result-
ing in higher or lower levels of NO production lead to variable
susceptibility to severe forms of malaria. The gene for NOS2 is
NOS2A. Attention has been focused on single nucleotide polymor-
phisms (SNPs) in NOS2A, G954C and C1173T, as well as a micro-
satellite pentanucleotide repeat, CCTTT(n).42 Additional SNPs and
promoter site mutations continue to be identified.
The NOS2 promoter SNP at site G954C in the promoter
region was first discovered in 1998 and found to be associated with
less severe malaria. This polymorphism was originally described
as the Lambarene mutation at site 969, but was subsequently
referred to as the 954 mutation by the same group.43 This SNP is
more prevalent among malaria-exposed populations than among
controls.44 Ex vivo studies demonstrated a 7-fold higher NOS2
activity level in patients with this mutation,43 and in vivo studies
demonstrated higher NO production in response to 15N-arginine
infusion.45 G954C has also been shown to be protective against
infection with malaria46 and associated with high NOx levels and
lower symptomology in malaria infection.47 Not all studies have
corroborated these findings, however.47–49 An additional NOS2 SNP,
C1173T, is associated with less symptomatic malaria and increased
fasting urine and plasma NOx.50
Large diversity in the CCTTT(n) repeat number is observed,
and although shorter alleles were initially associated with worse
malaria symptoms and CM death, more recent findings demonstrate
increased disease severity with greater allele length51,52 or no corre-
lation between length and disease severity,42,50 suggesting a complex
relationship between the microsatellite repeat and disease outcome.
NO SYNTHASE 1 AND NO SYNTHASE 3
NOS1 and NOS3 have received less attention than NOS2
because they are constitutively expressed, although some evidence
supports cytokine recruitment of NOS1 in viral encephalitis.53 Yet,
although significant enrollment of these enzymes in P. falciparum
infection has not yet been demonstrated, variation in activity may
be relevant. Two NOS1 SNPs are associated with altered NOS1
expression, G84A and C276T. The former is located in the pro-
moter region of NOS1 and is associated with decreased basal activ-
ity54 and higher risk for CM.55 NOS3 mutations are correlated with
protection against CM as well as plasma NOx levels.56,57
Collectively, these findings provide strong biochemical
evidence for a protective role of NO in CM via immune response
modulation as well as robust, although not invariably positive,
epidemiologic and genetic support for this effect. Coupled with
this elucidation of the relationship between NO and Plasmodium
infection is an increasing availability of iNO therapy worldwide.
Although engineering challenges remain in the reliable production
and transportation of NO to resource-limited settings, iNO can be
produced cheaply via electrical and chemical mechanisms, and
methods of production and delivery suited to the developing world
are under active investigation.58,59
Millennia of evolutionary pressure exerted by malaria have
helped to shape the human organism. The role of NO in the host’s
response to infection has become a focus of intense investigation over
the past 20 years as the interactions between NO, malaria, the immune
system and the endothelium become increasingly understood. The
weight of evidence suggests a protective role of NO produced by
inducible NOS in severe and cerebral malaria. Demonstration of
NO’s immunomodulatory role as well as measurement of NOx
levels, NOS promoter mutations and disease severity have provided
compelling evidence that NO is essential in preventing or moderating
the severe manifestations of P . falciparum infection. After more than
70,000 years of hominid–Plasmodium coevolution, the time has
come to test directly whether inhaled NO can give infected children
the time they need to survive cerebral malaria.
The authors acknowledge the contributions of Elisabeth
Kemigisha and David Bangsberg, as well as the support of the MSF
Epicentre Mbarara Research Base.Authors’ contributions: BB and
RB contributed to the writing and editing of the manuscript. PDB,
YB, JM, RC and WZ contributed to the planning of the project and
editing of the manuscript.
The Pediatric Infectious Disease Journal • Volume 31, Number 12, December 2012 Inhaled Nitric Oxide and Malaria
© 2012 Lippincott Williams & Wilkins www.pidj.com | e253
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Trends in Drug Resistance Prevalence in HIV-1–infected Children in Madrid: 1993 to 2010 Analysis: ERRATUM
In the article that appeared on page e213 of volume 31, issue 11, the figure legends were incorrect for Figures 1 and 2. The figures and
legends are reprinted below correctly.
de Mulder M, Yebra G, Navas A, et al. Trends in Drug Resistance Prevalence in HIV-1–infected Children in Madrid: 1993 to 2010 Analysis. Pediatr Infect Dis
FIGURE 1. Prevalence of DRM positions for each drug class represented over 5% in the selected population.
FIGURE 2. Temporal trends of DRM prevalence according to drug class. PI indicates protease inhibitors; RT, reverse tran
scriptase; NRTI, nucleoside reverse transcriptase inhibitors; NNRTI, nonnucleoside reverse transcriptase inhibitors. Asterisk
indicates statistical differences between P1 (1993–1999) and P4 (2008–2010).