Non-invasive measurement of methemoglobin (MetHb)

as marker of disease severity in children with malaria

T.M. Gresnigt, intern Medisch Spectrum Twente, Enschede

E-mail: t.m.gresnigt@student.rug.nl

Supervisor: Prof. Dr. M. P. Grobusch

E-mail: m.p.grobusch@amc.uva.nl

Center of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Academic

Medical Center, University of Amsterdam, The Netherlands

Institute of Tropical Medicine, University of Tuebingen, Tuebingen, Germany

Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon

Co-Supervisor: Prof. Thomas Hänscheid

E-mail: t.hanscheid@fm.ul.pt

Institute of Molecular Medicine, University of Lisbon, Portugal

UMCG supervisor: Dr. P.F. van Rheenen

E-mail: p.f.van.rheenen@umcg.nl

Paediatric Gastroenterologist, University Medical Center Groningen

Study site:

Medical Research Unit, Albert-Schweitzer Hospital, Lambaréné, B.P. 118, Gabon

2

Prologue

02/01/1960, Tortona (Italy)

The Republic of Upper Volta was established on December 11, 1958, as a self-governing colony within the

French Community. One year after attaining autonomy, in December 1959, the president of Upper Volta,

Maurice Yaméogo, invited Fausto Coppi, Raphaël Géminiani, Jacques Anquetil, Louison Bobet, Roger

Hassenforder and Henry Anglade to ride a race in Ouagadougou against local riders and to go hunting

afterwards.

Fausto Coppi was about to end his amazingly successful career,

being already forty years old. For him, there wasn’t anything left

to win. He had been world champion, he had won the Tour de

France – Europe’s largest annually returning sports event – twice,

although the Second World War unfortunately paused his career

for six consecutive years. A war which brought him to Northern

Africa where he was taken prisoner by the British.

He was kept prisoner of war in a camp in Algeria and later on in

Tunisia. At the very end of the war the British moved him to Italy

where he was finally released.

His next trip to Africa would not be a lot better, although the

omens were not bad at all. In December 1959 he just went for

some racing with close colleagues of whom a few also won the

Tour de France, and some hunting afterwards - Coppi’s biggest

passion apart from cycling.

Géminiani remembered:

‘I slept in the same room as Coppi in a house infested by

mosquitoes. I had got used to them but Coppi had not. Well, when

I say we 'slept', that is an overstatement. It was like the safari had

been brought forward several hours, except that for the moment

we were hunting mosquitoes. Coppi was swiping at them with a

towel. Right then, of course, I had no clue of what the tragic

consequences of that night would be. Ten times, twenty times, I

told Fausto 'Do what I'm doing and get your head under the

sheets; they can't bite you there.’ Fausto Coppi at the Stelvio

Two weeks later Coppi started to have fevers, more or less during Christmas 1959, back home in Novi Ligure

with his wife Giulia and his little boy Faustino. He already lost weight, got pale, and the fevers kept coming.

That day he got a call from Gémiani. His temperature got up to 41.6 degrees Celsius and he told Coppi he’d been

delirious and couldn't stop talking for a while. He told him as well that he imagined seeing people all around,

but that he didn't recognise one of them. Things that must have sound familiar to Coppi.

In the Pasteur Institute in Paris Gémiani was successfully treated for malaria, plasmodium falciparum strain.

Coppi denied that he had malaria, and so did his doctor Ettore Allegri. Coppi had a bronchial pneumonia, just

that, according to Dr. Allegri. Gémiani got angry and said he would bring the quinine, which he was successfully

treated with himself in Paris, to Tortona just by himself if Dr. Allegri did not want to treat Coppi properly.

On second January 1960 Fausto Coppi died, forty years old, of falciparum malaria.

Back then he did not know he would be elected in 1999 as being the most popular Italian sportsman of the 20th

century.

As being a very enthusiastic cyclist I have been at what used to be his house in Castellania in 2007, and I even

managed to talk with one of the guys Coppi has been imprisoned with in Africa. Impressive story, and a great

encounter for me. I think Coppi still counts like an example for a lot of young cyclists, even nowadays.

3

Abstract

Study Site

Medical Research Unit, Albert-Schweitzer Hospital, Lambaréné, B.P. 118, Gabon

Background

Malaria infection interferes with hemoglobin by disruption of red blood cells and by

conversion of hemoglobin to methemoglobin (MetHb). Methemoglobin is the oxidized form

of hemoglobin, which does not bind oxygen. Based on a murine model of a previous study, it

is expected that in case of cerebral malaria, hemoglobin is oxidized to MetHb which then

releases the highly inflammatory heme, which in turn triggers the release of cytokines and

other mediators and contributes to the picture of ‘cerebral malaria’. The aim of this study is

to establish a correlation between MetHb levels and differences in the severity of malaria, and

whether MetHb could therefore be a marker for disease severity in malaria patients.

Patients, Material and Methods

Children aged 12 to 120 months were recruited at the Medical Research Unit of the Albert

Schweitzer Hospital (MRU-HAS) in Lambaréné/Gabon, as well as at the outpatient clinic of

the paediatric ward of the HAS and a research outpost in Fougamou. For the control group,

healthy volunteers were recruited at the kindergarten of the HAS; children with other diseases

were recruited at the outpatient clinic of the paediatric ward. This cross-sectional study

comprises three groups, consisting of a control group and two groups of patients with malaria,

subdivided into uncomplicated malaria and severe malaria, which is defined as severe anemia

(Hb < 6 g/dl) or cerebral malaria (BCS<4). MetHb levels were non-invasively determined on

presentation using a Masimo® Rad-57 Pulse oximeter.

Results 103 children with malaria were measured, of which 14 had severe disease (13,6%); 9 severe

anemia (8,7%) and 5 cerebral malaria (4,9%). 88 controls were measured. MetHb levels of

children with malaria, non severe (1,8 ± 0,3) and severe (2,1 ± 0,7) are demonstrated to be

significantly elevated compared to controls (1,6 ± 0,3). We did not demonstrate a significant

correlation between parasitemia and MetHb values, nor between Hb and MetHb values.

Conclusion

In conclusion, this study has demonstrated that, although small in number, the MetHb level is

significantly elevated in severe cases, and cerebral malaria cases in particular compared to a

control group and an uncomplicated malaria group. To elucidate the value of MetHb as a

potential (prognostic) marker of disease severity and hence the value of routine measurement

of MetHb in children with malaria, further research is warranted.

4

Nederlandse samenvatting

Studieplaats

Medical Research Unit, Albert-Schweitzer Hospital, Lambaréné, B.P. 118, Gabon

Achtergrond

Een infectie met malaria interfereert met hemoglobine door de verstoring van de rode

bloedcel en tevens door conversie van hemoglobine naar methemoglobine (MetHb).

Methemoglobine is de geoxideerde vorm van hemoglobine, die niet in staat is om zuurstof te

binden. Gebaseerd op een model in muizen uit een voorgaande studie wordt verwacht dat bij

cerebrale malaria hemoglobine wordt geoxideerd tot MetHb, wat dan het zeer inflammatoire

haem vrij doet komen, wat op zijn beurt het vrijkomen van cytokines en andere mediatoren

induceert en daarbij aan het beeld van ‘cerebrale malaria’ bijdraagt. Het doel van deze studie

is om een correlatie aan te tonen tussen MetHb niveaus en het verschil in ernst van de malaria,

en tevens of MetHb daardoor mogelijkerwijs kan fungeren als een marker voor een ernstiger

verloop van de ziekte bij malaria patiënten.

Patiënten, Materiaal en Methode

Kinderen van 12 tot 120 maanden werden gerekruteerd in de Medical Research Unit van het

Albert Schweitzer ziekenhuis (MRU-HAS) in Lambaréné/Gabon, zowel op de polikliniek

kindergeneeskunde als in een buitenpost van het ziekenhuis in Fougamou. Voor de controle

groep werden gezonde vrijwilligers gerekruteerd in de crèche, en tevens werden er kinderen

met andere pathologie gerekruteerd op de polikliniek kindergeneeskunde. Dit

dwarsdoorsnede onderzoek omvat drie groepen, bestaande uit een controle groep en twee

groepen met patiënten met malaria, onderverdeeld in ongecompliceerde malaria en ernstige

malaria, wat gedefinieerd werd als ernstige anemie (Hb < 6 g/dl) of cerebrale malaria

(BCS<4). MetHb niveaus werden non invasief gemeten met een Masimo® Rad-57 Pulse

oximeter.

Resultaten 103 kinderen met malaria werden gemeten, van wie 14 ernstige verschijnselen hadden

(13,6%); 9 hadden ernstige anemie (8,7%) en 5 hadden cerebrale malaria (4,9%). Er werden

88 controles gemeten. MetHb niveaus van kinderen met malaria, ongecompliceerd (1,8 ± 0,3)

en ernstig (2,1 ± 0,7) waren aantoonbaar significant verhoogd vergeleken met controles

(1,6 ± 0,3). We hebben geen significante correlatie aangetoond tussen de parasitaire load en

de MetHb niveaus, en ook niet tussen de Hb en MetHb niveaus.

Conclusie

Concluderend heeft deze studie aangetoond dat, al hoewel het aantal relatief klein is, dat

MetHb niveaus significant verhoogd zijn in geval van ernstige malaria, en cerebrale malaria

in het bijzonder vergeleken met een controle groep en een ongecompliceerde malaria groep.

Om de waarde van MetHb als mogelijke marker van de ernst van de malaria infectie te

verduidelijken, en daarbij ook de waarde van routine bepaling van MetHb bij kinderen met

malaria, is verder onderzoek aangewezen.

5

List of abbreviations

BCS: Blantyre Coma Scale

CO: Carbon monoxide

CNS: Central Nervous System

DIC: Disseminated Intravascular Coagulation

HAS: Hôpital Albert Schweitzer

Hb: Hemoglobin

HO-1: Heme-Oxygenase 1

IQR Interquartile Range

MetHb: Methemoglobin

MRU: Medical Research Unit

P.: Plasmodium

RBC Red blood cell

ROS: Reactive Oxygen Species

SD Standard Deviation

WHO: World Health Organisation

TNF: Tumor Necrosis Factor

6

Index

Prologue 2

Abstract 3

Nederlandse samenvatting 4

List of abbreviations 5

Index 6

Introduction 7

Patients, Material and Methods 14

Results 18

Discussion 20

Conclusion 22

Figures 23

References 26

7

Introduction

Malaria is a mosquito-borne infectious disease of humans caused by parasites of the genus

Plasmodium (phylum Apicomplexa). About a hundred different species of Plasmodium spp.

infect a wide range of vertebrates. Five species of plasmodia can infect and be transmitted by

humans; P. falciparum, P. malariae, P. ovale (subspecies curtisi and wallikeri), P. vivax and

P. knowlesi1. While P. vivax is responsible for the largest number of malaria infections

worldwide, infections by P. falciparum account for about 90% of the deaths from malaria2.

Disease caused by Plasmodium vivax, ovale and malariae is generally a milder disease that is

rarely fatal. Plasmodium knowlesi is a zoonosis that causes malaria in macaques but can also

infect humans3.

Distribution and burden of disease

Worldwide malaria is the most important parasitic disease and is endemic in 105 countries.

About 3.3 billion people, almost half of the world’s population, are at risk of malaria. In the

last decade the incidence of malaria has begun to fall on a global scale, but it is rising again in

some areas, mainly due to antimalarial drug and insecticide resistance. There were an

estimated 225 million cases of malaria worldwide in 20094 (see figures at page four for spatial

distribution of Plasmodium falciparum and the clinical burden in Gabon as well as

worldwide). The estimated case number in Gabon was 677.000 cases in 2007 in a population

which is about double that size (see table 1, page 8).

Gloabally, an estimated 655,000 people died from malaria in 2010,5 a 5% decrease from the

781,000 who died in 2009, accounting for 2.23% of deaths worldwide.4 Ninety percent of

malaria-related deaths occur in sub-Saharan Africa, with the majority of deaths being young

children. Plasmodium falciparum is responsible for the vast majority of deaths associated with

the disease6. Especially children and pregnant women are vulnerable.

The basics of malaria parasitology

The malaria parasite is transmitted man to man by the female Anopheles mosquito during the

blood meal. After the vector has injected the parasites into the host, they undergo a clinically

silent replication phase in the liver (hepatic phase) after which the new generation of parasites

invade red blood cells (erythrocytic phase). In the blood, they multiply until they eventually

cause erythrocyte rupture, liberating new parasites, parasite products and any remaining,

undigested haemoglobin. The duration of this cycle (see figure 19) depends on the

Plasmodium spp; and in the case of P. falciparum it takes around 48 hours. P. falciparum

does not have any dormant liver stage (hypnozoites), whilst P. ovale and P. vivax do have,

causing relapses.

8

Plasmodium falciparum distribution and clinical burden globally and in Gabon

Global clinical burden of P. falciparum in 2007. 7

The spatial distribution of Plasmodium Clinical burden of Plasmodium falciparum map in 2007 in

falciparum malaria endemicity map in Gabon.7

2010 in Gabon.7

Lambaréné The map at the left shows estimated levels of Plasmodium falciparum malaria endemicity within the limits of

stable transmission. The mapped variable is the age-standardised P. falciparum Parasite Rate (PfPR2-10) which

describes the estimated proportion of 2-10 year olds in the general population that are infected with P.

falciparum at any one time, averaged over the 12 months of 2010. 7

The map at the right shows the mean predicted clinical burden of Plasmodium falciparum malaria in terms of

the number of clinical cases in people of all ages per year per 5km2.

7

Estimated P. falciparum clinical cases (thousands) by country in 2007, Gabon:

Clinical cases (1000s) 95% credible intervals (1000s) Population of Gabon (1000s)

estimated (2009)

677.0 346-1,085 1,475

Table 1. Malaria Atlas Project7

Malaria – pathophysiological basics and clinical picture

Symptomatic Plasmodium falciparum disease (malaria) usually arises 7 to 14 days after

infection8, but onset of symptoms may be delayed for months.

Malaria is not a simple disease of fever and chills. In fact, in a malarious area it can present

with such a variety of manifestations that malaria may have to be considered as a differential

diagnosis for a wide range of clinical problems.

9

All the clinical features of malaria are caused by the erythrocytic schizogony in the blood, and

therefore clinical symptoms occur only during the erythrocytic phase. The growing parasite

progressively consumes and degrades intracellular proteins, principally hemoglobin, resulting

in formation of the malarial pigment hemozoin and hemolysis of the infected red cell.

This also alters the transport properties of the red cell membrane. The red cell becomes more

spherical, less deformable and therefore more rigid. The rupture of red blood cells by

merozoites releases certain factors and toxins, which directly induces the release of cytokines

such as TNF and interleukin-1 from macrophages, resulting in chills and high-grade fever.

This occurs once in 48 hours, corresponding to the erythrocytic cycle.

Most commonly, symptoms in malaria are rather unspecific, including fever (with high spikes

when the erythrocytes rupture) headaches, abdominal aches, vomiting, diarrhoea, myalgia and

general fatigue. Children often present in prostration10

Anaemia is a fairly common problem encountered in malaria. Whilst not constituting a major

problem with most cases of imported malaria, as the direct parasitic effect is limited even in

severe disease in most cases, it is a major problem in young children who become

increasingly anemic following several consecutive malaria episodes in the face of co-

infections with intestinal helminths (and also in a subgroup with HIV). The anemia is mostly

normocytic and normochromic, and multifactorial: splenic sequestration contributes most,

followed by direct bone marrow suppression of the red cell line. Repeated hemolysis of

infected red cells further contributes to the reduction of hemoglobin levels.

Thrombocytopenia is also fairly common in malaria; most importantly, the remaining

thrombocytes retain full functionality, i.e. malaria patients, as a rule of thumb, ‘don’t bleed’,

except for that complications such as DIC is encountered in case of bacterial co-infection in

severe disease. Thrombocytopenia may be related to the sequestration of the platelets in the

spleen. Often an enlarged liver as well as an enlarged spleen are seen in patients with malaria.

The liver is usually firm and may be tender. It is oedematous, coloured brown, grey or even

black as a result of deposition of malaria pigment. The enlargement is caused by hepatic

sinusoids which are dilated and contain hypertrophied Kupffer cells and parasitized red cells.

Splenomegaly is a common feature in all types of malaria. The early enlargement of the

spleen is due to engorgement, oedema of the pulp and later due to lymphoid and reticulo-

endothelial hyperplasia with an increased hemolytic and phagocytic function of the organ.

Frequent relapses and re-infections lead to pulp sclerosis and dilated sinuses. Splenic rupture

is a serious complication of malaria, and happens most commonly in vivax malaria. However,

splenic rupture is rather rare in falciparum malaria. Other features of malaria comprise signs

of hypoglycemia and hyperlactatemia. The lactate levels in cerebral spinal fluid (CSF) are

high in patients with cerebral malaria, and significantly higher in fatal cases than in survivors.

Also hemoglobinuria with renal failure may occur.

In a comparably small proportion of cases, malaria can progress to severe forms and

eventually death. Severe malaria has been defined by the WHO and include severe anaemia

(Hb<5g/dL) and cerebral malaria11

. In the most severe cases of the disease, fatality rates can

exceed 20%, even with intensive care and treatment.12

Although survivors usually recover

fully; over the longer term, developmental impairments have been documented in children

who have suffered episodes of severe malaria.13

10

Figure 1 Malaria cycle

9(CDC)

P. falciparum infection, if treated promptly and appropriately, generally follows a relatively

mild course though. However, without effective therapy, it can become life-threatening,

especially in young children. Two important features distinguish falciparum infection from

the ‘benign’ malarias and account for the differences in severity:

- only P. falciparum has the ability to invade red blood cells of all ages, and with repeated

cycles of development within the red cells, the parasite numbers exponentially grow into very

high parasite burdens if the infection is uninhibited by treatment or host immunity.

- only falciparum malaria demonstrates ‘sequestration’ in the microvasculature of red cells

containing mature parasites. Sequestration results mainly from cytoadherence of parasitized

red cells to endothelial cells in the post capillary venules of critical organs including the brain,

in the face of decreased deformability of both parasitized and unparasitized red cells,

autoagglutination of parasitized cells, and following suit to adherence of unparasitized red

cells to parasitized red cells (rosetting) .14

Sequestration of the growing P. falciparum parasites in these deeper tissues provides them the

microaerophilic venous environment that is better suited for their maturation.15

The adhesion

11

to endothelium allows them to escape clearance by the spleen and to hide from the immune

system. These factors help the falciparum parasites to undergo unbridled multiplication,

thereby increasing the parasite load to very high numbers. Due to the sequestration of the

growing parasites in the deeper vasculature,only the ring-stage trophozoites of P. falciparum

are seen circulating in the peripheral blood (see figure 2), while

the more mature trophozoites and schizonts are bound in the deep

microvasculature, hence seldom seen on peripheral blood

examination.15

The adhesiveness is viz genuinely better for the

more mature parasites, because the parasites need the reduced

oxygen tension of the post gas exchange capillary to replicate.

If the cytoadherence-rosetting-sequestration of infected and

Figure 2 uninfected erythrocytes in the vital organs goes on uninhibited,

Ring stages in P. falciparum ultimately blocks blood flow, limits the local oxygen supply,

it hampers mitochondrial ATP synthesis, and consequently

lactate levels will rise due to anaerobic glycolyis and stimulates cytokine production - all

factors contributing to the development of severe disease.16,17

As a result complications such as cerebral malaria, hypoglycemia, metabolic acidosis, renal

failure, and respiratory distress are most commonly seen in P. falciparum infections. 15,17

Severe malaria thus often manifests as a serious multisystem disease and can lead to death if

untreated. The onset of severe disease can be rapid, with death, particularly in children,

occurring in a matter of hours.8

Cerebral malaria is the most important complication and cause of death in severe P.

falciparum infection. In falciparum malaria, 10% of all admissions and 80% of deaths are due

to the CNS involvement. Manifestations of cerebral dysfunction include any degree of

impaired consciousness and focal and generalized convulsions. The neurological dysfunction

can manifest suddenly, following a generalized seizure or gradually over a period of hours.

It is important to meticulously assess CNS involvement, and cerebral malaria is mainly

assessed by the Glasgow Coma Scale for adults; in case of children most often by the

Blantyre Coma Scale. Cerebral malaria is, according to the WHO severe malaria criteria,

defined as a Glascow Coma Scale <11/15 for adults or a Blantyre Come Scale <4/5 in pre-

verbal children not attributable to any other cause (seizure within 30 minutes, hypoglycemia

or sedative drugs or non-malarial cause) in a patient with P. falciparum malaria. Lumbar

puncture is encouraged to rule out bacterial meningitis, if suspected.

Cerebral malaria carries a mortality of around 15% in children. Hence, identification of

people that have malaria and that might eventually develop cerebral malaria is of importance

to institute adequate, intensive treatment as early as possible.

Despite considerable research efforts, the pathogenesis of severe malaria is still not fully

understood. Early on it was thought that the pathogenesis of malaria was merely caused by the

destruction of RBC during the parasite replication; although this did not explain well the

appearance of cerebral symptoms. Later on it was discovered that infected RBC sequester in

the capillaries of internal organs, including the brain, which led to the idea that this

“mechanical” obstruction of clogging of the cerebral microcirculation by the parasitized red

12

cells was the cause of cerebral malaria18

. Vascular permeability is found to be mildly

increased, however, no definite evidence of cerebral edema has been found on imaging

studies. 80% children with cerebral malaria have raised intracranial pressure, due to increased

cerebral blood volume and biomass rather than increased permeability.

Today, the discovery of cytokines and other mediators, as well as a better understanding of

the inflammatory processes led to the generally accepted theory that severe malaria is the

result of an unchecked, “out-of-balance” inflammatory process19

. However, it remains unclear

what does trigger this inflammation and how it is exactly controlled.

Methemoglobin and its possible in severe disease

One possible explanation may be the hemoglobin/methemoglobin/heme hypothesis20

.

Other investigators have shown recently, in a murine model (with all its limitations), that

cerebral malaria was associated with the heme-oxygenase system and the presence/levels of

methemoglobin and free heme 21

. Higher levels of methemoglobin and heme were correlated

with severe malaria.

Hemoglobin molecules contain iron within a porphyrin heme structure. The iron in

hemoglobin is normally found in the (ferrous) Fe++ state. The iron moiety of hemoglobin can

be oxidized to the (ferric) Fe+++ state to form MetHb.22

MetHb has a decreased affinity for

oxygen, resulting in an increased affinity of oxygen to other heme sites and overall reduced

ability to release oxygen to tissues. The oxygen–hemoglobin dissociation curve is therefore

shifted to the left. When methemoglobin concentration is elevated in red blood cells, tissue

hypoxia can occur. Because RBCs are bathed in oxygen, a certain amount of physiologic

methemoglobin formation occurs continuously.22

Several endogenous reduction systems exist

to maintain MetHb in the reduced state, and in normal individuals only about 1% of total

hemoglobin is MetHb at any given time.22

Symptoms of methemoglobinemia are written

down in figure 3.

The most common cause of a raised level of methemoglobin is ingestion or skin exposure to

an oxidizing agent. The second most common cause of methemoglobinemia is idiopathic, but

related to systemic acidosis. Another common causes are inborn errors of the metabolism,

especially glucose-6-phosphate

dehydrogenase (G6PD) deficiency.23

MetHb can be determined with a pulse

oximeter. However, a pulse oximeter

measures light absorbance at only 2

wavelengths: 660 nm and 940 nm.24,25

The device measures the pulsatile- and

background light absorbance to create a

pulse-added absorbance at each

wavelength .

This pulsatile absorbance corresponds to

arteriolar contributions to absorbance Figure 3

above the tissue and venous background Symptoms associated with MetHb blood concentrations23

and primarily reflects arteriolar Reproduced from Wright et al.

haemoglobin absorbance. Both oxy- and

deoxyhemoglobin absorb light at 660 and 940 nm; it is the ratio of the absorbance at the 2

wavelengths from which a pulse oximeter determines oxygen saturation. MetHb absorbs light

almost equally at both 660 and 940 nm.23

Therefore a more sophisticated pulse oximeter is

13

required for non invasive measurement of MetHb.

Malaria is associated with major RBC destruction,

mainly caused by consumption and subsequent rupture

of infected RBCs and to a reduction on the life-span of

uninfected RBC. Thus, a significant amount of

haemoglobin (Hb), HbFe2+

, is released. In the presence

of reactive oxygen species (ROS), Hb is readily oxidized

into methemoglobin (MetHb), HbFe3+

. MetHb is very

unstable and rapidly releases free heme, which is thought

to trigger a wide range of pro-inflammatory signals that

activate the immune system26

. Preliminary data from

mouse models showed that MetHb appears to be an

important part of the pathogenesis of cerebral malaria 27

(figure 4).

The homeostasis of Hb/MetHb is maintained by the RBC

MetHb reductase system28

, while the Hb/heme

equilibrium is maintained by various cellular defense

mechanisms, such as haptoglobin, albumin, and

hemopexin26

. However, in situations of hemolysis, as

they occur in malaria, physiological scavengers may

become saturated, and unable to exert their protective

effects. HO-1 is inducible by numerous stimuli,

including heme, and works as an additional protective

system of tissue/cell against free heme-mediated

Figure 4 21 oxidative stress and inflammation.

Pathogenesis of ‘cerebral malaria’ and the mechanism underlying the protective actions of HO-1 and CO.

Pamplona et al. showed that cerebral malaria was associated with the expression and activity

of the heme-oxygenase 1 (HO-1) system21

. They showed that mice with higher activity of

HO-1 are protected from cerebral malaria; an effect that can also be achieved by

administering carbon monoxide (CO). In fact, the anti-inflammatory effects of CO and the

HO-1 system have been shown in many other inflammatory conditions. Pamplona et al. found

that mice with cerebral malaria had increased levels of MetHb and free heme in the peripheral

blood. This led to the hypothesis that possibly haemoglobin is oxidized to MetHb which than

releases the highly inflammatory heme, as reviewed recently by the proponents.

Furthermore, a previous study measured MetHb in humans with and without malaria in

Africa29

. Significant differences were reported between individuals that had no malaria,

uncomplicated malaria and those with severe malaria (with cerebral malaria patients showing

the highest levels of MetHb). Anecdotal data from European intensive care units suggest that

MetHb levels are raised in patients with cerebral malaria (M.P. Grobusch, personal

communication). Consequently, methemoglobin as a molecule, and thus measuring levels of

MetHb may be very important for several reasons. MetHb may be causally involved in the

chain of events that leads to severe malaria and especially cerebral malaria. Therefore the

measurement of MetHb levels in human blood may allow to determine which patients are at

increased risk of developing severe/cerebral malaria. The aim of this study is to investigate

the correlation between MetHb levels and differences in the severity of malaria and whether

MetHb could therefore be a prognostic marker for disease severity in malaria patients.

14

Patients, Material and Methods

Study design

We designed a cross-sectional study comprising three groups, consisting of a control group

and two groups of patients with malaria, subdivided into uncomplicated malaria and severe

malaria.

Study setting

After obtaining informed oral consent from the primary caretaker, children were recruited

from ongoing trials at the Medical Research Unit of the Albert Schweitzer Hospital (MRU-

HAS) in Lambaréné, as well as at the outpatient clinic of the paediatric ward of the Albert

Schweitzer Hospital and a research outpost in Fougamou, 80 kilometers south of Lambaréné.

Study population

The inclusion and exclusion criteria are specified in table 2.

For the control group healthy volunteers were recruited at the kindergarten of the Albert

Schweitzer Hospital; children with other diseases were recruited at the outpatient clinic of the

paediatric ward.

Time frame

The study took place during the long rainy season in Gabon (February-May 2012), in which

period malaria incidence peaks.

Table 2 – Inclusion and exclusion criteria of study subjects

Inclusion Exclusion

Children aged 1-10 years Children <1 years, or > 10 years

Children diagnosed with malaria confirmed

by thick blood smear identification of

malarial parasites of either Plasmodium spp.

Children with sickle cell

Children with hemoglobinopathies

Children with comorbidity like tuberculosis,

measles, meningitis, leptospirosis.

Children with snake envenoming

Children with documented G6PD deficiency

Materials

MetHb levels were determined by non-invasive pulse-oximetry using a Masimo® Pulse

Oximeter (Rad -57 ‘rainbow’, Masimo Inc., Irvine/CA/USA). This pulse oximeter uses eight

wavelengths of light rather than the two wavelengths used by conventional pulse oximeters.

The Rad-57 can noninvasively and immediately measure both MetHb and

carboxyhemoglobin, as well as conventional SpO2.

Definitions

Uncomplicated malaria was defined as fever (temperature ≥ 38.0 degrees Celsius, measured

tympanically) plus a positive thick blood smear (any parasitemia in 100 visual field screened).

Severe malaria is defined as fever plus a positive thick blood smear, whereby malarial disease

15

is defined according to the WHO guidelines. In this study, severe malaria was defined as

malaria complicated by severe anemia (Hb<6,0 g/dl) or cerebral malaria (Blantyre Coma

Scale <4) (see table 3).

As controls, patients with other diseases, involving directly or indirectly the CNS (for

example viral or bacterial meningitis) and healthy individuals were recruited, age-sex

matched.

Measurements

MetHb levels were determined on presentation. Twenty-four hour measurements were

obtained in case of hospitalisation.

After obtaining informed consent from the primary caretaker, the MetHb level was

determined non-invasively, using a Masimo® Pulse Oximeter.

The procedure consists of placing a small measurement probe on the third finger of the right

hand and keeping it there for about one minute. The results can be read off the instrument.

Measurements were carried out in triplicate, and thereafter the average was calculated.

©Photo: T.M. Gresnigt

Furthermore the Blantyre Coma Scale was determined as well as the hemoglobin (ABX

pentra 60, Horiba Medical, Montpellier/France) level and the parasitemia determined by a

thick blood smear. After fixating the thick blood smear with methanol, and staining it for

twenty minutes with Giemsa (Merck, Darmstadt, Germany), the parasitemia was determined

by light microscopy by the Lambaréné method for counting slides30

. Slides of the thick blood

smear were read in duplicate by two different, independent investigators.

16

Table 3 Blantyre Coma Scale

The Blantyre Coma Scale is a modification of the Pediatric Glasgow Coma Scale and was designed in 1987 to

assess malarial coma in children; it is suitable to use in preverbal children31

Blantyre Coma Scale

Eye movement Best motor response Best verbal response

2 – Localizes painful stimulus

(e.g. pressure with blunt

end of pencil on sternum

or supraorbital ridge)

2 – Cries appropriately with

pain, or, if verbal, speaks

1 – Watches or follows

(e.g. mother’s face)

1 – Withdraws limb from

painful stimulus

1 – Moan or abnormal cry

with pain

0 – Fails to watch or follow 0 – No response or

inappropriate response

0 – No vocal response to pain

Data management, data storage and analytical strategy

The demographic characteristics as patient ID, age and sex were documented on a case report

form, as well as the BCS score, hemoglobin level and parasitemia and the obtained data from

the three measurements. Afterwards this was anonymously entered into a database (Microsoft

Office Excel, Microsoft, Redmond/WA/USA) for analysis.

Statistical analysis

Sample size was calculated based on an approximate sample size calculation for means of

unpaired data, with a power (1-β) of 80% and a two sided significance level (α) of 5%. The

sample size (m) is 62 patients in each group, according to the formula:

m=( 2(Ζα+Ζ2β)2σ

2/δ

2 ).

Analysis of the data was performed with SPSS version 15.0 (IBM Corporation, Armonk/

NY/USA).

MetHb measurements have been corrected for outliers, which we have determined as a

difference of ≥ 0,4 of one of the measurements within the three performed measurements per

included child. The parasitemia was transformed to logarithmic10 scale for analysis.

To detect differences in MetHb between the three groups the Student t-test was used. Within

all comparisons, the differences on a level of p < 0.05 were considered statistically

significant.

Financing, Grants and Conflict of Interest

No particular grant has been applied for. The travel-related cost were covered by the sending

institution, cost for housing was covered by the hosting institution. Other project-related cost

were covered by Prof. Dr. M.P. Grobusch’s Lambaréné research group budget.

The equipment has been provided by industry, with no further obligation. The sponsor had no

influence on study design, the conduction of the study, the data analysis nor the interpretation

of the data. The authors declare no conflicts of interest.

17

Ethics

This study was approved by the institutional review board of the Albert Schweitzer Hospital.

The data were collected and recorded by the investigators in such a manner that subjects can

not be identified, directly or through identifiers linked to the subjects. The obtained data are

treated confidentially.

A blood sample and a thick blood smear are routinely performed for febrile children who

show up at the paediatric ward. No study-specific additional invasive measurements were

performed.

18

Results

We have non-invasively measured the MetHb of 103 children with malaria using the

Masimo® Rad-57 Pulse Oximeter, and measured, in an identical fashion, 88 controls.

Fourteen of the 103 [13,6%] malaria cases were severe, of which nine had severe anemia (Hb

≤ 6,0 g/dl) [8,7%], and five had cerebral malaria [4,9%] (For baseline characteristics of the

three separate groups see table 4). We measured 88 controls, sex- and age-matched, without

malaria. There were no significant differences between the groups based on age, sex or

parasitemia. All data were collected in a database (Microsoft Office Excel, Microsoft,

Redmond/ WA/USA).

Table 4 Baseline characteristics of the groups

Controls

[n: 88]

Non severe

Malaria

[n: 89]

Severe

malaria

[n: 14]

Male (n) 54

[61,4%]

52

[58,4%]

10

[71,4%]3

Age (months)

Male

Female

Total

48.8

44.0

46.9

51.5

50.4

51.0

40.9

39.8

40.6

Parasitemia

(pf/µL)

- 13416 ±

(IQR: 83649) 1,2

26400 ±

(IQR: 61730) 1,2

Values are expressed as mean, or median ± IQR 1 the parasitemia of nine cases is missing [ 8,7%]

2Data have been log10 transformed.

3 One cerebral case was not measured at admission.

The BCS and the Hb do not appear in the baseline characteristics table, since they define the

groups. The only group in which the BCS varied was the cerebral malaria group, obviously.

In the cerebral malaria group one of the cases was admitted with a BCS of 5, but dropped

rapidly to a score of 3. Another cerebral case was not measured at admission, but arrived with

a BCS of 2 in the HAS after referral from a neighbouring hospital, where first admission was

done.

The mean ± SD of the Hb in the non severe malaria group is 9,3 ± 1,7. In the severe anemia

group the mean is 4,7 ± 1,0. For the cerebral cases this is 8,2 ± 1,6. The Hb of 14 cases

[13,6%] is missing.

In table 5 the mean and median MetHb for all separate groups are demonstrated. Compared to

the control group, the difference in MetHb levels for both the non severe group as well as the

severe group is demonstrated to be of significant. The small number of cerebral cases give a

mean of 2,5 ± 1,2 SD.

19

Table 5 Mean/median MetHb levels

Cases

(n)

Median

MetHb

level

Mean

MetHb

level

Maximum

MetHb

level

Minimum

MetHb

level

SD of

MetHb

level

P value

Non-severe

malaria 89

1.8 1.8 2.6 0.9 0.3

<0,011

Severe

malaria 14

1.9 2.1 4.4 1.3 0.7

0,031

Controls 88 1.6 1.6 3.4 1.1 0.3

Total 191 1.7 1.7 4.4 0.9 0.4

To calculate the p-values a student t-test was performed

1 Compared to controls

Displayed in figure 5 is the distribution of MetHb levels of the different groups. Visible is a

normal distribution, with in the severe group two outliers at 2.6 and 4.4. Although the severe

cases are rather small in number, they tend to show higher MetHb values than both the non-

severe group and the controls. Due to the number of cases in that very group, we have decided

upon not separating the severe group in the particular subgroups, as we have defined as severe

anemia and cerebral malaria.

Figure 6 demonstrates the correlation between parasitemia of the children with malaria and

their measured MetHb levels. We demonstrate no significant correlation between these

values. An elevated parasite load does not significantly correlate with an increase in MetHb

levels.

The same trend is shown in figure 7, which shows the correlation between the Hb levels of the

children with malaria and their measured MetHb levels. Also here, we do not demonstrate a

significant correlation between these values.

20

Discussion

MetHb levels are significant higher in the group of severe malaria, and in the cerebral malaria

cases in particular. In addition, MetHb levels do not show a correlation with neither the

parasitemia nor the Hb levels. Our groups are not significant different based on sex, age, Hb

nor parasitemia, see table 3. These results tend to suggest that MetHb could be a potential

marker for disease severity in children with malaria.

However, until now the measurement of MetHb has never been established as a standard in

the diagnostic phase of malaria, although Anstey et al. already suggested a correlation. They

found that the degree of methemoglobinemia correlated with disease severity, and reported

the highest values in patients with cerebral malaria with death or neurological sequelae29

. Uko

et al. also demonstrated that a high level of MetHb correlates with severe malaria, using the

method of Evelyn and Malloy to determine the MetHb.32

Methemoglobin in the blood cannot be detected by conventional pulse oximetry. Therefore

the determination of MetHb levels is usually done via direct measurement of MetHb levels in

the blood. Another option is advanced CO-oximetry, which currently serves as the golden

standard to measure MetHb.33

Mainly because of its cost, CO-oximetry is not used widely in

hospitals 34

, and does not seem to be applicable in a rural African hospital setting.

Another test, the Evelyn-Malloy test, is also frequently used to confirm methemoglobinemia.

Feiner and Bickler reported however that the non invasive Rainbow's methemoglobin

readings are acceptably accurate over an oxygen saturation range of 74%-100% and a

methemoglobin range of 0%-14%35

. Therefore we decided to use the Masimo® Rainbow

pulse oximeter to determine MetHb non-invasive, since it is accurate35

, cheap, easy to

perform and well applicable in a rural African hospital.

Our hypothesis is, based on a murine model of a previous study 20,21

, that haemoglobin is

oxidized to MetHb which than releases the highly inflammatory heme, which in turn triggers

the release of cytokines and other mediators and hence causes cerebral malaria. Therefore we

mainly expected elevated MetHb values in children with cerebral malaria, rather than in the

ones with severe anemia or uncomplicated malaria.

Our results raise the question whether MetHb is a marker specific for cerebral malaria or a

marker of disease severity per se, or rather for cerebral involvement of any kind. Therefore we

also included several controls with neurological disease. During the pilot of this study a child

with measles meningitis was measured, and showed high MetHb values (T. Hänscheid,

personal communication). These anecdotal data were supported by findings during this study

in the HAS. The highest MetHb values were observed in a two year old control case which

arrived in a hypoglycaemic coma, possibly due to a bacterial meningitis, although this

diagnosis has not been confirmed.

Moreover, MetHb levels seem to be elevated directly post-ictal. A possible explanation for

this could be the ischemia directly after seizures. Acute symptomatic seizures or status

epilepticus is common after hypoxic ischemic insults. Seizures are commonly reported in

African children with CM and occur in over 60% after admission36,37,38

. Also status

epilepticus is common.36,39,40

Many patients with seizures are hypoxic and hypercarbic from

21

hypoventilation and are at risk of aspiration.36,38

The causes of seizures are unclear. Most are not associated with fever at the time of the

seizure.38

Electroencephalography shows that many originate over the temporo-parietal

regions (which is a watershed area), suggesting that ischaemia and hypoxia may play a role.37

One of the major questions in addition which remains regarding the pathogenesis of CM is

how the parasites cause neuronal dysfunction, since parasites are largely confined to the

intravascular space.41

Consequently, since it is still not clear how the chain of events that leads

to severe malaria and especially cerebral malaria fully works, it is hard to predict who is at

risk to develop cerebral malaria in case of P. falciparum malarial disease. The relevance of

the findings in our study is that with the measurement of MetHb, and the finding of elevated

levels, one can be more suspicious on children who are at increased risk of developing

cerebral malaria. Hence the main value of MetHb as a marker in the diagnostic phase of

malaria would be if it is applicable as a prognostic marker for neurological sequelae in

malarial children. Non invasive measurement is in a rural, poor resource setting the most

applicable and cost effective way to determine these values.

Our study has several limitations. Firstly and most prominently, the number of severe cases in

general and cerebral malaria cases in particular [N=5; 4,9%] is rather small and smaller than

the initially calculated sample size. Although shown to be of significance, the power of these

numbers is hence suboptimal.

Besides that, several of the children included with malaria took part in ongoing trials as the

RTS,S vaccine candidate study or the GMZ2 vaccine candidate study in the HAS-MRU.

Children measured at the follow-up of the RTS,S study with fever and a positive thick smear

and without clinical signs of severe features of malaria, were ambulant treated, and therefore

the Hb and other haematological markers were in some cases [13,6%] not determined.

However, those subjects are clinically not expected at all to have a Hb below 6,0 g/dl.

Participation in one of the clinical trials of the vaccine candidate studies does not interfere

with our study, and hence we do not expect this to influence the results.

We also did not obtain the exact age of all control subjects expressed in months.

To rule out measurement errors, measurements were performed in triplicate, and the average

was calculated afterwards. No wide differences were observed. If one measurement within the

three measurements differed more than 0,4, this measurement was regarded as an outlier, as

mentioned before in the statistical analysis. A possible explanation for the interdigital

variation of the measurements could be a difference in nail bed pigmentation, which possibly

interferes with the different wavelengths of the Masimo® Rainbow pulse oximeter.

The device fitted well for the entire measured group aged from one to ten years old.

To elucidate the value of MetHb as a potential (prognostic) marker of disease severity and

hence the value of routine measurement of MetHb in children with malaria, further research is

warranted.

22

Conclusion

The aim of this cross-sectional study was to establish a correlation between MetHb levels and

differences in the severity of malaria and whether MetHb could therefore be a marker for

disease severity in malaria patients. The study demonstrates that, although small in number,

the MetHb level is significantly elevated in severe malaria, and cerebral malaria cases in

particular compared to a control group and an uncomplicated malaria group.

The non invasive measurement of MetHb could be addressed as an adequate diagnostic tool to

indicate who will possibly develop to cerebral disease. However, an increased number of

subjects seems to be required to validate MetHb as a disease marker for malaria, and non-

invasive measurement as the most viable diagnostic tool in a resource-poor setting. For future

research to establish MetHb as a marker of disease severity in children with malaria, it seems

recommendable to conduct a follow-up study with a higher inclusion number of cerebral

malaria cases, possibly at an IC unit; hence it is easier to obtain 24 hour measurements to

follow the course of MetHb values over time. In due course, it should be investigated whether

MetHb could serve as a prognostic marker, or even be applicable as measurement for

treatment.

Therefore, to elucidate the value of MetHb as a potential (prognostic) marker of disease

severity and hence the value of routine measurement of MetHb in children with malaria,

further research is warranted.

23

Figure 5 Distribution of MetHb values

24

Figure 6 Correlation parasitemia – MetHb levels Parasitemia data have been log10 transformed

25

Figure 7 Correlation Hb – MetHb levels

26

References

1 Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale

— the "bashful" malaria parasites. 2007 Trends Parasitol. 23 (6): 278–83.

2 Mendis K, Sina B, Marchesini P, Carter R. The neglected burden of Plasmodium

vivax malaria 2001 .Am J Trop Med Hyg 64 (1–2 Suppl): 97–106.

3 Singh B, Kim Sung L, Matusop A et al. A large focus of naturally acquired

Plasmodium knowlesi infections in human beings. 2004 Lancet 363: 1017–24.

4 World Malaria Report summary. World Health Organization.

5 World Malaria Report 2011 summary. World Health Organization.

6 Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of

clinical episodes of Plasmodium falciparum malaria. 2005. Nature 434 (7030):

214–7.

7 Hay, S.I., Okiro, E.A., Gething, P.W., Patil, A.P., Tatem, A.J., Guerra, C.A. & Snow,

R.W. (2010). Estimating the Global Clinical Burden of Plasmodium falciparum

Malaria in 2007. Public Library of Science Medicine, 7(6)

8 Trampuz A, Jereb M, Muzlovic I, Prabhu R Clinical review: Severe malaria. 2003.Crit

Care 7 (4): 315–23

9 Centers for Disease Control and Prevention's Public Health Image Library /Alexander

J. da Silva, PhD/Melanie Moser, identification number #3405

10 Grobusch, M.P.; Kremsner, P.G.: Uncomplicated malaria. Current Topics in

Microbiology and Immunology (2005) 295: 83-104

11 WHO. Management of Severe Malaria, (2000). A Practical handbook.2nd

ed. Geneva:

World Health Organization; 2000.

12 Kain K, Harrington M, Tennyson S, Keystone J Imported malaria: prospective

analysis of problems in diagnosis and management. 1998 Clin Infect Dis 27 (1):

142–9.

13 Carter JA, Ross AJ, Neville BG, Obiero E, Katana K, Mung'ala-Odera V, Lees JA,

Newton CR. Developmental impairments following severe falciparum malaria in

children. 2005 Trop Med Int Health 10 (1): 3–10.

14 May Ho, Nicholas J. White. Molecular mechanisms of cytoadherence in malaria.

Am. J Physiol Cell Physiol 1999;276(6):C1231-C1242

15 Louis H. Miller, Dror I. Baruch, Kevin Marsh, Ogobara K. Doumbo. The pathogenic

basis of malaria. Nature February 2002;415(7):673-679.

16 Ian A Clark, Alison C Budd, Lisa M Alleva, William B Cowden. Human malarial

disease: a consequence of inflammatory cytokine release. Malaria Journal

2006;5:85

17 Qijun Chen, Martha Schlichtherle, Mats Wahlgren. Molecular Aspects of Severe

Malaria. Clinical Microbiology Reviews, July 2000;13(3);439-450.

18 Berendt AR, Ferguson DJ, Gardner J, Turner G, Rowe A, McCormick C, et al.

Molecular mechanisms of sequestration in malaria. Parasitology 1994; 108: 19 -28.

19 Clark IA, Rockett KA. The cytokine theory of human cerebral malaria. Parasitology

Today 1994; 10: 410 – 2.

20 Pamplona A, Hanscheid T, Epiphanio S, Mota MM, Vigário AM. Cerebral malaria

and the hemolysis/methemoglobin/heme hypothesis: shedding new light on an old

disease. Int J Biochem Cell Biol. 2009; 41:711-6.

21 Pamplona, A., Ferreira, A., Balla, J., Jeney, V., Balla, G., Epiphanio, S., Chora, A.,

Rodrigues, C. D., Gregoire, I. P., Cunha-Rodrigues, M., et al. Heme oxygenase-1

27

and carbon monoxide suppress the pathogenesis of experimental cerebral malaria.

Nature Medicine 2007, 13, 703-710.

22 Jaffe ER, Hultquist DE: Cytochrome b5 reductase deficiency and enzymopenic

hereditary methemoglobinemia, in Scriver CR, Beaudet AL, Sly WS, et al (eds):

The Metabolic and Molecular Basis of Inherited Disease, ed 7. New York:

McGraw-Hill, 1995:2267-2280.

23 Wright RO, Lewander WJ, Woolf AD: Methemoglobinemia: Etiology, pharmacology,

and clinical management. Ann Emerg Med November 1999;34:646-656.

24 Ralston AC, Webb RK, Runciman WB: Potential errors in pulse oximetry. III: Effects

of interference,dyes, dyshemoglobins and other pigments. Anaesthesia

1991;46:291-294.

25 Watcha MF, Connor MT, Hing AV: Pulse oximetry in methemoglobinemia. Am J Dis

Child 1989;143:845-847.

26 Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, et al.

Different faces of the heme – heme oxygenase system in inflammation.

Pharmacological Reviews 2003; 55.551-71.

27 Coltel N, Combes V, Wassmer SC, Chimini G, Grau GE. Cell vesiculation and

immunopathology: implications in cerebral malaria. Microbes and infection 2006;

8:2305 – 16

28 Hsiech HS, Jaffe ER. The metabolism of methemoglobin in human erythrocytes. In:

Surgenor DMN, editor. The Red Blood Cell. New York; Academic Press II; 1975,

p. 799-824.

29 Anstey NM, Mushtaq J, Hassanali Y, Mlalasi J, Manyenga D, Mwaikambo ED. 1996.

Elevated levels of methaemoglobin in Tanzanian children with severe and

uncomplicated malaria method in venous blood. Trans Roy Soc Trop Med Hyg 90:

147-151

30 T. Planche, S. Krishna, M. Kombila, K. Engel, J. F. Faucher, E. Ngou-Milama, P. G.

Kremsner. Comparison of methods for the rapid laboratory assesment of children

with malaria. Am. J. Trop. Med. Hyg., 65(5), 2001, pp. 599–602

31 Molyneux ME Taylor TE et al. Clinical features and prognostic indicators in

paediatric cerebral malaria: A study of 131 comatose Malawian children. Q J Med.

1989; 71: 441-459.

32 Uko EK, Udoh AE, Etukudoh MH. Methaemoglobin profile in malaria infected

children in Calabar. Niger J Med. 2003 Apr-Jun;12(2):94-7.

33 Skold A, Cosco DL, Klein R. Methemoglobinemia: pathogenesis, diagnosis, and

management. South Med J. 2011 Nov;104(11):757-61

34 Wright RO, Lewander WJ, Woolf AD. Methemoglobinemia: etiology, pharmacology,

and clinical management. Ann Emerg Med 1999;34:646-656

35 Feiner JR, Bickler PE. Improved accuracy of methemoglobin detection by pulse CO-

oximetry during hypoxia. Anesth Analg. 2010 Nov;111(5):1160-7.

36 Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C, Marsh K. Seizures and

status epilepticus in childhood cerebral malaria. Q J Med 1996;89(8):591-7.

37 Crawley J, Smith S, Muthinji P, Marsh K, Kirkham F. Electroencephalographic

and clinical features of cerebral malaria. Arch Dis Child 2001;84(3):247-53.

38 Waruiru CM, Newton CR, Forster D, et al. Epileptic seizures and malaria in Kenyan

children. Trans R Soc Trop Med Hyg 1996;90(2):152-5.

39 Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic

indicators in paediatric cerebral malaria: a study of 131 comatose Malawian

28

children. Q J Med 1989;71(265):441-59.

40 Richard Idro, Neil E Jenkins and Charles RJC Newton Pathogenesis, clinical features

and neurological outcome of cerebral malaria Lancet Neurology, 2005; 4(12):

827 – 40

41 Gitau EN, Newton CR. Review Article: Blood-brain barrier in falciparum malaria.

Trop Med Int Health 2005;10(3):285-92.