dafpus 9 age-dependent effect of plasma nitric oxide on parasite density

Age-dependent effect of plasma nitric oxide on parasite density

in Ghanaian children with severe malaria

Jakob P. Cramer1, Andreas K. Nussler2, Stephan Ehrhardt3, Jana Burkhardt1, Rowland N. Otchwemah4,

Philipp Zanger5, Ekkehart Dietz6, Sabine Gellert7, Ulrich Bienzle1 and Frank P. Mockenhaupt1

1 Institute of Tropical Medicine Berlin, Charite – University Medicine Berlin, Germany2 Department of Surgery, Charite – University Medicine Berlin, Germany3 Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany4 School of Medicine and Health Sciences, University for Development Studies, Tamale, Ghana5 Northern Region Malaria Project, NORMAP, Tamale, Ghana6 Division for International Health, Charite – University Medicine Berlin, Germany7 Bernsteinklinik Binz, Binz auf Rugen, Germany

Summary Nitric oxide (NO) has toxic properties against Plasmodium falciparum. While high blood levels have

been associated with protection against severe malarial disease, they may also contribute to the

pathophysiology of cerebral malaria and severe anaemia. Promoter variants in the inducible nitric oxide

synthase (iNOS) gene have been shown to influence NO concentrations and disease manifestation.

However, findings are conflicting. We examined associations of plasma NO metabolites (NOx) with

symptoms of severe malaria, particularly malarial anaemia and cerebral malaria, and with iNOS

promoter variants. In 210 Ghanaian children with severe malaria, we measured plasma nitrite, nitrate,

and S-nitrosothiol, and genotyped the iNOS promoter variants )954G fi C, )1173C fi T, and the

)2.5 kb (CCTTT)n microsatellite. NOx levels decreased with age. In young children (<24 months), high

NOx was associated with reduced parasite density. This was not seen in patients of 24–48 months of age

and reversed in older children. Subgroup analysis revealed that in children with severe anaemia but

without cerebral involvement (prostration, impaired consciousness, convulsions), high NOx levels

correlated with low parasitaemia (P ¼ 0.02). In these children, elevated NOx levels were also associated

with the iNOS )954C fi T/(CCTTT)8 haplotype (P ¼ 0.03). No association between NOx or iNOS

genotypes and cerebral malaria was observed. Our findings suggest that in young children with severe

malaria NOx reduces parasitaemia. This effect wanes at higher ages and may reflect a predominance of

unspecific immune responses to infection in early childhood. This finding may have importance for the

understanding of associations between iNOS variants and severe malaria in regions of differing disease

manifestation.

keywords nitric oxide, iNOS promoter polymorphisms, Plasmodium falciparum, severe malaria

Introduction

Nitric oxide (NO) forms part of the immune response but

at the same time contributes to the pathogenesis of

infectious diseases (Nussler & Billiar 1993). In vitro, NO is

parasiticidal to Plasmodium falciparum (Rockett et al.

1991; Mellouk et al. 1994) and high levels are considered

to contribute to rapid parasite clearance (Kremsner et al.

1996; Chiwakata et al. 2000). Likewise, high serum NO

levels, accompanied by increased expression of the

inducible nitric oxide synthase (iNOS) gene, may protect

against severe malaria (Anstey et al. 1996; Chiwakata

et al. 2000). However, fatal cerebral malaria has been

attributed to increased local NO production causing direct

neurotoxicity or vasodilatation and raised cerebral pres-

sure (Weiss et al. 1998; Clark et al. 2003). Furthermore,

NO is involved in the pathogenesis of malarial anaemia by

suppressing haematopoiesis and inducing erythrocyte

destruction (Mannick et al. 1994; Kolb & Kolb-Bachofen

1998; Gyan et al. 2002).

Several iNOS promoter variants may alter gene expres-

sion and NO production. Two single nucleotide polymor-

phisms, )954G fi C and )1173C fi T, have been

reported to increase NO synthesis in Gabonese and in

Kenyan children, respectively (Kun et al. 2001; Hobbs

et al. 2002). However, their role in clinical disease is

controversial (Kun et al. 1998; Hobbs et al. 2002; Cramer

et al. 2004). The same applies to a pentanucleotide

Tropical Medicine and International Health

volume 10 no 7 pp 672–680 july 2005

672 ª 2005 Blackwell Publishing Ltd

(CCTTT) microsatellite )2.5 kb of the transcription start

site. Short microsatellite alleles with CCTTT(<11) occurred

more frequently in fatal cerebral malaria in The Gambia

(Burgner et al. 1998) whereas we and others found longer

alleles with CCTTT(‡13) to be associated with severe

malaria (Ohashi et al. 2002; Cramer et al. 2004).

In this study, we examined the role of peripheral blood

NO levels in Ghanaian children with severe malaria, in

particular with severe malarial anaemia (SMA) and with

cerebral involvement (prostration, multiple convulsions,

and/or impaired consciousness). Moreover, associations of

iNOS promoter variants with NO levels were analysed.

Patients and methods

Plasma samples of 210 children with severe malaria were

available. Patients belonged to a group of 290 children

aged 6 months to 9 years with severe malaria according to

the current WHO (2000) definition who were recruited

at the end of the rainy season 2002 (August–November)

at the Teaching Hospital in Tamale, northern Ghana.

Clinical and parasitological data of these children are

described in detail elsewhere (Mockenhaupt et al. 2004a).

In the study area, climate and vegetation are savanna-type

and malaria is hyperendemic (Mockenhaupt et al.,

unpublished observations). Children were treated with

artesunate (5 mg/kg of body weight; Plasmotrim, Mepha

Pharma, Switzerland) for 5 days receiving double dose on

first day. Supportive care was provided as required. All

patients with severe anaemia received blood transfusions.

Samples were taken upon admission and venous blood

was collected into EDTA. Plasma was separated by

centrifugation and stored at )20 �C. DNA was extracted

by commercial kits (QIAmp blood kit, Qiagen, Germany).

Haemoglobin (Hb), glucose, and lactate were measured as

described previously (Mockenhaupt et al. 2004a). Parasites

were counted per ‡200 white blood cells on Giemsa-

stained thick blood films and P. falciparum was ascertained

by specific PCR assays (Djimde et al. 2001). Severe

anaemia was defined as an Hb level <5 mg/dl, hyperpar-

asitaemia as a parasite density >250 000 parasites/ll, andimpaired consciousness as a Blantyre coma score of £2.Malnutrition was defined as a weight-for-age (WAZ) score

less than )2 based on National Center for Health Statistics

(Hyattsville, USA) reference data. For subgroup analysis,

patients were categorized into subgroups: children with

cerebral involvement (prostration, multiple convulsions,

and/or impaired consciousness) but no severe anaemia and

patients with SMA but no cerebral involvement.

Plasma levels of stable NO end products (NOx) were

assessed by measuring nitrite/nitrate and S-nitrosothiols

(RSNO). Nitrite/nitrate was determined by a modified

Griess assay after incubation with nitrate reductase

(Nussler et al. 2002). RSNO was measured fluorometri-

cally using the 2,3-diaminonaphthalene reagent (Nussler

et al. 2002). Values of nitrite/nitrate and S-nitrosothiols

were combined and referred to as reactive nitrogen

intermediates (NOx).

Genotyping of the iNOS promoter polymorphisms

)954G fi C, )1173C fi T, and the )2.5 kb (CCTTT)npentanucleotide microsatellite (GenBank accession no.

AF017634) was performed as previously described (Xu

et al. 1997; Kun et al. 2001; Hobbs et al. 2002; Cramer

et al. 2004).

NOx and parasite densities were normalized by log10transformation and geometric mean parasite density

(GMPD) and 95% confidence interval (95% CI) were

calculated. Continuous variables were compared between

clinical symptoms and outcome by Student’s t-tests or

Mann–Whitney U tests. Linear regression analysis and

Fisher’s transformation test were used for continuous

variables. Proportions were compared by chi-square tests.

Multiple regression analysis and logistic regression analysis

were performed as appropriate in order to correct for

confounders of NOx levels (encoding: age; sex, female, 0;

male, 1; residence, urban, 0; rural, 1; malnutrition, absent,

0; present, 1). A P-value <0.05 was considered as

statistically significant.

Ethical approval

Informed consent was obtained from the patient’s parents

or legal guardians. The study protocol was reviewed and

approved by the Ethics Committee, University for Devel-

opment Studies, Tamale, Ghana, and the institutional

guidelines were followed.

Results

The median age of the 210 children (109 girls, 101 boys)

was 24 months (range 6–102). Mean rectal temperature

(±SD) was 38.6 �C (±1.1). GMPD was 30 130 parasites/ll(95% CI: 20 910 ) 43 416; Table 1). Ninety-three

children (44%) were malnourished. Hyperparasitaemia

was found in 50 children (24%). The three most frequent

symptoms/conditions were severe anaemia (113/210,

54%), hyperlactataemia (87/210, 41%), and prostration

(73/210, 35%; Table 2). Twenty-six (13%) children died

and four (2%) absconded.

The geometric mean of plasma nitric oxide metabolites

(NOx) was 41 lmol/l (95% CI: 34–49). log10 NOx levels

decreased with age (R ¼ )0.23; P ¼ 0.001; Figure 1a) and

correlated negatively with log10 parasite density

(R ¼ )0.22; P ¼ 0.002; Figure 1b). This held true in

Tropical Medicine and International Health volume 10 no 7 pp 672–680 july 2005

J. P. Cramer et al. Nitric oxide levels and severe malaria

ª 2005 Blackwell Publishing Ltd 673

multiple linear regression analysis (age, regression coeffi-

cient b ¼ )0.005; standard error (SE) ¼ 0.002; P ¼ 0.006;

sex, b ¼ )0.045; SE ¼ 0.076; P ¼ 0.6; residence,

b ¼ 0.145; SE ¼ 0.086; P ¼ 0.09; malnutrition,

b ¼ 0.014; SE ¼ 0.079; P ¼ 0.9; log10 parasite density,

b ¼ )0.086; SE ¼ 0.035; P ¼ 0.01). NOx was higher in

children living in rural (geometricmean, 61 lmol/l; 95%CI:

43–87) than in urban areas (geometric mean, 33 lmol/l;

95%CI: 28–40; P ¼ 0.001). Malnutrition occurred in 50%

and 41% of children of rural and urban residence, respec-

tively, but had no influence on NOx levels neither in

univariate (mal- andnormally nourished children, geometric

mean, 46 lmol/l; 95% CI: 35–60; vs. 37 lmol/l; 95% CI:

30–48; P ¼ 0.3) nor in multivariate analysis (data not

shown). This was also true for nitrite/nitrate as well as

S-nitrosothiol levels separately (data not shown). Yet, the

above-mentioned effect of age on NOx was significant in

malnourished (R ¼ )0.35; P ¼ 0.0006) but not in normally

nourished children (R ¼ )0.13; P ¼ 0.16; Figure 1c).

In analysis of variance, the effect of NOx on parasite

density differed significantly with age (age · NOx,

F ¼ 8.6; P ¼ 0.004). Therefore, we stratified into age

groups of children <24 months (n ¼ 74), ‡24 to

<48 months (n ¼ 91), and ‡48 months (n ¼ 45). High

NOx was associated with low parasite density in children

<24 months (R ¼ )0.39; P ¼ 0.0007). This association

was less pronounced in children ‡24 to <48 months of age

(R ¼ )0.26; P ¼ 0.01) and reversed in children

‡48 months (R ¼ 0.24; P ¼ 0.1; Figure 1d). In multivari-

ate analysis including sex, residence, malnutrition, and the

interaction term of age and NOx, this finding was

confirmed (age <24 months, R ¼ )0.51; P ¼ 0.008; ‡24to <48 months, R ¼ )0.36; P ¼ 0.2; ‡48 months,

R ¼ 0.76; P ¼ 0.0003).

Then, associations of NOx with anaemia were analysed.

NOx levels were significantly higher in patients with SMA

than in children without (geometric mean, 49 lmol/l; 95%

CI: 38–64; vs. 33 lmol/l; 95% CI: 26–42; P ¼ 0.03). After

correcting for age, sex, residence, malnutrition, and para-

sitaemia, this association waned (b ¼ 0.17; SE ¼ 0.058;

P ¼ 0.8).

In the next step, patients were categorized into the

subgroups of children with cerebral involvement (prostra-

tion, multiple convulsions, and/or impaired consciousness)

but no severe anaemia (n ¼ 85) and patients with SMA but

no cerebral involvement (n ¼ 61). Fivty-two children had

both and 12 children had neither (Figure 2). Clinical and

parasitological parameters are summarized in Table 1.

Hyperparasitaemia, hypoglycaemia, and – at borderline

statistical significance – hyperlactataemia occurred more

frequently in children with cerebral involvement than in

those with SMA (Table 2). None of the children with SMA

died compared with 11 deaths (13%) in the group with

cerebral involvement (P ¼ 0.003). Geometric mean plasma

NOx was slightly lower in children with cerebral involve-

ment than in patients with SMA (Table 2). In children with

SMA but not in those with cerebral involvement, high

NOx levels were associated with low parasite density

(R ¼ )0.30; P ¼ 0.02). This was confirmed in multivariate

analysis (b ¼ )0.211, SE ¼ 0.073, P ¼ 0.005; age,

b ¼ )0.013, SE ¼ 0.005, P ¼ 0.009; sex, b ¼ )0.001,SE ¼ 0.154, P ¼ 1; urban residence, b ¼ 0,214,

SE ¼ 0.154, P ¼ 0.2; malnutrition b ¼ 0.088,

SE ¼ 0.142, P ¼ 0.5). No further associations of NOx

levels with disease symptoms and conditions within these

two subgroups were detected.

Finally, the influence of iNOS promoter variants on

plasma NOx levels was analysed. Neither in the group of

all 210 children nor in the subgroup of children with

cerebral involvement, associations of NOx levels with

iNOS promoter variants were detected. However, in the

SMA subgroup (n ¼ 61), all children with the )954G fi C

Table 1 Clinical and parasitological characteristics of 210 severe malaria patients

Patient characteristics All (n ¼ 210)

Cerebral involvement

(n ¼ 85) SMA (n ¼ 61)


vs. SMA* (P)

Age [months, median (range)] 24 (6–102) 36 (12–102) 19 (8–84) <0.0001

Female sex [n (%)] 109 (52) 49 (58) 24 (39) 0.01

Weight [kg, mean (SD)] 10.9 (3.3) 11.9 (3.3) 9.5 (2.8) <0.0001

WAZ [mean (SD)] )1.7 (1.2) )1.6 (1.3) )1.9 (1.3) 0.1Temperature [�C, mean (SD)] 38.6 (1.1) 38.8 (1.0) 38.3 (1.1) 0.004

Haemoglobin [g/dl, mean (SD)] 5.5 (2.3) 7.5 (2.0) 3.9 (0.8) <0.0001

Glucose [mg/dl, mean (SD)] 72.8 (34.0) 76.2 (41.6) 80.0 (17.0) 0.4Lactate [mmol/l, mean (SD)] 5.4 (3.8) 4.9 (3.2) 3.9 (2.3) 0.06

GMPD [/ll, (95% CI)] 30 130 (20 910–43 416) 77 268 (46 784–127 616) 9183 (4750–17 756) <0.0001

SMA, severe malarial anaemia; SD, standard deviation; GMPD, geometric mean parasite density; WAZ, weight-for-age.

* Applying chi-square, Student’s t, or Mann–Whitney U tests as appropriate.




Table

2Distributionofsingle

symptoms,conditions,andoutcomeaswellasNOxlevelsin

210childrenwithseveremalaria

Conditions/symptoms

All

Cerebralinvolvem

ent

SM

ACerebralinvolvem

entvs.SM

A

n(%

)

NOx(lmol/l)

GM

(95%

CI)

n(%

)

NOx(lmol/l)

GM

(95%

CI)

n(%

)

NOx(lmol/l)

GM

(95%

CI)

No.(chi-square)

NOx

(Student’st)

OR

(95%

CI)

Pt

P

All

210

41(34–49)

85(40)

34(26–45)

61(29)

51(36–73)

)1.8

0.08

Definingsymptoms

Severeanaemia

113(54)

49(38–64)

––

61(100)

Prostration*

73(35)

38(28–52)

44(52)

38(26–55)

––

Multiple

convulsions*

43(20)

33(23–48)

34(40)

28(19–41)

––

Impaired

consciousness*

39(19)

41(26–63)

23(27)

28(16–49)

––

Other

symptoms

Respiratory

Distress

51(24)

39(28–55)

24(28)

35(21–59)

9(15)

103(41–255)

2.3

(0.9–5.8)

0.05

)2.1

0.047

Jaundice

23(11)

47(30–73)

8(9)

74(28–191)

3(5)

27(13–56)

2.0

(0.5–10.0)

0.3

1.2

0.3

Circulatory

collapse

10(5)

27(12–61)

5(6)

25(2–258)

1(2)

63.8

(0.4–87.1)

0.2

––

Haem

oglobinuria

2(1)

37(26–54)

1(1)

31

0(0)

–Undefined

0.4

––

Hyperparasitaem

ia50(24)

35(25–48)

33(39)

33(24–46)

2(3)

11(3–43)

18.7

(4.1–118.8)

<<0.0001

1.5

0.4

Hypoglycaem

ia35(17)

46(27–80)

17(20)

32(17–61)

1(2)

13

15.0

(2.0–311.3)

<0.001

––

Hyperlactataem

ia87(41)

39(29–52)

34(40)

29(19–44)

16(26)

50(22–110)

1.9

(0.9–4.1)

0.08

)1.3

0.2

Hyperpyrexia

17(8)

27(15–46)

7(8)

30(16–55)

3(5)

42(3–654)

1.7

(0.4–8.9)

0.4

)0.4

0.7

Fataloutcome

26(12)

46(25–82)

11(13)

30(14–64)

0(0)

–Undefined

0.003

––

SM

A,severemalarialanaem

ia;GM

,geometricmean;OR,oddsratio;95%

CI,95%

confidence

interval.

*Summarizedascerebralinvolvem

ent.




polymorphism (n ¼ 10) also possessed eight CCTTT

copies supporting the presence of a respective haplotype

)954G fi C/(CCTTT)8. These children exhibited signifi-

cantly higher NOx levels (122 lmol/l; 95% CI: 42–351)

than those with other genotypes (43 lmol/l; 95% CI:

30–81; P ¼ 0.03; Figure 3a). Stratified by age groups, this

effect was only found in children below 24 months of age

(433 lmol/l; 95% CI: 228–819; vs. 54 lmol/l; 95% CI:

33–86; P ¼ 0.004; Figure 3b). In multivariate analysis

adjusting for age, sex, residence, and malnutrition, the

)954G fi C/(CCTTT)8 haplotype was independently

associated with increased NOx levels (b ¼ 0.442;

SE ¼ 0.2; P ¼ 0.03).

Discussion

In the present group of Ghanaian children with severe

malaria, severe anaemia was the predominant mani-

festation followed by prostration and respiratory distress.

Impaired consciousness was comparatively rare which

confirms previous findings in highly endemic areas (Snow

et al. 1994; WHO 2000). We assume that severe

anaemia reflects chronic and repeated infections during

the preceding rainy season whereas cerebral involvement

may rather represent acute disease onset (Mockenhaupt

et al. 2004a). This is supported by the higher proportion

of hyperparasitaemia, hypoglycaemia, and hyperlactatae-

mia in the latter condition.

Plasma NOx levels in this study correspond well with

those described earlier in children with severe malaria

(Kremsner et al. 1996; Gyan et al. 2002). Also, the decline

of NOx with age has been seen in other endemic regions

and attributed to differing NO production by macroph-

ages, endothelial cells, and/or hepatocytes in different age

groups (Anstey et al. 1999).

Malnutrition has been shown to increase plasma NO

(Fechner et al. 2001). This could not be seen in the present

study and, thus, cannot explain the differences between

children of rural and urban origin. Irrespective of its origin,

measured NO may be associated with parasite load and

(a)

0.50.75

11.251.5

1.752

2.252.5

2.753

3.25

Log 10

NO

x

Log 10

NO

x

10 20 30 40 50 60 70 80 90 100 110Age (months)

(b)

0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5 6 7Log10 parasite density

(c)

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

Log 10

NO

x

Age < 24 months Age > = 24 < 48 months Age > = 48 months

MalnourishedNormally nourished

(d)

4

4.2

4.4

4.6

4.8

5

5.2

Log 10

par

asite

den

sity

< 25 µmol/l > = 25 < 40 µmol/l > = 40 µmol/l

Age > = 48 monthsAge > = 24 <48 monthsAge <24 months

Figure 1 Associations of plasma NOx levels with patients’ characteristics. (a) High plasma NOx correlates with lower age

(R ¼ )0.23; P ¼ 0.001), and (b) with parasite density (R ¼ )0.22; P ¼ 0.002). (c) Effect of age on NOx in malnourished

(R ¼ )0.35; P ¼ 0.0006) and in normally nourished children (R ¼ )0.13; P ¼ 0.16). (d) Influence of NOx on parasite density inchildren <24 months (R ¼ )0.39; P ¼ 0.0007), ‡24 < 48 months (R ¼ )0.26; P ¼ 0.01), and ‡48 months (R ¼ 0.24; P ¼ 0.1).




disease manifestation. Confounding factors, however,

possibly influence the assessment of the role of iNOS on

NO levels. To limit possible influence of nutritional status,

multivariate analyses were corrected for malnutrition.

Influences of additional NO sources such as isoenzymes as

well as dietary intake or the presence of other microbes

cannot be elucidated. This should be taken into account

when interpreting our results.

In vitro, NO has been shown to be parasiticidal

(Rockett et al. 1991; Mellouk et al. 1994) and in vivo

it accelerates parasitological cure (Kremsner et al. 1996).

In the present study, increasing NOx levels were

associated with decreasing parasite density. This is

consistent with the findings from Gabon (Kremsner et al.

1996) but contrasts the results from coastal Ghana and

from Papua New Guinea (Gyan et al. 2002; Boutlis et al.

2003). Age stratification revealed that this effect was

present only in young children, decreased at higher ages,

and, eventually, reversed. In the non-immune child, fever

and cytokines constitute supposably the main immune

defence mechanism showing antiparasitic effects but also

contributing to clinical disease (Kwiatkowski 1991).

In accordance with current concepts on host defence

strategies in the youngest children (Smith et al. 1999),

this may reflect a tendency to rapidly eliminate parasit-

aemia while specific immune mechanisms are not yet

fully developed. Our findings indicate that NO contri-

butes to this defence mechanism. The protective effect

then may become less important when the growing child

acquires specific immune protection after repeated

infections.

Cerebral malaria in contrast to malarial anaemia is

considered to result from localized pathology (e.g. Mane-

erat et al. 2000). Albeit further processes and mediators

involved, the distinctive pathophysiologies may originate

from different stimuli for NO-production as well as

different NO sources and production sites. Peripheral

blood mononuclear cells and possibly hepatocytes are the

sources of NO in systemic parasite infections (Anstey et al.

1996). Endothelial cells, astrocytes, and neurones contrib-

ute to localized NO production (Maneerat et al. 2000)

which is thought to be a major determinant of cerebral

malaria (Clark et al. 1995; Weiss et al. 1998). NO is

involved in the pathogenesis of malarial anaemia by

suppressing haematopoiesis and direct toxicity to erythro-

cytes (Mannick et al. 1994; Kolb & Kolb-Bachofen 1998;

Gyan et al. 2002). Haemozoin-induced iNOS expression in

peripheral blood mononuclear cells is elevated in children

with malarial anaemia (Keller et al. 2004). Age-adjusted,

we found higher peripheral NOx levels in severe anaemia

and a negative association with parasite density in this

condition but not in cerebral involvement. This finding

likely reflects the above-mentioned pathophysiological

differences between severe anaemia and cerebral malaria

but also the incongruity of peripherally measured and

localized, i.e. cerebral NO production.

The role of iNOS promoter variants in severe malaria

remains controversial (Kun et al. 1998; Hobbs et al. 2002;

85 (40%) 61 (29%)52 (25%)

Severe malarial anaemia

Severe malarial anaemia and cerebral involvement


No cerebral involvement and no severe malarial anaemia

12 (6%)

Figure 2 Distribution of cerebral involvement (prostration, multiple convulsions, and/or impaired consciousness) and severe malarialanaemia in 210 children with severe malaria according to WHO (2000) criteria.




Burgner et al. 2003). In northern Ghana, iNOS promoter

variants did not protect against disease progression from

asymptomatic parasitaemia to severe disease (Cramer et al.

2004). In Gabon, the iNOS )954G fi C promoter variant

has been reported to increase iNOS expression (Kun et al.

2001). Here, the )954G fi C polymorphism alone as well

as the haplotype )954G fi C/(CCTTT)8 were associated

with increased NOx levels in children with severe anaemia.

Yet, this was mainly because of significantly higher NOx

levels in children younger than 24 months. Therefore, the

age effect of the genotype corresponds to the above-

described age-dependent phenotype. The question why a

certain genotype confers different effects in different age

groups remains obscure. An age-dependent influence on

malaria disease manifestation has also been observed in

other genetic traits, e.g. sickle cell trait and a+-thalassaemia

(Guggenmoos-Holzmann et al. 1981; Mockenhaupt et al.

2004b). This, again, supports the hypothesis that host

defence strategies differ in non-immune infants and semi-

immune children. In addition, age-dependency of anti-

parasitic effects of NOx could be involved in the conflicting

results on associations between iNOS variants and protec-

tion from severe disease (Kun et al. 1998;Hobbs et al. 2002;

Burgner et al. 2003) considering geographical differences in

age distribution and diseasemanifestation of severemalaria.

In conclusion, our results support previous findings

demonstrating antiparasite effects of NO. We show that

this effect is age-dependent and occurs predominantly in

the youngest children. The roles of plasma NOx and iNOS

promoter variants seem to differ in children with SMA and

in those with cerebral involvement suggesting different

pathogenetic backgrounds of these two malaria disease

syndromes.

Acknowledgements

We thank Susanne Roewer for assistance in the laboratory

in Berlin and Dr Laari for storing samples in the laboratory

in Tamale. P. Zanger was supported by a German

Academic Exchange Service (DAAD) fellowship. We also

thank the staff of the Northern Region Malaria Project

(NORMAP) and all participating children as well as their

parents. Financial support was provided through Charite

grant 2003-676 and 2004-585.

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(a)

0

0.5

1

1.5

2

2.5

3

3.5

Log 10

NO

xLo

g 10 N

Ox

Homozygous –954G / without–954G→C/(CCTTT)8

Heterozygous–954G→C / with –954G→C/(CCTTT)8

(b)

0

0.5

1

1.5

2

2.5

3

3.5

Homozygous–954G / without –954G→C/(CCTTT)8

Heterozygous–954G→C / with–954G→C/(CCTTT)8

Age ≥ 48 months Age ≥ 24 < 48 months Age < 24 months

Figure 3 Plasma NOx levels in children with severe anaemiaaccording to iNOS )954/(CCTTT)n genotype and age. (a) log10NOx levels in the severe anaemia subgroup with homozygous

)954G and with heterozygous )954G fi C (all children with the)954G fi C polymorphism in this subgroup also harboured eight

CCTTT copies): 43 lmol/l; 95% CI: 30–81; vs. 122 lmol/l; 95%

CI: 42–351 respectively; P ¼ 0.03. (b) Children <24 months of age

with the )954G fi C/(CCTTT)8 genotype had significant higherlevels of NOx than those harbouring the homozygous )954G(433 lmol/l; 95% CI: 228–819; vs. 54 lmol/l; 95% CI: 33–86

respectively; P ¼ 0.004).




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Authors

Jakob P. Cramer (corresponding author), Frank P. Mockenhaupt, Jana Burkhardt and Ulrich Bienzle, Institute of Tropical Medicine,

Spandauer Damm 130, 14050 Berlin, Germany. Tel.: +49 30 30116 838; Fax: +49 30 30116 888; E-mails: [email protected],

[email protected]; [email protected]; [email protected]

Andreas K. Nussler, Department of Surgery, Charite – University Medicine Berlin, Campus Virchow, Augustenburger Platz 1, 13353

Berlin, Germany. E-mail: [email protected]

Stephan Ehrhardt, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.

Tel.: +49 40 42818 373; Fax: +49 40 42818 394; E-mail: [email protected]

Rowland N. Otchwemah, School of Medicine and Health Sciences, University for Development Studies, PO Box TL 1350, Tamale,

Ghana. Tel./Fax: +233 71 22046; E-mail: [email protected]

Philipp Zanger, Northern Region Malaria Project NORMAP, PO Box TL 2153, Tamale, N/R, Ghana. E-mail: [email protected]

Ekkehart Dietz, Division for International Health, Institute for Social Medicine, Epidemiology, and Health Economics, Charite –

University Medicine Berlin, Fabeckstrasse 60-62, 14195 Berlin, Germany. Tel.: +49 30 8445 1293; Fax: +49 30 8445 1280;

E-mail: [email protected]

Sabine Gellert, Bernsteinklinik Binz, Proraer Strasse 27, 18609 Binz auf Rugen, Germany. Tel.: +49 38393 47 190;

Fax: +49 38393 47 189; E-mail: [email protected]




dafpus 9 age-dependent effect of plasma nitric oxide on parasite density

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