ORIGINAL

Niger J Paed 2012; 39 (2):51- 55

Nwaneri DU

Ibadin MO

Haemoglobin genotype of children with

severe malaria seen at the University of

Benin Teaching Hospital, Benin city,

Nigeria

DOI:http://dx.doi.org/10.4314/njp.v39i2.2

Had abnormal Hb genotype. Preva-

lence of severe malaria in children

with abnormal Hb was 20/24

(83.3%) as against 58/68(85.3%)

observed in those with HbAA. Sig-

nificantly fewer incidence of heavy

malaria parasitaemia (3+ and 4+)

was observed in children with ab-

normal Hb genotype. Heavy para-

site density was the most important

features of severe malaria in chil-

dren with HbAA (p= 0.013) as

against altered sensorium, prostra-

tion, and haemoglobinuria in chil-

dren with abnormal Hb genotype (p

Received:19th April 2011

Accepted: 23rd October 2011

Abstract: Introduction: Types of

haemoglobin (Hb) genotype have

been found to be crucial to the rate

of red blood cell parasite invasion,

multiplication, and destruction as

well as outcome of malaria disease.

In a bid to provide more informa-

tion on the relationship between Hb

genotype and level of protection

conferred by genotype against se-

vere forms of malaria, this study

was undertaken. This is done

through evaluation of forms of Hb

genotype in children with severe

malaria seen in University of Benin

Teaching Hospital (UBTH), Benin

City.

(

)

Nwaneri DU

Ibadin MO

Department of Child Health,

University of Benin Teaching

Hospital, PMB 1111, Benin City,

Edo State, Nigeria.

E-mail: damiannwaneri@yahoo.com

Tel: +2348056321577,

+

23480139172309

=

0.003, 0.041, and 0.023 respec-

tively). Children with HbAA were

also about 3 times more likely to

die from severe malaria (p = 0.567,

O.R = 2.96) when compared with

their counterparts with abnormal

Hb.

Patients and methods: This cross-

sectional study was carried out on

children (6 - 60 months old) admit-

ted for severe malaria using stan-

dard World Health Organization

Conclusion: Study supports

a

(

WHO) guidelines. Diagnosis of

higher prevalence of severe malaria

in children with HbAA when com-

pared with those with abnormal Hb

genotype. Altered sensorium, pros-

tration and haemoglobinuria were

the significant presenting features

of severe malaria in children with

abnormal Hb genotype in this

study.

malaria was by microscopic demon-

stration of parasitaemia or serology

(

in those with negative parasitae-

mia). Hb genotype was done using

the Hb electrophoresis.

Results: Ninety-six well nourished

children; (56(58.3%) males and 40

(

2

41.7%) females) mean age (± SD)

9.22 ± 16.02 months were re-

cruited for the study. Sixty-eight

73.4%) of the 92 subjects had Hb

genotype AA while 24(26.1%)

Key words: children, genotype,

haemoglobin, mortality, severe ma-

laria

(

Introduction

about 50.0% by the age of one year and persists at a

5.6

high level during childhood. In Benin City, Nigeria

69.0% of Under-5s who had fever without specific l₆ocal-

izing signs of infection were found to have malaria.

More than 300-500 million people suffer from_-m₄ alaria

1

annually; of which two million deaths occurs. About

9

0.0% of all malaria related deaths in the world today

occur in Africa, and the_-₃se deaths occur in children under

A major phase in malaria parasite life cycle is red blood

cell invasion and multiplication, consequently leading

2

7

the age of five years. This is because majority of the

infections in Africa are caused by Plasmodium falci-

parium, ₂_-t₃he most dangerous of the four Plasmodium

species . Among the unprotected children in rural ar-

eas of Southern Nigeria, the infection rate rises rapidly

from 0. 2.0% during the first three months of life to

to red blood cells destruction. However host immunity

and types of haemoglobin (Hb) genotype have been

found to be crucial to the rate of parasite invasion, multi-

plica₇tion, and destruction as well as outcome of the dis-

ease .

5

2

The relationship between Hb genotype and level of pro-

tection conferred against severe forms of malaria re-

mains unclear. It was reported by Okam in 2002 that

secutively into the study. Ethics and Research Commit-

tee of UBTH, Benin City gave approval for the study.

Written informed consent was also obtained from each

parent or care-giver of the subjects recruited in the

study. Researcher administered validated questionnaire

was used to obtained socio-demographic data of each

subject, anthropometry, symptoms before presentation,

drug treatment and outcome. Outcome was defined as

discharged home or dead.

8

children with heterozygous sickle cell traits have lower

parasite rates and less fatal infections as compared to

children with HbAA. These children with heterozygous

Hb when infected with Plasmodium falciparum are more

likely to survive the acute illness due to the presence of

human leukocyte antigen (HLA) which has been sug-

gested to play crucial role in the defense of host against

malaria infection and reduce susceptibility to severe

Specimen collection/laboratory procedure

1

0

form of malaria . The HLA-Bw53-restricted cytotoxic

T lymphocytes are reported to recognize a conserved

epitope of Plasmodium falciparum liver-stage antigen

type 1 which is a crucial stage malaria disease progres-

sion10. hence children with he_-t₁e₀rozygous Hb are pro-

Diagnosis of malaria was supported by demonstration of

parasitaemia by microscopy on venous blood or serol-

ogy (in those with negative parasitaemia who had over-

whelming features of severe malaria including response

to anti-malarial drugs only). Thick smear was used to

determined the parasite density and was₆_,d₁₇escribed by

7

tected against severe malaria . However, Konotey-

1

1,12

13

Ahulu . and Jones in their separate studies observed

that children with sickle cell disease are not immuned to

cerebral malaria. These findings were corroborated by

1

WHO criteria for malaria parasite density . Thin film

was₁₇made to identify the species of the malaria para-

1

4,15

.

those observed by Luzzatto in some of his studies

site.

These authors concluded that these groups of individuals

die quicker from cerebral malaria and₁w₁_-₁o₅uld therefore

need chemoprophylaxis against malaria.

Hb genotype was done by electrophoresis using cellu-

lose acetate paper₁₈(Shandon Scientific Co Ltd) as de-

scribed by Khon. Two millimeter of venous blood

washed in a solution of 0.9% normal saline by adding

9.0 mls of normal saline to 1ml of blood. The solution

thus formed was centrifuged at 5000 rpm for 5minutes.

The supernatant was discarded living the cells. About

3mls of water was added to 1ml of the cell to effect lysis

of the red cells. 0.1ml of the haemolysate was then ap-

plied to the cellulose acetate strip at pH 8.4 and the strip

was placed across the bars of electrophoretic chamber

with a positive and negative electrode. The rate of mi-

gration on this electrophoresis machine was used to clas-

sify the different genotypes.

In a bid to provide more information on the relationship

between Hb genotype and level of protection against

malaria, this study was undertaken; involving the

evaluation of types of Hb genotype in children (6 - 60

months old) with severe malaria seen in Children Emer-

gency Room (CHER) of the University of Benin Teach-

ing Hospital (UBTH), Benin City.

The study also entailed the determination of the preva-

lence of severe malaria in children with abnormal Hb

genotype, comparison of the prevalence, presentation

and outcome of severe malaria between children with

abnormal Hb and those with HbAA genotypes.

All laboratory results were recorded in a pro-forma.

Data Analysis

Data obtained were entered into a Microsoft Excel 2007

and analyzed using Statistical Package for Social Sci-

ences (SPSS) 13.0 software. Quantitative variables were

summarized using means and standard deviations. The

significance of association between proportions was

tested using chi-square or Fisher's exact tests (where

appropriate) while student t-test was used for compari-

son of means. Binary logistic regression was done to

obtain the independent predictors of severe malaria in

the subjects using the Hb status (abnormal Hb genotype

versus HbAA genotype) as dependent variables. Abnor-

mal genotype is defined as all individual with heterozy-

gous ₈ Hb genotype (example HbAS, HbSS, HbAC,

Patients and Methods

The cross-sectional study was carried out in Children's

Emergency Room (CHER) of UBTH, Benin City, Nige-

ria between January and April 2009. CHER is a 15 bed-

ded unit of the paediatrics department of the hospital.

There were at least three nurses on duty per shift. A con-

sultant paediatrician (with sub-specialty interest on

emergency paediatrics is incharge of all medical affairs

in CHER. There were also two senior registrars and at

least one registrar and a paediatrics casualty officer who

work on shift basis. The nursing staff and the medical

doctors are equipped to take care of common paediatrics

emergencies using the principle of patient triage. The

unit serves as a transition unit for patients admitted in

the main paediatrics ward and has an average patient

load of 150 per month.

1

etc). The level of significance of each test was set at p

< 0.05.

Subjects were children aged 6 to 60 months admitted for

severe malaria based on standard World Health Organi-

zation (WHO) guidelines . They were recruited con-

Results

1

6

Ninety- six well nourished children; 56(58.3%) males

5

3

and 40(41.7%) females were recruited into the study.

The mean age was 29.22 ± 16.02 months (range 6- 60

months). Age and gender distribution of subjects is pre-

sented in Table 1.

Binary logistic regression model using Hb genotype

status (abnormal Hb versus HbAA) as the dependent

variable showed that symptoms such as altered sensor-

ium, prostration, and haemoglobinuria were the signifi-

cant presenting features of severe malaria in children

with abnormal Hb when compared with HbAA children

Table 1: Age and gender distribution of the subjects

(

p = 0.003, 0.041, and 0.023 respectively). Of note was

Age (Years) Male (%) Female (%) Total (%)

that pallor and level of anaemia (pack cell volume at

presentation) were not significant presenting features of

severe malaria in neither patients with abnormal Hb nor

those with HbAA (Table 2).

6

1

2

3

4

-12

7(12.5)

26(46.4) 13(36.1)

14(25.0)

3(5.4)

6(10.7)

0(0.0)

7(7.6)

3-24

5-36

7-48

9-60

39(42.4)

23(25.0)

8(8.7) F o u r

9(25.0)

5(13.9)

9(25.0)

Table 2: Binary logistic regression models of severe

malaria presentation in children with Hb genotype

(

15(16.3)

abnormal Hb versus HbAA) as dependent variable.

Total

56(100.0) 36(100.0) 92(100.0)

(

4.2%) of the 96 subjects were excluded from the study

Abnormal Hb HbAA

Symptoms

Convulsion

n=20(%)

16(80.0)

n=58(%)

β

t

O.R p-value

because their genotype was unequivocal due to history

of recent blood transfusion, thus the total number of

subjects whose genotypes were obtained was 92. This

comprised 56(60.9%) males and 36(39.1%) females.

Sixty-eight (73.9%) of the 92 subjects had genotype AA

while 24(26.1%) had abnormal Hb [Abnormal Hb in-

cluded HbAS 16/24(66.7%); HbSS 7/24(29.2%) and

HbAC 1/24(4.1%)]. Mean age of children with abnor-

mal Hb (32.10 ±17.13 months) was comparable to the

43(74.1) -0.06 -0.50 1.40 0.617

28(48.2) -0.65 -3.10 3.20 0.003

23(39.7) 0.15 1.07 0.50 0.287

42(72.4) 0.08

31(53.4) 0.47

2(3.4) -0.38 -2.33 18.70 0.023

3(5.2) -0.10 -0.60 12.20 0.552

8(13.8) -0.12 -0.96 4.20 0.339

12(20.7) -0.12 -0.98 0.96 0.332

Altered sensorium 15(75.0)

Coma

Pallor

5(25.0)

16(80.0)

13(65.0)

8(40.0)

8(10.0)

4(20.0)

0.74 1.50 0.463

2.09 1.60 0.041

Prostration

Haemoglobinuria

Jaundice

Vomiting

Diarrhoea

3

genotype. The anthropometric measurements between

the two groups were also comparable.

0.64 ± 25.21 months obtained in those with HbAA

β = measure of how strongly each variable influences the de-

pendent variables, O.R = odds ratio, p-value,

Standard Error = 0.000 in all the indepenent variables.

Prevalence of severe malaria in subjects with abnormal

Hb was 20/24(83.3%) as against 58/68(85.3%) observed

in children with HbAA (Fisher's exact; p = 0.75, OR =

Seven (29.2%) of the subjects with abnormal Hb geno-

type were on malaria chemoprophylaxis. All the subjects

0

.86).

(

abnormal Hb or HbAA) were treated with intra-venous

Plasmodium falciparum was the sole specie of malaria

parasite identified.

quinine.

Table 3 shows the outcome of severe malaria in children

with abnormal Hb and those with HbAA. The odds ratio

shows that children with HbAA are 3 times more likely

to die from severe malaria (p = 0.567, O.R = 2.96) when

compared with their counterpart with abnormal Hb.

However none of the symptoms were significant predic-

tors of outcome of severe malaria in neither children

with normal Hb genotype nor those with abnormal Hb

genotype (Table 4). The model fitting information (R2 =

Figure1 shows that subjects with abnormal Hb had sig-

nificantly lower proportion of malaria parasite densities

of 3+ and 4+ (p=0.013) as compared with those with

HbAA genotype. Malaria parasite density independently

was a significant feature of severe malaria in children

with HbAA using Hb status of subjects as dependent

variable (β = -0.286, t = -2.30, R2 = 0.215, p = 0.024).

Figure 1: Types of Hb genotype in relation to malaria

parasite density in subjects.

0

.13, χ2 = 10.23, df = 10, p = 0.42).

Table 3: Outcome of severe malaria in children with

abnormal Hb and HbAA

Outcome

Abnormal Hb(%) HbAA (%) Total (%)

Discharged

Died

Total

20 (100.0)

0(0.0)

20(100.0)

54(93.1) 74(94.9)

4(6.9) 4(5.1)

58(100.0) 78(100.0)

Fisher's Exact; p = 0.567, O.R = 2.96

χ2 = 10.75, df = 3, p = 0.013

5

4

Table 4: Multiple logistic regression models of predic-

tors of outcome of severe malaria (using symptoms at

presentation as independent variables) in children with

normal Hb and those with abnormal Hb genotypes

usually have lower parasites densities when compared

with children with HbAA. The higher parasite density

found in children with HbAA as observed in this present

study was one of the major features of severe malaria in

children with HbAA (R2 = 0.215, p = 0.024) and could

offer some explanations for the slightly higher preva-

lence of severe malaria in children with HbAA as

against those with abnormal Hb genotype.

Symptoms at

presentation

2

β

SE

χ

Exp (β) p-value

Convulsion

Altered sensorium -8.95 7846.02 0.00 0.00

Coma

Pallor

Prostration

Haemoglobinuria -1.99

Jaundice

Vomiting

Diarrhea

Constant

-17.1

0.00 0.00 3.91

0.99

1.00

0.36

1.00

Altered sensorium, prostration and haemoglobinuria

were the significant presenting features of severe ma-

laria in children with abnormal Hb in this study. These

symptoms are easily recognizable by care-givers at

home and hence may warrant their early presentation in

health institutions, with attendant improved outcome.19

To buttress this fact is the better outcome of severe ma-

laria in children with abnormal Hb in this study.

Whereas all children with severe malaria and who had

abnormal Hb genotype survived and were discharged

home, 7.0% of those with HbAA died from severe ma-

laria when compared with children with abnormal Hb

-7.10 7844.52 0.00 0.00

-17.81 0.00 0.00 1.84

-16.51 5430.91 0.00 1.48

1.52 0.00 0.14

1.30 0.00 8.45

16.54 5520.91 0.00 1.53

-1.25 1.38 0.83 0.29

12.61 7847.11 0.00

18.25

SE = Standard Error, β = measure of how strongly each predic-

tor variable influences the outcome variables, Exp (β) = Expo-

nential (β)

(

O.R= 2.96). HbAS specifically is associated with pro-

tection against mortality in children (2 - 16 months)

which is the period they are at most risk of severe falci-

parium malaria. Some authors observed that HbAS is

associated with protection against severe anaemia in the

presence of any level of malaria parasitaemia, hence

these c_-h₉ildren have lower risk of severe anaemic epi-

Discussion

7

sodes. Therefore individuals (especially children) in-

The study revealed a slightly lower prevalence (83.3%)

of severe malaria in children with abnormal Hb when

compared with the prevalence of 85.3% observed in

children with HbAA. This does not support the assertion

of selective protective effect against severe malaria con-

ferred on individuals with abnormal Hb (HbAS, HbSS

and HbSC). This finding is in contrast to the documenta-

fected with Plasmodium falciparum are more likely to

survive th₇e_,₈_,a₁₀c_,₁u₁te malaria illness if they have the HbAS

genotype.

9

tion by Aidoo et al in 2002 which noted that children

Conclusion

with genes for HbAS and HbSS had significantly fewer

episodes of severe malaria when compared with children

This study conforms high prevalence of severe malaria

and high parasite density in children with HbAA as

against those with abnormal Hb. Presence of altered

sensorium, prostration and haemoglobinuria are signifi-

cant presenting features of severe malaria in children

with abnormal Hb genotype and may be relied upon in

effecting early presentation to competent health facility.

9

with HbAA genotype. Aidoo et al views had been cor-

7

roborated by Pasvol et al and Okam.8The high degree

of polymorphism in human leukocyte antigen genes had

been suggested to account for the natural selection

against susceptibilit₀y to a variety of infectious pathogens

1

including malaria. This phenomenon is also said to

play a crucial role in the defense of individual₁s₀_-₁w₁ ith

abnormal Hb against severe malaria parasitaemia.

It

Authors contributions

may also however be argued that children with HbSS

are on malaria chemoprophylaxis and as such, are less

likely to have high parasite burden and frequ₀ent epi-

Ibadin MO, Nwaneri DU

Conception and design,

Drafting the manuscript, Analysis and interpretation

of data

Nwaneri DU Acquisition of data

Ibadin MO Revising the manuscript, for intellectual

content

1

sodes of malaria including the severe forms. In this

present study nearly one-third of subjects were on ma-

laria chemoprophylaxis, yet they presented with severe

malaria hence debunking the protective effect of anti-

malarial chemoprophylaxis against severe malaria.

Ibadin MO, Nwaneri DU Final approval of the com-

pleted manuscript

This study also showed that children with abnormal Hb

have significantly lower incidence of heavy malaria

parasitaemia when compared with their counterpart with

HbA₉A. This finding is in consonance with that of Aidoo

et al in 2002 who observed, that children with abnor-

mal Hb significantly had reduced risks of heavy malaria

parasite densities (>10,000 parasites/uL) that is, they

Conflict of interest: None

Funding: None

5

Acknowledgment

Department of Microbiology and Haematology UBTH,

Benin City for sample analysis (malaria parasite and Hb

electrophoresis).

The authors wish to thank Dr (Mrs) Isreal-Aina Yetunde

and Dr Adigweme Ikechukwu for their assistance in data

collection. Special thanks to Laboratory Scientists in the

References

7

8

.

Pasvol G, Weatherall DJ. The red

cell and the malaria parasite. Brit J

Haematol 1980; 46: 165-170.

13. Jones KDJ. Malarial chemoprophy-

laxis. BMJ 2008; 337: a1875.

14. Luzzatto L. Genetics of red cells

and susceptibility to malaria.

Blood 1979; 54: 961-976.

15. Luzzatto L. Malaria. In: Recent

advances in haematology 1985; 4:

109-126.

16. World Health Organisation: Action

programme on severe and compli-

cated malaria. Trans R Soc Trop

Med Hyg 2000; 94: 190.

17. Monica Cheesebrough. Examina-

tion of blood for malaria parasite

in district laboratory Practice in

tropical countries. Part 1, second

edition. Cambridge University

press 2005.

18. Khon J. Separation of haemoglo-

bins on cellulose acetate. J Clin

Path 1969; 22: 109-111.

19. Tripathy R, Parida S, Mishra DP,

Tripathy D, Das MC, Maguire JH,

et al. Clinical manifestations and

predictors of severe malaria in

Indian children. Paediatr 2007;

120: e454-e460.

1

.

WHO World malaria report

008. http://malaria.who.int/

wmr2008/malaria2008.pdf

2

. Okam M. Sickle cell and thalassae-

mic disorders. Available at http://

sickle.bwh.harvard.edu/index.html.

Accessed on 19/02/2011.

2

. Snow R, Craig H, Newton C,

Steketer R. The public health

burden of plasmodium falcipa-

rum malaria, deriving the num-

bers, working paper no:11,

Fogarty international centre,

National institute of health,

9. Aidoo M, Terlouw DJ, Kolczak

MS, McElroy PD, Ter-Kuile FO,

Kariuki S, et al. Protective effects

of the sickle cell genes against

malaria morbidity and mortality.

Lancet 2002; 359: 1311-1312.

2

003; 1-75.

3

. WHO guidelines on prevention

of the reintroduction of ma-

laria/who regional office for

the eastern Mediterranean.

1

0. Hananantachai H, Patarapotikul J,

Ohashi J, Naka I, Looaresuwan S,

Tokunaga K. polymorphisms of

the HLA-B and HLA-DRB1 genes

in Thai malaria patients. Jpn J

Infect Dis 2005; 58: 25-28.

Publication series no: 34, ISSN

1

020-0428.

4

5

. Malaria Centers for Disease

Control and Prevention, 1600

Clifton Rd. Atlanta, GA 30333,

U.S.A. June 27, 2001.

. Bruce-Chwatt LJ. Malaria in

African infants and children in

Southern Nigeria. Ann Trop

Med Parasitol 1952; 46, 173-

11. Konotey-Ahulu FID. A non-sense

mutation and protection from se-

vere malaria. Lancet 2001; 358:

9

27-928.

1

2. Konotey-Ahulu FID. Malaria and

sickle cell: 'Protection or no pro-

tection?'- Confusion reigns. Avail-

able at http://ucc.edu.gh/konotey/

scell_protection. Accessed on

06/06/2010.

1

75.

6

.

Akpede GO, Sykes, RM. Rela-

tive contribution of bacteremia

and malaria to acute fever

without localizing signs of in-

fections in under-5 children. J

Trop Pediatr 1992; 38:295-298.