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Tropical Medicine and International Health

doi:10.1111/j.1365-3156.2011.02738.x

volume 16 no 5 pp 551554 may 2011

Short communication

Standardised versus actual white cell counts in estimating thick


film parasitaemia in African children under five
Piero Olliaro1,2, Abdoulaye Djimde3, Corine Karema4, Andreas Martensson5,6, Jean-Louis Ndiaye7,
Sodiomon B. Sirima8, Grant Dorsey9 and Julien Zwang10
1 UNICEF UNDP WB WHO Special Programme for Research & Training in Tropical Diseases, Geneva, Switzerland
2 Centre for Tropical Medicine and Vaccinology, University of Oxford, Oxford, UK
3 Malaria Research and Training Center, University of Bamako, Bamako, Mali
4 National Malaria Control Programme, Kigali, Rwanda
5 Infectious Diseases Unit, Karolinska Institutet, Stockholm, Sweden
6 Division of Global Health, Karolinska Institutet, Stockholm, Sweden
7 Department of Parasitology, Cheikh Anta Diop University, Dakar, Senegal
8 Centre National de Recherche et de Formation sur le Paludisme, Ministe`re de la Sante, Ouagadougou, Burkina Faso
9 Department of Medicine, University of California San Francisco, San Francisco, CA, USA
10 Drugs for Neglected Diseases Initiative, Geneva, Switzerland

Summary

In patients with malaria, parasitaemia is usually estimated by assuming 8000 white cell counts (WCC)
per microlitre of blood. In a sample of 3044 African children under 5 years of age with uncomplicated
falciparum malaria, parasitaemia estimated using standardised WCC was compared to parasitaemia
calculated based on each childs own WCC. The two methods produced comparable results. However,
WCC were >8000 in under-fives with an inverse relationship with age, resulting in the standard
approximation method significantly underestimating parasitaemia in the youngest age group and
overestimating parasitaemia in the oldest age groups.
keywords malaria, parasitaemia, white cell counts

Plasmodium falciparum parasitaemia is normally estimated


by counting on microscopic examination of Giemsa-stained
blood smears the number of parasites after a pre-determined number of white blood cells (normally 100500) has
been counted and assuming a standard white cell count
(WCC, normally 8000 ll). The following formula is
generally applied: parasites ll = [(number of parasites
counted number of leucocytes counted) 8000 leucocytes ll] [World Health Organization (WHO) 2010].
This approximation is expedient as WCC normally
cannot be determined in most primary health care settings
of the malaria-endemic areas. However, WCC vary greatly
and are age-dependent. As opposed to western standards
(which have 5.011.9 WBC 109 l for all children under
6 years of age), a study in Mozambique reported intervals
of 2.216.6 in children 12 years old, 5.85.6 in 2- to
3-year-olds, 5.514.7 in 3- to 4-year-olds and 5.614.4 in
4- to 5-year-olds (Quinto et al. 2006). Several studies,
predominantly in malaria-infected adults, concur that

2011 Blackwell Publishing Ltd

WCC increase in acute malaria (Erhart et al. 2004;


McKenzie et al. 2005). Therefore, this estimation may
generate systematic errors and lead to incorrect conclusions. The present analysis was conducted to assess the
correlation between parasitaemia as generally estimated
and parasitaemia calculated based on a patients WCC.
Children under 5 years of age were selected because they
are the most vulnerable group and constitute the study
population in the majority of clinical trials of antimalarial
drugs in sub-Saharan Africa. Data on age, parasitaemia
and white blood cell total and differential counts were
extracted from a database of seven randomised controlled
trials of artesunate plus amodiaquine vs. various comparator drugs for treating acute uncomplicated falciparum
malaria at 14 sites in 10 sub-Saharan African countries (on
average 217 patients per site or 304 patients per country).
These studies were conducted between February 1999 in
Kenya and December 2006 in Senegal. All were approved
by local or national and WHO ethics committees. The
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Tropical Medicine and International Health

volume 16 no 5 pp 551554 may 2011

P. Olliaro et al. Standardised vs. actual white cells to estimate malaria parasitaemia

designs of these clinical trials were similar and based on the


WHO (2006) protocol, as described by Zwang et al.
(2009). Here, analyses were restricted to the patients under
5 years of age with recorded WCC at baseline (n = 3044,
i.e. 26% of the 11 700 patients enroled in the trials).
In the original studies, parasitaemia (number ll) was
quantified by a standard approximation method (40
number of parasites per 200 WCCs on thick film, i.e.
assuming 8000 WCCs ll) in six of the seven studies
included in this analysis (Adjuik et al. 2002; Martensson
et al. 2005; Karema et al. 2006; Dorsey et al. 2007;
Djimde et al. 2008; Sirima et al. 2009). The 7th study
counted P. falciparum, assuming an average WCC of
7500 ll (Ndiaye et al. 2009). For the purpose of coherence, we also recalculated the estimated parasitaemia based
on 8000 WCCs for the present study.
Parasitaemia on admission was recalculated for all studies
assuming 200 WCCs for all patients vs. each patients own
actual WCC on admission. The Wilcoxon signed rank test
was used to compare both methods of estimating parasitaemia on paired patient data; the Wilcoxon rank test was
used for non-paired data. For multivariate analysis, we used
a linear model between age (in years) and parasitaemia (logtransformed) with a random effect for study site. P-values
under 0.05 were considered significant. The statistical
programme used was STATA (version 10; Stata Corp.).
The geometric mean of the actual pre-treatment parasitaemia was 18 334 per ll overall (Table 1). Combining
data from all sites over the entire 04 year age range, no
significant difference (+2%, P = 0.277) was detected
between estimated and actual parasitaemia. However,
statistically significant differences were detected when data
were stratified by age and site. Using paired analysis,
parasitaemia estimated using standardised WCC was
significantly lower than when computed using actual WCC
in children under 1 year of age ()14%, P = 0.001) and
slightly higher in 4-year-olds (+8%, P = 0.076). Differences were detected at 11 of the 14 sites between the two
methods, with five sites overestimating (Zanzibar and

Age
(year)
<1
1
2
3
4
Total

WBC
( 109 l)

Parasitaemia
calculated on
actual WCC

Parasitaemia
estimated on
standardised
8000 WCC

220
370
863
866
725
3044

10 167
9634
9095
8507
8072
8827

15
18
19
17
18
18

13
17
19
18
19
18

125
365
905
920
090
334

031
822
988
749
603
681

Rwanda where WCC were lower) and six underestimating


parasitaemia (no significant difference at three sites)
(Table 2). In the sites with significant differences between
the two methods, the overall percentage overestimation of
the geometric mean was twice (+37%) as much as that of
the underestimation ()19%) corresponding approximately
to the difference in WCC (10.6 and 6.6 109 l, respectively, P = 0.001) and in age (2 vs. 3 years, P = 0.001).
Overall in these sites, the difference in parasitaemia
estimated based on actual (17 852 ll) and standardised
WCC (18 210 ll) was not significant (+2%, P = 0.313,
paired analysis).
Using a linear random effects model, there was a trend
for older children having higher log-transformed parasitaemia when using standardised WCC (r = 0.019, P = 0.058)
but when using the actual WCC, age was not related to
parasitaemia (r = )0.001, P = 0.954) (Figure 1).
This study shows that, in children under 5 years of age
with acute uncomplicated malaria living in sub-Saharan
African countries with moderate to intense malaria transmission, the parasite biomass needed for malaria to be
clinically manifest is not significantly different across the
age range 14 years when calculated using the patients
actual WCC. Overall, parasitaemia on presentation did not
change significantly with age. One must not forget
although that these, like any other trials, had set limits of
parasitaemia to qualify for inclusion. Here, the upper limit
of parasitaemia was 200 000 ll in all studies but the
lowest limit varied from 500 ll (Dorsey et al. 2007) to
1000 ll (Adjuik et al. 2002; Ndiaye et al. 2009; Sirima
et al. 2009) to 2000 ll (Martensson et al. 2005; Karema
et al. 2006; Djimde et al. 2008). Therefore, it is not
possible to categorically set the pyrogenic threshold of
P. falciparum parasitaemia in African children under
5 years of age from this dataset.
The admission parasitaemia as customarily estimated in
the published series assuming 8000 WBCs ll increased
with age, which would be interpreted as a sign of mounting
immunity with older children being able to tolerate higher

Relative
difference
(%)

)16
)3
0
4
8
2

0.001
0.195
0.238
0.395
0.076
0.277

Table 1 Pre-treatment parasitaemia in


African malaria patients under 5 years of
age estimated (as reported in the publication) based on standardised and actual
white cell counts (WCC) by age (geometric
mean)

P-values result from a Wilcoxon paired analysis.

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2011 Blackwell Publishing Ltd

Tropical Medicine and International Health

volume 16 no 5 pp 551554 may 2011

P. Olliaro et al. Standardised vs. actual white cells to estimate malaria parasitaemia

Table 2 Estimated (as reported in the publication) and actual [recalculated using actual white cell counts (WCC)] pre-treatment
parasitaemia by site (geometric mean)
WCC ( 109 l)

Age (years)

Site

Mean

SD

Mean

SD

Parasitaemia
calculated on
actual WCC

Burkina Faso-Pouytenga
Cameroon-Mendong
Gabon-Lamberene
Kenya-Migori
Madagascar-Tsiroanomandidy
Mali-Bancoumana
Mali-Bougoula
Rwanda-Kicukiro
Rwanda-Mashesha
Rwanda-Rukara
Senegal-Mlomp
Uganda-Kampala
Zanzibar-Kivunge
Zanzibar-Micheweni
Total

582
72
71
28
55
94
669
174
218
250
204
249
278
100
3044

2.0
2.9
3.2
2.4
3.3
3.0
2.3
2.9
2.8
2.3
3.1
3.2
2.4
2.0
2.5

1.2
0.9
0.9
1.6
0.8
0.9
1.2
1.0
1.0
1.0
0.9
0.8
1.2
1.2
1.2

11.5
8.2
8.2
9.9
7.7
10.6
10.4
6.8
5.4
4.0
11.2
9.1
6.3
6.5
8.8

4.8
2.1
2.9
4.2
2.9
3.6
4.4
2.6
1.6
1.6
4.6
3.8
3.1
1.5
4.5

18
57
26
30
10
31
19
27
14
17
25
11
11
13
18

797
345
492
864
145
787
164
999
331
641
846
682
777
432
334

Parasitaemia
estimated on
standardised
8000 WCC

14
54
27
27
10
23
15
35
21
37
20
11
16
17
18

0.001
0.146
0.920
0.007
0.409
0.001
0.001
0.001
0.001
0.001
0.001
0.020
0.001
0.001
0.277

151
353
597
402
456
813
935
295
942
676
129
035
479
026
681

P-values result from a Wilcoxon paired analysis.

30

(a)

150 000

WCC day 0 (109/l)

Actual parasitaemia (u/l) day 0

200 000

100 000

(b)

20

10

50 000

0
<1

2
Age (year)

<1

2
Age (year)

Figure 1 Relationship between age and parasitaemia (calculated on actual white cell counts) (a) and white cells counts (b) on admission.
The red dotted horizontal line represents the geometric mean of the estimated parasitaemia for the entire age range. WCC, white cells
counts.

parasitaemia. However, this appears to be an artefact, as it


does no longer hold true when each subjects own WCC
are used to calculate parasitaemia no significant association between age and parasitaemia was observed. This
discrepancy occurs because although the two methods
agree overall for all under-fives, WCC are higher in
younger children.

2011 Blackwell Publishing Ltd

These changes in WCC with age cause the standard


approximation method to significantly underestimate parasitaemia in infants. The equipoise between the two
methods of estimation in our study was at 2 years of age;
thereafter, parasitaemia became (non-statistically significant) higher with standardised than with actual WCC. It
should also be noted that here the average WCC were
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Tropical Medicine and International Health

volume 16 no 5 pp 551554 may 2011

P. Olliaro et al. Standardised vs. actual white cells to estimate malaria parasitaemia

approximately 8800 and >8000 (the figure assumed in the


standard method) at all ages.
A cross-sectional study in 240 Nigerian children aged
18 years found that the assumed parasite counts with the
standard method overestimated parasitaemia but did not
provide details as to possible age-related differences
(Jeremiah & Uko 2007). Also, children in that study were
asymptomatic, and parasite counts were considerably
lower than those recorded in the symptomatic children of
our study.
Most laboratories in malaria-endemic rural areas would
not have the capacity to measure WCC; our data indicate
that in routine practice using standard WCC counts seems
altogether acceptable. Moreover, with WCC declining with
age, the standardised 8000 WCC generates adequate
estimates in older children and adults.
However, clinical trials of uncomplicated falciparum
malaria should express parasitaemia calculated on the basis
of the patients own baseline WCC. The differences
observed between the two methods, although overall
minimal, are important especially in the younger children.
Using actual WCC will increase precision in estimating
parasitaemia and improve our understanding of malaria
and response to treatment, including haematological
changes in acute and convalescent malaria. A larger sample
size is desirable to confirm that this observation can be
widely generalised.
Acknowledgements
We thank the patients and staff of all trial sites who
participated in these trials. JZ received a grant from DNDi
(Drugs for Neglected Diseases initiative) to do this analysis.
The sponsor did not have any role in the design and
conduct of the analysis, interpretation of results or writeup of the manuscript. We would like to thank Ric Price for
advice. PO is a staff member of the WHO; the authors
alone are responsible for the views expressed in this
publication, and they do not necessarily represent the
decisions, policy or views of the WHO.
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Corresponding Author Piero Olliaro, Special Programme for Research & Training in Tropical Diseases (TDR), World Health
Organization, Avenue Appia 20, 1211 Geneva 27, Switzerland. E-mail: olliarop@who.int

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2011 Blackwell Publishing Ltd

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