# Malaria Parasite Density Estimated with White Blood Cells Count Reference Value Agrees with Density Estimated with Absolute in Children Less Than 5 Years in Central Ghana

Authors: Dennis Adu-Gyasi; Kwaku Poku Asante; Sam Newton; Sabastina Amoako; David Dosoo; Love Ankrah; George Adjei; Seeba Amenga-Etego; Seth Owusu-Agyei
Journal: Malaria Research and Treatment
 (2015)

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## Abstract

Introduction. The estimation of malaria parasite density using a microscope heavily relies on White Blood Cells (WBCs) counts. An assumed WBCs count of 8000/µL has been accepted as reasonably accurate in estimating malaria parasite densities due to the challenge to accurately determine WBCs count.Method. The study used 4944 pieces of laboratory data of consented participants of age group less than 5 years. The study compared parasite densities of absolute WBCs, assumed WBCs, and the WBCs reference values in Central Ghana. Ethical approvals were given by three ethics committees.Results. The mean (±SD) WBCs and geometric mean parasite density (GMPD) were 10500/µL (±4.1) and 10644/µL (95% CI 9986/µL to 11346/µL), respectively. The difference in the GMPD compared using absolute WBCs and densities of assumed WBCs was significantly lower. The difference in GMPD obtained with an assumed WBCs count and that of the WBCs reference values for the study area, 10400/µL and 9200/µL for children in different age groups, were not significant.Discussion. Significant errors could result when assumed WBCs count is used to estimate malaria parasite density in children. GMPD generated with WBCs reference values statistically agreed with density from the absolute WBCs. When obtaining absolute WBC is not possible, the reference value can be used to estimate parasite density.


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## Body

## 1. Background

Malaria is caused by one or a combination of four species ofPlasmodia and leads to over one million deaths, of which over 75% occur in African children under 5 years infected mainly withPlasmodium falciparum (Pf).PfandPlasmodium vivax (Pv) are identified as thePlasmodium species responsible for causing the most severe form of malaria [1].In epidemiological studies, intervention studies, and clinical trials, malaria microscopy is routinely relied upon as a primary endpoint measurement of the level of malaria infection [2]. This is expressed as parasite density and is classically defined as the number of asexual forms of parasite relative to a blood volume (e.g., microliter) [3]. In the four basic counting techniques using microscopy [3], White Blood Cells (WBCs) are relatively used in estimatingPlasmodium parasitaemia by counting the number of parasites against a predetermined number of WBCs on Giemsa stained blood smears.Complete blood counts, particularly WBCs count, can be performed with new generation automated haematology analysers [4] and/or manually using stained microscope smears and the Neubauer chamber and counters [5, 6]. Some health facilities frequently use manual convention methods to determine the complete blood counts of a patient for management as a result of a high cost of purchasing and maintaining fully automated or semiautomated haematology analyzers. Another cost burden using an automated system includes the ability to ensure prompt validation, maintenance, and implementation of rigorous quality systems. Automated machines may therefore not be the preferred choice to quantify WBCs in resource poor areas.Due to the frequent lack of facilities in some malaria endemic countries to quantify WBCs, an assumed WBCs count of 8000/μL of blood has been accepted by World Health Organization as reasonably accurate [7] to estimate malaria parasite densities. Assumed WBCs count of blood may generate systematic errors which could produce incorrect conclusions in patient management or during clinical research that uses malaria parasite counts as an end point [3, 8].Medical laboratories need to set relevant reference ranges for WBCs and other common laboratory parameters for clinical management of patients or during malaria clinical research [9]. Kintampo Health Research Centre has established reference values for common haematological and biochemical laboratory parameters for its study area covering an area of about 7200 km2 [10].The team compared the geometric mean parasite densities (GMPD) calculated using absolute WBCs, assumed WBCs, and WBCs reference values of the participants enrolled in the malaria studies in middle Ghana to assess the impact of using these as malaria parasite density estimators.


## 2. Methods

### 2.1. Ethics Statement

The studies used for this data analysis were approved by the ethical review committees of the Ghana Health Service, London School of Hygiene and Tropical Medicine, Kintampo Health Research Centre Institutional Ethics Committee, Noguchi Memorial Institute of Medical Research, and The Ghana Food and Drugs Authority. Written informed consent was sought from all mothers whose children participated in the studies.


### 2.2. Site Description

The data analysis was carried out on laboratory data from a study area with a profile presented by Adu-Gyasi et al., 2012 [8]. Briefly, the area is located within the forest-savannah transitional ecological zone in Ghana with perennial malaria transmission [11].Pf is the predominatingPlasmodium species whileP. malariae andP. ovale are in the minority.


### 2.3. Description of Data Sources

The data was obtained from children under 5 years who were recruited into malarial studies carried out by Kintampo Health Research Centre between the periods of October 2008 and March 2011. In the first study, children were enrolled and followed actively every month for two years. The children were referred to the hospital for care whenever they were unwell to determine the incidence of malaria [12]. The second study which was a clinical trial was to determine the effect of providing Micronutrient Powder with or without iron on the incidence of malaria among children under three years living in a high malaria-burden area in Ghana [13].


### 2.4. Blood Sample Collection and Processing

In each of the studies, blood samples were collected by finger-prick into 0.5 mL microtainers containing ethylenediaminetetraacetic acid (K2EDTA-BD, USA) from the participants on their scheduled visit days and on any other day participants visited the health facilities with illness. Samples were collected and transported according to the Standard Operating Procedures (SOPs) established by the Kintampo Health Research Centre. Briefly, participant’s finger was rubbed, and the tip was cleaned with disposable alcohol swab and allowed to dry. Safety lancet was used to prick and drops of blood were collected into labelled test-tubes to the required volume. Collected samples were used for full blood count analysis and for making blood smears. Examination of thick and thin smears was carried out as described by Adu-Gyasi et al., 2012 [8].


### 2.5. Estimation of Parasite Density

Parasite densities for all participants were calculated using assumed WBCs count of 5000/μL, 6000/μL, 8000/μL, and 10000/μL of blood. In addition, we used the WBCs reference values established for children aged less than one year (10000/μL of blood) and also for children less than five years (9200/μL) [14].


### 2.6. Data Entry, Cleaning, and Analyses

Data obtained from the database of the malarial studies was checked for completeness and consistency and all queries were resolved. All parasite negative blood slide results were removed before analysis. Analyses were done using STATA (version 12; Stata Corp., TX, USA) and GraphPad PRISM version 5.0 (GraphPad Software, Inc.) particularly for geometric mean and intervals. Geometric means at 95% CI that did not overlap were considered significant.


## 2.1. Ethics Statement

The studies used for this data analysis were approved by the ethical review committees of the Ghana Health Service, London School of Hygiene and Tropical Medicine, Kintampo Health Research Centre Institutional Ethics Committee, Noguchi Memorial Institute of Medical Research, and The Ghana Food and Drugs Authority. Written informed consent was sought from all mothers whose children participated in the studies.


## 2.2. Site Description

The data analysis was carried out on laboratory data from a study area with a profile presented by Adu-Gyasi et al., 2012 [8]. Briefly, the area is located within the forest-savannah transitional ecological zone in Ghana with perennial malaria transmission [11].Pf is the predominatingPlasmodium species whileP. malariae andP. ovale are in the minority.


## 2.3. Description of Data Sources

The data was obtained from children under 5 years who were recruited into malarial studies carried out by Kintampo Health Research Centre between the periods of October 2008 and March 2011. In the first study, children were enrolled and followed actively every month for two years. The children were referred to the hospital for care whenever they were unwell to determine the incidence of malaria [12]. The second study which was a clinical trial was to determine the effect of providing Micronutrient Powder with or without iron on the incidence of malaria among children under three years living in a high malaria-burden area in Ghana [13].


## 2.4. Blood Sample Collection and Processing

In each of the studies, blood samples were collected by finger-prick into 0.5 mL microtainers containing ethylenediaminetetraacetic acid (K2EDTA-BD, USA) from the participants on their scheduled visit days and on any other day participants visited the health facilities with illness. Samples were collected and transported according to the Standard Operating Procedures (SOPs) established by the Kintampo Health Research Centre. Briefly, participant’s finger was rubbed, and the tip was cleaned with disposable alcohol swab and allowed to dry. Safety lancet was used to prick and drops of blood were collected into labelled test-tubes to the required volume. Collected samples were used for full blood count analysis and for making blood smears. Examination of thick and thin smears was carried out as described by Adu-Gyasi et al., 2012 [8].


## 2.5. Estimation of Parasite Density

Parasite densities for all participants were calculated using assumed WBCs count of 5000/μL, 6000/μL, 8000/μL, and 10000/μL of blood. In addition, we used the WBCs reference values established for children aged less than one year (10000/μL of blood) and also for children less than five years (9200/μL) [14].


## 2.6. Data Entry, Cleaning, and Analyses

Data obtained from the database of the malarial studies was checked for completeness and consistency and all queries were resolved. All parasite negative blood slide results were removed before analysis. Analyses were done using STATA (version 12; Stata Corp., TX, USA) and GraphPad PRISM version 5.0 (GraphPad Software, Inc.) particularly for geometric mean and intervals. Geometric means at 95% CI that did not overlap were considered significant.


## 3. Results

Of the 39851 results received over the study period, data of 4944 participant results were consistent and complete for the purpose of our analysis. Of the total, 50.6% (2498/4944) were males and 49.4% (2443/4944) were females.The mean (±SD) WBCs and GMPD of the 4944 positive samples were 10500/μL (±4.1) and 10644/μL (95% CI 9986/μL to 11347/μL) of blood, respectively. There was no significant difference between the mean (SD) WBCs 10600/μL (4.0) for males and 10500/μL (±4.1) for females (P
=
0.255). Neither was there a significant difference between the GMPD 11058/μL (95% CI 10114/μL to 12091/μL) of blood for males (Table 1) and 10217/μL (95% CI 9324/μL to 11197/μL) of blood for females (Table 2).Table 1
Comparison of parasite densities using absolute WBCs, assumed WBCs, and WBCs reference value among males of the population.
Absolute WBCs
WBCs count of 5000/µL
WBCs count of 6000/µL
WBCs count of 8000/µL
WBCs count of 9200/µL
WBCs count of 10000/µL
WBCs count of 10400/µL
Number of observations,N
2498
2498
2498
2498
2498
2498
2498
Minimum
10
10
12
16
18
20
21
25% percentile
2040
1050
1260
1680
1932
2100
2184
Median
12497
6798
8158
10877
12509
13597
14141
75% percentile
71645
35750
42900
57200
65780
71500
74360
Maximum
2081035
1705766
2046920
2729226
3138610
3411533
3547994
Mean
68255
35019
42023
56031
64435
70038
72840
Std. deviation
139655
79080
94896
126528
145508
158160
164487
Std. error (mean)
139655
79080
94896
126528
145508
158160
164487
Lower 95% CI of mean
62775
31917
38300
51067
58726
63833
66386
Upper 95% CI of mean
73734
38122
45746
60995
70144
76244
79293
Geometric mean
11058
5596
6715
8953
10296
11191
11639
Lower 95% CI of geo. mean
10114
5116
6139
8186
9414
10232
10642
Upper 95% CI of geo. mean
12091
6120
7344
9792
11261
12240
12730Table 2
Comparison of parasite densities using absolute WBCs, assumed WBCs, and WBCs reference value among females of the population.
Absolute WBCs
WBCs count of 5000/µL
WBCs count of 6000/µL
WBCs count of 8000/µL
WBCs count of 9200/µL
WBCs count of 10000/µL
WBCs count of 10400/µL
Number of observations,N
2443
2443
2443
2443
2443
2443
2443
Minimum
13
10
12
16
18
20
21
25% percentile
1843
957
1149
1531
1761
1914
1991
Median
10325
5375
6450
8600
9890
10750
11180
75% percentile
73851
35866
43039
57385
65993
71731
74600
Maximum
2790960
1993543
2392252
3189669
3668119
3987086
4146569
Mean
70980
38690
46428
61904
71190
77380
80475
Std. deviation
158340
102849
123418
164558
189242
205697
213925
Std. error (mean)
3204
2081
2497
3329
3829
4162
4328
Lower 95% CI of mean
64698
34610
41532
55375
63682
69219
71988
Upper 95% CI of mean
77262
42770
51324
68433
78698
85541
88962
Geometric mean
10217
5243
6292
8389
9647
10486
10906
Lower 95% CI of geo. mean
9324
4783
5739
7653
8800
9566
9948
Upper 95% CI of geo. mean
11197
5748
6897
9196
10576
11495
11955With an assumed WBCs count of 8000/μL, a GMPD of 8679/μL (95% CI, 8140/μL to 9253/μL) of blood was estimated (Table 3).Table 3
Comparison of parasite densities using absolute WBCs, assumed WBCs, and WBCs Reference value.
Absolute WBCs
WBCs count of 5000/µL
WBCs count of 6000/µL
WBCs count of 8000/µL
WBCs count of 9200/µL
WBCs count of 10000/µL
WBCs count of 10400/µL
Number of observations,N
4944
4944
4944
4944
4944
4944
4944
Minimum
10
10
12
16
18
20
21
25% percentile
1947
1013
1215
1620
1863
2025
2106
Median
11316
6081
7298
9730
11190
12163
12649
75% percentile
72777
35780
42936
57248
65835
71560
74423
Maximum
2790960
1993543
2392252
3189669
3668119
3987086
4146569
Mean
69618
36851
44221
58961
67805
73701
76649
Std. deviation
149152
91603
109924
146565
168550
183206
190534
Std. error (mean)
2121
1303
1563
2085
2397
2606
2710
Lower 95% CI of mean
65459
34297
41156
54875
63106
68593
71337
Upper 95% CI of mean
73776
39405
47286
63048
72505
78809
81962
Geometric mean
10644
5424
6509
8679
9981
10848
11282
Lower 95% CI of geo. mean
9986
5088
6105
8140
9361
10175
10581
Upper 95% CI of geo. mean
11347
5783
6940
9253
10641
11567
12029In using the WBCs reference values established among children in the study area, (10400/μL), a GMPD of 11282/μL (95% CI, 10581/μL to 12029/μL) of blood and a WBCs count of 9200/μL produced a GMPD of 9981/μL (95% CI, 9361/μL to 10641/μL) of blood.The difference in the GMPD calculated using absolute WBCs compared to densities estimated with assumed WBCs was significantly lower for 8000/μL. However, GMPD for assumed WBCs count of 10.0 × 109/L, WBCs reference values of 10400/μL, and 9200/μL estimated a geometric mean parasite density of 10848/μL (95% CI, 10175/μL to 11567/μL), 11282/μL (95% CI, 10581/μL to 12029/μL), and 9981/μL (95% CI, 9361/μL to 10641/μL) of blood that was not significantly different from estimates obtained with the absolute WBCs (Table 3). The interval of GMPD obtained when an assumed WBCs of 8000/µL, 6000/µL and 5000/µL were used and that from absolute WBCs count did not overlap. However, the intervals of GMPD from absolute WBCs count and that obtained with assumed WBCs counts of 9200/µL, 10000/µL and 10400/µL overlapped showing no significant difference in the GMPD (Table 3).A correlation analysis of the malaria parasite densities with the WBCs reference value and assumed WBCs compared to the absolute WBCs showed a significant level of correlation (P
<
0.0001) with all the assumed WBCs count (Table 4).Table 4
Correlation analysis of malaria parasite densities using assumed and reference value WBCs count compared with absolute WBCs.
Variable
Mean
Standard deviation
Minimum
Maximum
Absolute WBCs
69618
149152
10
2790960
WBCs of 5000/µL
36851
91603
10
1993543
WBCs of 6000/µL
44221
109924
12
2392252
WBCs of 8000/µL
58961
146565
16
3189669
WBCs of 9200/µL
67805
168550
18
3668119
WBCs of 10000/µL
73701
183206
20
3987086
WBCs of 10400/µL
76649
190534
21
4146569
Absolute WBCs
WBCs count of 5000/µL
WBCs count of 6000/µL
WBCs count of 8000/µL
WBCs count of  9200/µL
WBCs count of 10000/µL
WBCs count of 10400/µL
Absolute WBCs
1.0000
WBCs of 5000/µL
0.8825
1.0000
WBCs of 6000/µL
0.8825
1.0000
1.0000
WBCs of 8000/µL
0.8825
1.0000
1.0000
1.0000
WBCs of 9200/µL
0.8825
1.0000
1.0000
1.0000
1.0000
WBCs of 10000/µL
0.8825
1.0000
1.0000
1.0000
1.0000
1.0000
WBCs of 10400/µL
0.8825
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
Significance level of correlation isP
<
0.0001.


## 4. Discussion

Malaria parasite density is necessary for patient management. This has become dominated by the convenient but inaccurate assumption of a constant WBCs count of 8000/μL of peripheral blood [3, 15] due to lack of capacity to measure patients absolute WBCs [16].The mean WBCs count from the participants withmalaria infection compares with the WBCs reference values, 10400/μL for children less than 1 year and 9200/μL for children up to 5 years [14]. The parasite densities from the WBCs reference values agree with the density obtained from using participant’s absolute WBCs.It is agreed that the best solution in estimating parasite densities would be to use the corresponding absolute WBCs count for each age group [17]. Though there is a significant level of correlation, estimating the parasite density ofPlasmodiumspecies with the assumed WBCs count of 8000/μL of blood [7], compared to the absolute and reference value WBCs count, would mean underestimating significantly the parasite density ofPlasmodiumspecies infections for patients in the study area. Therefore, establishing regional based reference WBCs as suggested [17] to estimate parasite densities in malarial infections will be appropriate. This was evident in the fact that the geometric mean parasite density, 11282/μL (95% CI, 10581/μL to 12029/μL) and 9981/μL (95% CI, 9361/μL to 10641/μL) of blood, estimated by using the established WBCs standard values of 10,400/μL and 9200/μL of blood for children less than 1 year and up to 5 years old, respectively, overlaps with the geometric mean parasite density obtained by using the mean absolute WBCs count, 10644/μL (95% CI 9986/μL to 11347/μL) of blood, of the participants. Results obtained when an assumed WBCs count of 10000/μL of blood was used to estimate the parasite density [8] were also consistent with that from the absolute WBCs count of participants. The parasitaemia obtained by the use of the other assumed WBCs count was significantly lower (Table 3) [8].


## 5. Conclusions

Since lack of resources in some settings in Africa makes it difficult to estimate malaria parasite density based on actual WBCs count of patients, where available, the study recommends the use of an established WBCs reference value for a known population and where the reference value could be implied. The reference value established in Kintampo has been predicted as a malaria density estimator when compared with the absolute WBCs of participants. In environments where reference values have not been established, the study affirms the use of an assumed WBCs count of 10000/μL of blood to estimate malaria parasite density as documented [8].



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