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ASSOCIATION OF INTRALEUKOCYTIC PLASMODIUM FALCIPARUM MALARIA
PIGMENT WITH DISEASE SEVERITY, CLINICAL MANIFESTATIONS, AND
PROGNOSIS IN SEVERE MALARIA
KIRSTEN E. LYKE, DAPA A. DIALLO, ALASSANE DICKO, ABDOULAYE KONE, DRISSA COULIBALY,
ANDO GUINDO, YACOUBA CISSOKO, LANSANA SANGARE, SEYDOU COULIBALY, BLAISE DAKOUO,
TERRIE E. TAYLOR, OGOBARA K. DOUMBO,
AND CHRISTOPHER V. PLOWE
Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland; Malaria
Research and Training Center, Bandiagara Malaria Project, University of Bamako, Bamako, Mali; Blantyre Malaria Project,
University of Malawi College of Medicine, Blantyre, Malawi; College of Osteopathic Medicine, Michigan State University,
East Lansing, Michigan
Abstract. Peripheral parasite density of Plasmodium falciparum is used as an indicator of malaria disease severity,
but does not quantify central sequestration, which is important in the pathogenesis of severe disease. Malaria pigment,
recognizable within the cytoplasm of phagocytic cells by light microscopy may represent a peripheral marker for parasite
biomass. One hundred seventy-two index cases of severe malaria and 172 healthy age-, residence-, and ethnicity-matched
controls with uncomplicated malaria in Bandiagara, Mali were analyzed prospectively for presence of malaria pigment.
The presence of polymorphonuclear cell (PMN) and monocyte pigment was strongly associated with severe disease
compared with uncomplicated malaria. Total PMN pigment burden in children with severe malaria was higher in those
with cerebral manifestations and with combined cerebral manifestations and severe anemia (hemoglobin ⱕ 5 g/dL) but
was not associated with hyperparasitemia (> 500,000 asexual forms/mm
3
). Additionally, pigmented PMNs/mm
3
was
associated with a fatal outcome in patients with severe malaria. This study validates the presence of malaria pigment in
monocytes and neutrophils as a marker for disease severity, and demonstrates that pigmented neutrophils are associated
with cerebral malaria and with death in children with severe malaria.
INTRODUCTION
The pathophysiology of severe Plasmodium falciparum ma-
laria is complex and results in a broad spectrum of disease
manifestations.
1,2
Sequestration of parasites is thought to be
central to these disease processes, but anemia, cytokine pro-
duction, and metabolic distortion may also contribute to the
spectrum of disease.
3–5
Parasite density generally correlates
with disease severity, but peripheral parasitemia does not
necessarily reflect the burden of sequestered parasites. In
hospital-based studies, malaria therapy taken prior to admis-
sion may further hamper efforts to properly diagnose and
characterize acute illness. Methods of estimating malaria dis-
ease severity and predicting prognosis may be useful in strati-
fying patients in the early stages of admission and assessment.
Hemozoin, also known as malaria pigment, is a product of
hemoglobin digestion by Plasmodia.
6
The existence of hemo-
zoin has been recognized for centuries, but the complexities
of its formation and its biologic and clinical relevance are
incompletely understood. As part of parasite erythrocytic in-
vasion, hemoglobin is proteolyzed releasing toxic heme. Due
to the absence of heme oxygenase, Plasmodia are unable to
cleave heme into open-chain tetrapyrrole to allow for cellular
excretion.
7
To detoxify soluble heme, a novel breakdown
product known as hemozoin is created intracellularly. This
digestive end-product of hemoglobin is sequestered in the P.
falciparum digestive vacuole within infected red blood cells
and released into host circulation during schizogeny. Hemo-
zoin is composed of Fe
III
-porphyrin units linked by propi-
onate oxygen-iron bonds into polymers accompanied by ad-
ditional host and parasite nucleic acids and lipids.
8,9
Once this
insoluble polymer is released into host circulation, scavenger
neutrophils and monocytes phagocytose the material. It is
easily visible by light microscopy, appearing as a black,
brown, or amber pigment or as a birefringent crystal under
polarized light
10,11
(Figures 1 and 2). Given that the typical
half-life of a neutrophil is 6−8 hours and that of a monocyte
is several days, the quantity and distribution of engulfed pig-
ment within these phagocytic cells may reflect the chronology
of a patient’s infection.
In the past decade, investigators have demonstrated the
utility of assessing pigment formation for disease character-
ization. The presence of pigment correlates with mortality of
severe malaria in Asian adults from an area hypoendemic for
disease
12
and with disease severity in African children.
13–15
We sought to expand and validate these clinical findings
through a matched case-control study in Malian children in
which we assessed the presence and quantity of malaria pig-
ment and its association with disease severity, clinical mani-
festations and prognosis.
METHODS AND MATERIALS
Study site and enrollment. Blood films were obtained from
516 Malian children (age range ⳱ 3 months to 14 years) on
enrollment into a case-control study evaluating risk and pro-
tective factors for severe malaria. The study site of Bandia-
gara is located in east central Mali and has intense seasonal
transmission (July−December) of P. falciparum malaria. The
dominant ethnic group is Dogon (80%) with Peuhl (10%),
Bambara (3%), and other ethnic groups (7%) also present.
One hundred seventy-two index cases of severe malaria from
Bandiagara and surrounding areas were admitted to the Ban-
diagara Malaria Project ward from July 2000 to December
2001. Cases were classified as severe malaria based on modi-
fied criteria put forth by the World Health Organization
1
(Table 1). More than one clinical diagnosis for severe malaria
was possible, but cerebral malaria and severe anemia were
considered to be the primary defining features when they
co-existed with other criteria. Each index case was age-, resi-
dence-, and ethnicity-matched to a case of uncomplicated ma-
laria and a healthy control. Age categories were defined as
3−5 months, 6−11 months, 1 year, 2 years, 3−4 years, 5−6
Am. J. Trop. Med. Hyg., 69(3), 2003, pp. 253–259
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
253
years, 7−8 years, 9−10 years, 11−12 years, and 13−14 years.
Residence was defined as one of eight distinct sectors of Ban-
diagara town or, in the case of children from outer villages,
the specific village of origin. Uncomplicated malaria was de-
fined as P. falciparum parasitemia and an axillary tempera-
ture ⱖ 37.5°C detected by active surveillance, or parasitemia
and symptoms leading to treatment-seeking behavior in the
absence of other clear cause of fever on passive surveillance.
Matched uncomplicated malaria controls were enrolled from
the population of children presenting to the daily Bandiagara
Malaria Project clinic. Healthy controls were enrolled after
traveling to the home of the child with severe malaria and
following a standardized routine of exiting the front entrance
of a compound and making consecutive left turns until an-
other compound with an eligible control was identified. Chil-
dren were enrolled as healthy controls if they were asymp-
tomatic for acute illness, had no evidence or history of chronic
illness, and if they were found to be aparasitemic upon ex-
amination. The study protocol was reviewed and approved by
the University of Mali and the University of Maryland Insti-
tutional Review Boards. Written informed consent was ob-
tained from parents or guardians of all study participants.
Assessment of pigment. Thick and thin blood films ob-
tained at enrollment prior to therapy were stained with Gi-
emsa. Peripheral parasite density was determined from thick
films based on the number of asexual forms/mm
3
per 300
leukocytes and an assumed white blood cell (WBC) count of
7,500 cells/mm
3
. Parasite density calculations were performed
immediately and results were used for severe disease stratifi-
cation. Thin smears were analyzed in groups with absolute
WBC and differential counts determined manually by two
microscopists. Acceptable degrees of variation have been
noted between automated and manual WBC counts and dif-
ferential counts with a high correlation between the two when
analyzed using a Coulter (Hialeah, FL) JR apparatus.
16,17
Malaria pigment was detected on thin films by counting 100
polymorphonuclear cells (PMNs) and 30 monocytes and de-
termining the quantity of cells containing pigment. A ratio
was then determined of total pigmented PMNs/100 or mono-
cytes/30. Microscopists were blinded to clinical presentation
and outcome. To determine interobserver variability, 10% of
the slides were re-examined by a second microscopist. To
standardize total pigment burden across variable absolute
WBCs and differential counts as determined from thin
smears, total PMN and monocyte pigment per mm
3
were cal-
culated as follows: Total pigmented PMN/mm
3
⳱ (number of
pigmented PMNs/100) × (absolute WBCs × percent of
PMNs). Similarly, total pigmented monocytes/mm
3
⳱ (num-
ber of pigmented monocytes/30) × (absolute WBCs × percent
of monocytes). When the percent of monocytes in the differ-
ential cell count was less than 1% but pigment was noted on
monocyte pigment count, an arbitrary monocyte differential
count of 0.5% (n ⳱ 9) was assigned.
Statistical analysis. Pooled analyses between clinical
groups were made using a two-sided Student’s t-test for con-
tinuous variables with equal variance and a chi-square test for
categorical variables using Stata version 7.0 (Stat Corp., Col-
lege Station, TX) and SPSS version 10.0 (SPSS, Inc., Chicago,
IL). For the purposes of analyzing differences between
matched pairs, McNemar’s test was used to assess absolute
pigment counts and pigment/mm
3
using Stata version 7.0. A
TABLE 1
Defining features of severe malaria as published by the World
Health Organization
Coma (Blantyre Coma Scale [BCS] ⱕ 2)
Seizure (one or more witnessed by the investigators)
Obtundation (depressed consciousness with BCS > 2)
Parasitemia ⱖ 500,000/mm
3
Lethargy or prostration (clinical judgment or child ⱖ 7 months
old unable to sit unassisted)
Severe anemia (hemoglobin ⱕ 5 g/dL)
Respiratory distress (intercostal muscle retraction, deep breathing,
grunting)
Hypoglycemia (glucose ⱕ 40 mg/dL)
Jaundice
Renal insufficiency as indicated by lack of urination for ⱖ 1day
Gross hematuria
Inability to eat or drink
State of shock (systolic blood pressure ⱕ 50 mm of Hg, rapid
pulse, cold extremities)
Repeated vomiting
FIGURE 1. Birefringent amber crystals (arrow) visualized by light
microscopy within a monocytic vacuole in a case of uncomplicated
malaria from Mali.
FIGURE 2. Amber intracytoplasmic hemozoin (arrows) visualized
by light microscopy within a polymorphonuclear cell in a case of
severe malaria with hyperparasitemia from Mali.
LYKE AND OTHERS254
level of statistical significance (two-sided) was set at P < 0.05.
A Kappa coefficient was used in assessing inter-observer vari-
ability in the reading of a 10% proportion of thin smears.
RESULTS
Clinical characteristics. One hundred seventy-two cases
each of severe malaria, uncomplicated malaria, and healthy
controls were enrolled in the study and evaluated. The patient
characteristics are listed in Table 2. Fifteen of 172 patients
with severe malaria (8.7%) died. Of those with severe ma-
laria, 49% were hyperparasitemic, 45% had cerebral malaria,
16% were severely anemic, and 2.4% met other criteria. Pa-
tients meeting the criteria for both cerebral malaria and se-
vere anemia (n ⳱ 15) were analyzed as a unique subset.
Anemia was more common among those with a fatal outcome
than in survivors (mean hemoglobin concentration ⳱ 5.9
g/dL versus 8.2 g/dL, respectively; P < 0.0001). Additionally,
geometric mean parasite densities in children with severe ma-
laria were significantly lower in those that died than in sur-
vivors (29,994 asexual forms/mm
3
versus 174,428 asexual
forms/mm
3
; P < 0.0001). When patients whose sole criterion
for severe malaria was hyperparasitemia (mortality ⳱ 0.01%)
were excluded, the geometric mean parasite density in survi-
vors was 69,278 asexual forms/mm
3
compared with 29,994
asexual forms/mm
3
in those who died (P ⳱ 0.06). No signifi-
cant differences were noted among study groups in age dis-
tribution, sex, or total WBC count.
Malaria pigment measurements. Of 172 cases enrolled in
each group, thin films were available for 163 of the severe
malaria cases, 163 of the uncomplicated malaria controls, and
164 of the healthy controls. Ten percent of the films were
counted by a second observer and the interobserver variabil-
ity was determined (kappa coefficient [] ⳱ 0.88 for PMN
agreement, ⳱ 0.77 for monocytes). Total hemozoin counts
are summarized in Table 3. The results are depicted graphi-
cally in a scatter plot in Figure 3.
The proportions of both neutrophils and monocytes with
malaria pigment were higher in patients with severe malaria
than with uncomplicated malaria (PMNs ⳱ 349 cells/mm
3
versus 64 cells/mm
3
; P < 0.0001, monocytes ⳱ 219 cells/mm
3
versus 94 cells/mm
3
; P ⳱ 0.003). Similarly, phagocytic cells
from uncomplicated malaria cases had more pigment than
those from healthy controls (PMNs ⳱ 64 cells/mm
3
versus 1.1
cells/mm
3
; P < 0.0001, monocytes ⳱ 94 cells/mm
3
versus 4.9
cells/mm
3
; P < 0.0001).
Using the matched design (age, residence, and ethnicity) of
the study population, we performed McNemar’s testing to
assess differences between individually matched pairs. An in-
creased amount of malaria pigment in phagocytic cells was
more likely in severe malaria cases compared with uncompli-
cated malaria cases (pigmented PMNs: odds ratio [OR] ⳱ 5.6,
95% confidence interval [CI] ⳱ 2.83−12.31, P < 0.0001, pig-
mented monocytes: OR ⳱ 2.85, 95% CI ⳱ 1.38−5.85, P ⳱
0.003). Within the severe malaria group, an average of 635
pigmented PMNs/mm
3
were noted at admission in subjects
who subsequently died due to severe disease, compared with
an average of 320 pigmented PMNs/mm
3
in the survivors (P
⳱ 0.02). A trend was noted, but did not achieve statistical
significance, when the same analysis was performed evaluat-
ing pigment presence in monocytes in severe malaria survi-
vors versus children who died of severe disease (407 cells/
mm
3
versus 198 cells/mm
3
; P ⳱ 0.09).
To evaluate association between presence of pigment and
disease syndrome, the severe malaria group was stratified into
the four predominant presenting diagnoses: cerebral malaria
(n ⳱ 77), severe anemia (n ⳱ 26), severe anemia and cerebral
malaria (n ⳱ 15), and hyperparasitemia (n ⳱ 85). For sub-
jects in both the cerebral malaria group and the combined
cerebral malaria/severe anemia group, significantly more pig-
mented PMNs/mm
3
were noted at presentation than in pa-
tients with severe malaria without cerebral symptoms (Table
4). No such association with clinical syndrome was found for
monocytic pigment. When stratified by parasitemia (those
with peripheral parasite densities ⱖ 500,000 asexual forms/
mm
3
and those with < 500,000) no significant differences were
noted either in amount of pigmented PMNs or monocytes/
mm
3
. A higher concentration of monocyte pigment was noted
in children with severe malaria who were not hyperpara-
sitemic compared with those who were (290 cells/mm
3
versus
143 cells/mm
3
; P ⳱ 0.04). Multivariate regression analysis
found no significant association between pigmented mono-
cytes/mm
3
and peripheral parasite density (P ⳱ 0.11). Simi-
larly, no association was noted between polymorphonuclear
cell pigment and level of parasitemia (P ⳱ 0.68). A separate
analysis was performed recalculating peripheral parasitemia
based on the absolute WBC count determined on a thin
smear rather than the standardized WBC count (7,500 cells/
mm
3
in this study) that is typically used in field trials. Periph-
eral parasite density results were not significantly different
than those calculated using the standard WBC count, and no
association was noted between PMN pigment and level of
parasitemia.
TABLE 2
Patient characteristics at enrollment in age-, residence-, and ethnicity-matched severe malaria, uncomplicated malaria, and healthy control groups
Characteristics
Severe malaria
Uncomplicated malaria Healthy controlSurvived Died
Number 157 15 172 172
Females (%) 74 (47) 8 (53) 85 (49.4) 76 (44.2)
Age in months (range) 39.4 (3–125) 27.5 (8–99) 39.4 (6–113) 37.8 (4–121)
Hemoglobin g/dL (range) 8.2 (2.6–12.9) 5.9 (2.6–7.4)† 9.3 (5.3–13.8)† 10.5 (6.2–14.5)†
WBC (mm
3
) 14,093 16,178 17,560 13,887
Parasite density
‡
174,428 29,927† 8,200† 0†
* Paired t-test were performed between healthy controls and uncomplicated malaria controls, between uncomplicated malaria and severe malaria patients, and between children that survived
and those that died of severe disease. WBC ⳱ white blood cell.
† P < 0.001.
‡ Geometric mean (asexual forms/mm
3
).
MALARIA PIGMENT, DISEASE SEVERITY, AND PROGNOSIS 255
DISCUSSION
Peripheral P. falciparum parasite density alone, while nec-
essary for the diagnosis of malarial infection, provides limited
information about disease severity, manifestations, or out-
comes. The distinction between malaria infection and disease
is complex. Our understanding is limited by incomplete
knowledge of how host factors including immunity, behavior,
genetics, and susceptibility interact with parasite pathogenesis
and genetic polymorphisms.
2
Diagnostic capabilities in the
field are often limited to stained thick and thin blood smears.
Results of these smears may be altered by partial or insuffi-
cient drug therapy. Methods of extracting additional informa-
tion from these smears to aid in diagnosis or disease stratifi-
cation could be useful in directing scarce treatment resources
to those who need them most.
Clinical evidence supports a role for hemozoin as an indi-
cator for disease severity in both children and adults and for
prognosis in adults.
12–15
Additionally, malaria pigment itself
may possess physiologic properties that contribute to the
course of disease. Natural pigment has been demonstrated in
vitro to induce production of both tumor necrosis factor-␣
and interleukin-1-; this effect is ameliorated by protease di-
gestion, suggesting the role of uncharacterized proteins.
18
Monocytic cell dysfunction has been demonstrated (inhibition
of oxidative burst, inability to digest hemozoin, or repeatedly
phagocytose).
19,20
In the present study, we used a matched case-control design
to explore hemozoin characteristics as it relates to severe ma-
larial disease. Because of the broad range of peripheral WBC
counts (1,700−44,500 cells/mm
3
), and the disparity between
neutrophil and monocyte percentages on absolute differential
assessment, we standardized hemozoin measurements among
groups by assessing the total amount of malaria pigment/
mm
3
. We first analyzed groups by quantifying the percentage
of pigmented monocyte and polymorphonuclear cells.
Consistent with previous studies, differences were noted be-
tween patients with severe and uncomplicated malaria and
aparasitemic controls. By analyzing the data as a calculated
amount of pigment/mm
3
, a significantly higher amount of pig-
ment for both neutrophils and monocytes was again observed
between the severe malaria group and the matched uncom-
plicated malaria controls, as well as between the uncompli-
cated malaria group and matched healthy controls. These re-
sults validate the correlation of malaria pigment and disease
severity.
Malaria pigment has been found to correlate with death in
adults, but this has not been substantiated in children. We
analyzed the role of hemozoin as a prognostic indicator of
severe malaria. After stratifying the severe malaria cases into
children who died versus those who survived, a significantly
greater amount of pigmented PMNs/mm
3
was observed in the
group who died. These findings mark the first time that neu-
trophilic pigment presence has proven a prognostic indicator
for severe malaria mortality in children. No difference was
detected in pigmented monocytes/mm
3
.
To illuminate differences between categories of severe ma-
laria, subjects were stratified into one of the three predomi-
nant admission diagnoses; cerebral malaria, severe anemia
and hyperparasitemia. A subset analysis was performed on
children with the combined diagnosis of cerebral malaria with
severe anemia. Children with cerebral malaria had more pig-
mented PMNs/mm
3
on admission. Conversely, more pig-
mented monocytes were noted in children with severe ane-
mia. Of the 26 children with severe anemia, 15 had concomi-
tant cerebral malaria. As in children with cerebral malaria
alone, those with both diagnoses had more pigmented PMNs/
mm
3
but no increase in pigmented monocyte/mm
3
.
In children with severe anemia as a sole diagnosis (n ⳱ 11),
pigmented monocytes were significantly increased; this was
also noted in children with severe anemia combined with
other manifestations of severe malaria. It is possible that the
low numbers of children with anemia as a sole diagnosis did
not provide adequate power to detect an association between
hemoglobin and pigmented PMNs/mm
3
. It is also possible
that the pathophysiology of acute cerebral malaria and severe
anemia may, in and of itself, have properties creating a milieu
for increased pigment formation independent of either mani-
festation alone.
The reasons for variability in monocyte and neutrophil pig-
ment in different disease manifestations of severe malaria
remain unclear. The proportion of neutrophils and monocytes
containing malaria pigment is affected by total parasite bur-
den and synchronicity of the parasite life cycle, and the clear-
ance kinetics of these pigmented cells may be inherently dif-
ferent. Cellular clearance kinetic studies performed by Day
and others have shown peripheral pigment-containing neu-
trophil clearance times of 72 hours (range ⳱ 49−95 hours)
and peripheral pigment-containing monocyte clearance of 216
hours (range ⳱ 180−240 hours). While clearance of pig-
mented monocytes appears to follow first-order kinetics, that
TABLE 3
Hemozoin characteristics noted in age-, residence-, and ethnicity-matched cases of severe malaria, uncomplicated malaria, and aparasitemic
healthy controls*
Characteristic Severe malaria
Uncomplicated
malaria Healthy control
Number 163 163 164
Pigment-containing PMNs
Patients with pigment (%) 140 (85.9) 88 (54) 3 (1.8)
Pigment/mm
3
(range) 349 (0–3,721) 64 (0–534) 1.1 (0–81)
Mean cells pigmented (%) 4.4 1.7 0.03
Pigment-containing monocytes
Patients with pigment (%) 139 (85.2) 114 (69.9) 10 (6.1)
Pigment/mm
3
(range) 216 (0–3,420) 94 (0–1,698) 4.9 (0–285)
Mean cells pigmented (%) 14.4 5.4 0.03
* PMNs ⳱ polymorphonuclear phagocytic cells. Pigment/mm
3
refers to the calculated amount of pigment-containing cells in 100 PMNs counted or in 30 monocyte cells counted on a blood
thin smear multiplied by the absolute white blood cell count and the percentage of PMNs or monocytes determined on a differential cell count.
LYKE AND OTHERS256
of pigmented neutrophils departs from first-order kinetics,
with increased rates of clearance at a lower cell density. The
presence of pigmented neutrophils, with the rapid turnover of
PMNs, may indicate a recent heavy parasitic burden and pro-
vide prognostic indications of disease, while longer-lived pig-
mented monocytes with longer clearance rates may reflect a
more protracted infection or repeated infections.
21
Malaria-induced anemia is multifactorial with hemolysis
occurring more frequently in non-immune children and dys-
erythropoiesis occurring more often in regions with frequent
and recurrent infections. The predominance of monocytic
pigment in our anemic children may suggest that the pro-
found anemia that occurred over a more protracted time pe-
riod.
22
The elevated pigmented polymorphonuclear cells in
children with both cerebral malaria and severe anemia com-
pared with those with cerebral malaria alone may relate to the
acuity of the illness with cerebral manifestations coupled with
acute hemolysis.
21
Thus, we hypothesize that pigmented
monocytes indicate a protracted or indolent infection, while
the factors leading to severe disease and death may be more
fulminant and reflected in pigmented neutrophils. Longitudi-
nal studies with serial pigment analysis would be better suited
to address these hypotheses.
The use of hyperparasitemia as a criterion for severe ma-
laria is recommended by the World Health Organization, but
the clinical course in children who present with this manifes-
tation appears less acute. At our study site, children whose
sole defining criterion for severe malaria was hyperpara-
sitemia appear to fall somewhere between uncomplicated ma-
laria and severe malaria in the spectrum of illness. We noted
FIGURE 3. Scatterplot graph of A, polymorphonuclear cell (PMN) or B, monocyte pigment/mm
3
distribution among study subjects with severe
malaria, uncomplicated malaria, or no malaria. The standard error bars show 95% confidence intervals about the means, which are shown by the
short cross-hatch bars.
MALARIA PIGMENT, DISEASE SEVERITY, AND PROGNOSIS 257
a lower peripheral parasite density in children who died com-
pared with children that survived, although the statistical sig-
nificance of this finding disappeared upon removing hyper-
parasitemia as a criterion for severe disease. Children with
hyperparasitemia alone are presumably healthier children
who were more likely to survive. It is possible that this group
should be considered separately from those with more severe
forms of malaria, which would require larger studies, prob-
ably including multiple sites.
Ongoing trials conducted through the Severe Malaria in
African Children network have a large sample size at multiple
study sites and should further illuminate the significance
of malaria pigment in severe malaria, including hyper-
parasitemia. Linear regression models taking into account hy-
perparasitemia or peripheral parasite density demonstrated
no association with either pigmented PMNs or monocytes.
An inconsistency was noted in that monocyte presence did
not appear to be associated with hyperparasitemia on regres-
sion analysis, although upon stratifying patients with periph-
eral parasitemias greater than and less than 500,000 asexual
forms/mm
3
, increased monocyte pigment was observed in pa-
tients without severe disease. While the signficance of this
finding is unclear, we have demonstrated that the degree of
parasitemia is not associated with increased amount of pig-
ment seen in leukocytes, consistent with our hypothesis that
the presence of pigment reflects overall sequestered parasite
burden.
In summary, in an age-, residence-, and ethnicity-matched
case-control study, we have found malaria pigment/mm
3
in
neutrophils to be associated with disease mortality in Malian
children three months to 14 years of age. We have validated
previously reported associations between the amount of neu-
trophilic malaria pigment and disease severity, and have es-
tablished an association with monocyte pigment when mea-
sured as pigment/mm
3
. In this study, we found that intra-
neutrophilic pigment was associated with cerebral malaria
and the combination of cerebral malaria and severe anemia,
while intra-monocytic pigment is associated with severe ane-
mia. These results do not appear to be biased by the degree of
peripheral parasitemia. Based on these results, the assessment
of intraleukocytic pigment appears to be valuable for more
detailed characterization of children who present with P. fal-
ciparum parasitemia and clinical symptoms consistent with a
malarial illness.
Received April 29, 2003. Accepted for publication July 1, 2003.
Acknowledgments: We thank the members of the Severe Malaria in
African Children Clinical Network for their work in the genesis of
this study and the protocol to which we adhered. We also thank
Timothy Mnalemba (Blantyre Malaria Project, Blantyre, Malawi) for
his patient teaching, which was invaluable in identifying pigment and
applying the protocols.
Financial support. This study was supported by contract no. N01-AI-
85346 from the National Institutes of Health.
Authors’ addresses. Kirsten E. Lyke and Christopher V. Plowe, Ma-
laria Section, Center for Vaccine Development, University of Mary-
land School of Medicine, 685 West Baltimore Street, HSF 480, Bal-
timore, MD 21201, Telephone: 410-706-3082, Fax 410-706-6205, E-
mail: cplowe@medicine.umaryland.edu. Dapa A. Diallo, Alassane
Dicko, Abdoulaye Kone, Drissa Coulibaly, Ando Guindo, Yacouba
Cissoko, Lansana Sangare, Seydou Coulibaly, Blaise Dakouo, and
Ogobara K. Doumbo, Malaria Research and Training Center, De-
partment of Epidemiology of Parasitic Diseases, Faculty of Medicine,
Pharmacy and Dentistry, University of Mali, BP 1805 Point G, Ba-
mako, Mali, Fax 223-2-8109. Terrie E. Taylor, Department of Internal
Medicine, College of Osteopathic Medicine, Michigan State Univer-
sity, B315-C W. Fee Hall, East Lansing MI 48824, Fax 517-432-1062.
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T
ABLE 4
Association of polymorphonuclear phagocytic cells (PMNs) and monocyte pigment with manifestations of severe disease in 163 patients*
Category Patients Cell
Mean pigmented cells/mm
3
(95% CI) P
Cerebral vs. non-cerebral 77 PMN 448 (298–598) 0.018†
260 (196–324)
Monocyte 226 (99–318) 0.80
208 (138–315)
Severe anemia vs. hemoglobin 26 PMN 516 (239–394) 0.066
ⱖ 5 g/dL 316 (230–802)
Monocyte 159 (97–221) 0.0002†
522 (236–808)
Anemia/cerebral‡ 15 PMN 318 (244–391) 0.008†
vs. neither 707 (171–1,244)
Monocyte 199 (128–269) 0.08
428 (45–810)
Hyperparasitemia 85 PMN 334 (236–431) 0.71
vs. Pf < 500,000§ 364 (238–489)
Monocyte 143 (50–235) 0.04†
290 (185–396)
*CI⳱ confidence interval.
† Denotes significance at P ⱕ 0.05.
‡ Implies concomitant severe anemia (hemoglobin ⱕ 5 g/dL) and a cerebral malaria diagnosis.
§ Pf ⳱ Plasmodium falciparum parasite density/mm
3
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