Severe Anemia in Malawian Children

T h e n e w e ng l a n d j o u r na l o f m e dic i n e
original article
Severe Anemia in Malawian Children
Job C.J. Calis, M.D., Kamija S. Phiri, M.D., E. Brian Faragher, Ph.D.,
Bernard J. Brabin, F.R.C.P.C.H., Imelda Bates, M.D., Luis E. Cuevas, M.D.,
Rob J. de Haan, Ph.D., Ajib I. Phiri, M.D., Pelani Malange, M.Sc.,
Mirriam Khoka, B.Sc., Paul J.M. Hulshof, M.Sc., Lisette van Lieshout, Ph.D.,
Marcel G.H.M. Beld, Ph.D., Yik Y. Teo, Ph.D., Kirk A. Rockett, Ph.D.,
Anna Richardson, B.Sc., Dominic P. Kwiatkowski, F.R.C.P.,
Malcolm E. Molyneux, F.R.C.P., and Michaël Boele van Hensbroek, M.D.
Abs tr act
Background
From the Malawi–Liverpool–Wellcome Severe anemia is a major cause of sickness and death in African children, yet the
Trust Clinical Research Programme, Col-
causes of anemia in this population have been inadequately studied.
lege of Medicine (J.C.J.C., K.S.P., A.I.P.,
P.M., M.K., M.E.M., M.B.H.) and the Col-
lege of Medicine (J.C.J.C., K.S.P., A.I.P., Methods
M.E.M., M.B.H.), Blantyre, Malawi; Emma We conducted a case–control study of 381 preschool children with severe anemia (he-
Children’s Hospital, the Global Child
Health Group (J.C.J.C., B.J.B., M.B.H.), moglobin concentration, <5.0 g per deciliter) and 757 preschool children without severe
the Department of Clinical Epidemiology anemia in urban and rural settings in Malawi. Causal factors previously associated
and Biostatistics (R.J.H.), and the De-
with severe anemia were studied. The data were examined by multivariate analysis
partment of Medical Microbiology, Labo-
ratory of Clinical Virology (M.G.H.M.B.) and structural equation modeling.
— al at the Academic Medical Center, Am-
sterdam; the Liverpool School of Tropical Results
Medicine (E.B.F., I.B., M.E.M.) and the
Child and Reproductive Health Group, Liv-
Bacteremia (adjusted odds ratio, 5.3; 95% confidence interval [CI], 2.6 to 10.9), ma-
erpool School of Tropical Medicine (B.J.B., laria (adjusted odds ratio, 2.3; 95% CI, 1.6 to 3.3), hookworm (adjusted odds ratio,
L.E.C., M.B.H.), Liverpool, United King-
4.8; 95% CI, 2.0 to 11.8), human immunodeficiency virus infection (adjusted odds
dom; the Division of Human Nutrition,
Wageningen University, Wageningen, the ratio, 2.0; 95% CI, 1.0 to 3.8), the G6PD?202/?376 genetic disorder (adjusted odds ratio,
Netherlands (P.J.M.H.); the Department 2.4; 95% CI, 1.3 to 4.4), vitamin A deficiency (adjusted odds ratio, 2.8; 95% CI, 1.3
of Parasitology, Leiden University Medical to 5.8), and vitamin B
Center, Leiden, the Netherlands (L.L.);
12 deficiency (adjusted odds ratio, 2.2; 95% CI, 1.4 to 3.6) were
and the Wellcome Trust Centre for Hu-
associated with severe anemia. Folate deficiency, sickle cell disease, and laboratory
man Genetics, Oxford, United Kingdom signs of an abnormal inflammatory response were uncommon. Iron deficiency was
(Y.Y.T., K.A.R., A.R., D.P.K.). Address re-
not prevalent in case patients (adjusted odds ratio, 0.37; 95% CI, 0.22 to 0.60) and
print requests to Dr. Calis at Emma Chil-
dren’s Hospital, Academic Medical Cen-
was negatively associated with bacteremia. Malaria was associated with severe ane-
ter, the Global Child Health Group, mia in the urban site (with seasonal transmission) but not in the rural site (where
Meibergdreef 9, Amsterdam 1105 AZ, the malaria was holoendemic). Seventy-six percent of hookworm infections were found
Netherlands, or at job.calis@gmail.com.
in children under 2 years of age.
N Engl J Med 2008;358:888-99.
Copyright © 2008 Massachusetts Medical Society.
Conclusions
There are multiple causes of severe anemia in Malawian preschool children, but folate
and iron deficiencies are not prominent among them. Even in the presence of malaria
parasites, additional or alternative causes of severe anemia should be considered.
888
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Severe Anemia in Malawian Children
Severe anemia (hemoglobin concen- patients were recruited by selecting the first child
tration less than 5.0 g per deciliter) is a ma- presenting at the outpatient department at the
jor cause of sickness and death among chil- same time on the next working day after presen-
dren in sub-Saharan Africa.1-4 In various settings, tation of the case patient. Community and hospi-
12 to 29% of hospitalized children are severely tal control patients were eligible for recruitment if
anemic,1-4 and the in-hospital case fatality rate in their hemoglobin level was at least 5.0 g per deci-
these children is 8 to 17%.1,3,4 Little is known about liter and they were between 6 and 60 months of
the causes of severe anemia in African children. age; no other matching was applied. Written in-
Most studies have been confined to the anemia formed consent was obtained from a parent or a
associated with malaria5 or with other individual guardian of each child in all three study groups.
factors.1,2,6 As a result, the treatment guidelines ad- The study was approved by the ethics commit-
vocated by the World Health Organization (WHO) tees of the College of Medicine, Malawi, and the
deal specifically with malaria, folate deficiency, Liverpool School of Tropical Medicine, United
and iron deficiency, which are widely held to be the Kingdom.
most common causes of severe anemia in African
children.7 To improve our understanding of severe Clinical assessment and management
anemia, we conducted a case–control study in Ma- On enrollment, a clinical research form, including
lawi to assess the causative factors in Malawian a medical and dietary history, sociodemographic
children with severe anemia.
data, and physical examination, was completed,
and samples of blood, urine, and stool were col-
Methods
lected. In case patients, if the clinical condition
permitted it, a bone marrow aspirate was obtained
Study sites
under local anesthesia. Children requiring admis-
We conducted the study in Malawi at Chikwawa sion were treated in a study ward. All conditions
District Hospital in a rural area where malaria in- were managed according to standard protocols.
fections occur throughout the year (approximately
170 infectious bites per person per year) and at Anthropometric measurements
Queen Elizabeth Central Hospital, a referral hos- Nutritional z scores were calculated according to
pital in urban Blantyre, where malaria is season- the WHO growth reference curves8 with the use
al, largely coinciding with the rainy season (ap- of Epi Info 2000. Weight-for-height, height-for-age,
proximately 1 infectious bite per person per year) and weight-for-age z scores of less than ?2 were
(Mzilahowa T: personal communication). Prede- considered to indicate wasting, stunting, and
fined catchment areas were used; the urban area underweight, respectively; z scores of less than
was confined to the city limits.
?3 were considered to indicate severe wasting, se-
vere stunting, or severe underweight.
Study design
Between July 2002 and July 2004, a consecutive Laboratory methods
sample of children (382 case patients) who present- Laboratory tests (hematologic, bacteriologic, and
ed at the outpatient department during working parasitologic) were performed within 24 hours af-
hours with a primary diagnosis of severe anemia ter collection, and aliquots were stored at ?80°C
(defined as a hemoglobin concentration of <5.0 g for later analysis. The laboratory staff were unaware
per deciliter) were recruited into a prospective of the children’s study groups.
case–control study. Additional inclusion criteria
were an age between 6 and 60 months and no blood Hematologic Studies
transfusion within the previous 4 weeks.
Hemoglobin concentration was measured on site
For each case patient, a community control with a HemoCue system. A complete blood count,
patient and a hospital control patient were en- including reticulocytes, was performed by a Coul-
rolled. Community control patients were recruited ter counter. In case patients, bone marrow slides
from apparently healthy residents living within were stained with Hematognost Fe (Merck) and
100 to 1000 m of the case patient; hospital control graded for iron content9; these results were used
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889
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T h e n e w e ng l a n d j o u r na l o f m e dic i n e
to validate peripheral blood markers of iron defi- or coagulase-negative staphylococci were consid-
ciency. The ratio of soluble transferrin receptor ered contaminants.
to log ferritin (TfR-F index)10 best predicted bone
marrow iron status, irrespective of the presence Virologic Studies
of infection; a TfR-F index greater than 5.6 was Whole-blood isolates17 were assessed for Epstein–
used to define iron deficiency with a sensitivity of Barr virus and cytomegalovirus infection by semi-
70% and a specificity of 75% (unpublished data).
quantitative PCR18 and for parvovirus by real-time
PCR.19 Infections were considered clinically im-
Chemical Studies
portant if the number of viral copies exceeded
Plasma levels of C-reactive protein, haptoglobin, 1000 per milliliter of blood. Testing for human
transferrin, iron, ferritin, folate, and vitamin B12 immunodeficiency virus (HIV) was performed by
were analyzed on Modular P800 and Modular An- two rapid tests (Determine, Abbott Laboratories;
alytics E170 systems (Roche). Inflammatory cy- and Uni-Gold, Trinity Biotech). Discordant or pos-
tokine profiles were measured by Cytometric Bead itive rapid-test results in children less than 18
Array on a FACSCalibur flow cytometer (Becton months of age were resolved by PCR.20
Dickinson). Serum vitamin A (retinol) and soluble
transferrin receptor were measured by high-per- Genetic Studies
formance liquid chromatography11 and enzyme- DNA was extracted with a Nucleon extraction kit
linked immunosorbent assay, respectively.
(Amersham Biosciences) and genotyped by prim-
er-extension mass spectrometry with the use of
Parasitologic Studies
MassARRAY (Sequenom).21 The presence of sickle
The number of Plasmodium falciparum asexual par- cell disease (homozygosity for hemoglobin S) and
asites per 200 white cells was counted and ex- single-nucleotide polymorphisms in the promoter
pressed as the number per microliter of blood. Ma- regions of the genes encoding interleukin-10 (IL10)
laria slides were read by two independent readers, (–1117, ?3585, and +4949)21 and tumor necrosis
with a third being used if the results differed by factor (TNF) (?238, ?308, and ?1031)22 was deter-
more than 25%. Malaria was defined by the pres- mined. The term G6PD?202/?376 is used to denote
ence of P. falciparum asexual parasites. Recent or boys who are hemizygous and girls who are homo-
current malaria was defined by the presence of zygous for both the G6PD202A and the G6PD376G
P. falciparum asexual parasites in erythrocytes or alleles, a condition that is strongly predictive of
malaria pigment in monocytes or macrophages.12 glucose-6-phosphate dehydrogenase deficiency.23
Hyperparasitemia was defined as more than The Hardy–Weinberg equilibrium was applied (cut-
100,000 parasites per microliter.2 Stool samples off, P<0.001), and there was no significant popu-
were examined for helminths by the Kato–Katz lation stratification. We chose the allele frequen-
method.13 Heavy hookworm infection was defined cy, dominant model, or haplotype that was most
by the presence of more than 1000 ova per gram strongly associated with severe anemia.
of feces. A polymerase-chain-reaction (PCR) test
was used to confirm the microscopical results and Statistical analysis
define the species (Ancylostoma duodenale or Necator The prevalence rates of each factor were compared
americanus).14 Urine specimens were examined for individually across the three groups with the use
Schistosoma haematobium by a semiquantitative con- of Fisher’s exact test and the chi-square test. The
centration method.15
combined association of characteristics related to
the risk of severe anemia (P?0.10, unless the char-
Bacteriologic Studies
acteristics were uncommon [<5%]) was examined
A bone marrow or venous blood sample (1 to 2 ml) by an unconditional multivariate logistic-regres-
was inoculated into BACTEC Myco/F-Lytic culture sion model corrected for potential confounding
vials and incubated in a BACTEC 9050 automated factors (age, sex, recent use [i.e., within the pre-
culture system (Becton Dickinson) for 56 days. Sub- vious 8 weeks] of antimalarial or hematinic agents,
culturing, susceptibility testing, and isolate identi- and a history of transfusions [i.e., before the pre-
fication were performed by standard techniques.16 vious 4 weeks]). Missing observations were in-
Cultures were checked for mycobacteria with the cluded in the analysis by creating missing-value
use of Ziehl–Neelsen staining of smears. Mixed categories. Alternative definitions for malaria,
growths or growths of micrococci, bacillus species, hookworm, and nutritional deficiencies and sta-
890
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Severe Anemia in Malawian Children
tus were tested. Attributable-risk percentages were
R esults
calculated with the use of adjusted odds ratios.24
The primary analysis compared all case patients We enrolled 1141 children over a 2-year period. Five
with the two control groups combined. To explore protocol violations occurred: two hospital control
the possibility that different patient characteris- children had severe anemia and were redesignat-
tics were important in the two study locations, ed as case patients, one case patient with a hemo-
secondary analyses were performed with stratifi- globin concentration of more than 5.0 g per deci-
cation according to location and with the com- liter was excluded, and two control patients under
munity and hospital control groups separated. 6 months of age were excluded. Table 1 summa-
More complex associations and alternative strat- rizes the characteristics of the 1138 children in-
egies for handling missing data (e.g., maximum- cluded in the analysis. Hemoglobin levels differed
likelihood imputation) were explored by structural significantly between the case patients and each
equation modeling.25 All reported P values are of the two control groups but were similar in the
two-sided. The data were analyzed with the use two control groups. Splenomegaly (in which >1 cm
of Stata (version 9), SPSS (version 12), and Amos of the spleen was palpable) and severe splenomeg-
(version 6.0) software.
aly (in which ?8 cm was palpable) were more com-
Table 1. Characteristics of the Study Groups.*
Community Control
Hospital Control
Case Patients
Patients
Patients
Characteristic
(N = 381)
(N = 380)
(N = 377)
Area — no. (%)†
Urban
205 (53.8)
203 (53.4)
201 (53.3)
Rural
176 (46.2)
177 (46.6)
176 (46.7)
Sex — no. (%)
Female
203 (53.3)
191 (50.3)
180 (47.7)
Male
178 (46.7)
189 (49.7)
197 (52.3)
Age — mo‡
20.4±12.8
25.3±13.1
22.5±12.1
Jaundice — no./total no. (%)§
19/379 (5.0)
1/380 (0.3)
0/376
Splenomegaly — no./total no. (%)§¶
237/372 (63.7)
108/363 (29.8)
86/349 (24.6)
Fever — no./total no. (%)?**
189/376 (50.3)
41/375 (10.9)
172/374 (46.0)
Hemoglobin — g/dl§††
3.6±0.8
9.9±1.9
9.6±2.2
Mean corpuscular volume — fl§‡‡
82.9±15.2
75.5±9.3
74.2±9.7
Reticulocyte count — ×10?9/liter‡§§
Median
53.2
76.8
64.5
Interquartile range
30.2–91.7
46.4–114.7
43.0–103.2
Admitted to hospital — no. (%)†
381 (100)
3 (0.8)
17 (4.5)
Died in hospital — no. (%)†
24 (6.3)
0
0
* Plus–minus values are means ±SD.
† No statistical tests were applied.
‡ P<0.05 for differences among all three groups (by Tukey post hoc test or Kruskal–Wallis test with Tukey multiple
comparisons).
§ Community and hospital control patients were significantly different from case patients (P<0.05 by Tukey post hoc test).
Case patients had a primary diagnosis of severe anemia (defined as a hemoglobin concentration of <5.0 g per deciliter).
¶ Splenomegaly was defined as more than 1 cm of palpable spleen below the left costal margin in the midaxillary line.
? Community control patients were significantly different from case patients and hospital control patients (P<0.05 by
Tukey post hoc test).
** Fever was defined as an axillary temperature of more than 37.5°C.
†† Hemoglobin values were recorded in 373 community control patients and 375 hospital control patients.
‡‡ Mean corpuscular volume was recorded in 316 case patients, 322 community control patients, and 314 hospital con-
trol patients.
§§ Reticulocyte values were recorded in 266 case patients, 284 community control patients, and 279 hospital control patients.
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T h e n e w e ng l a n d j o u r na l o f m e dic i n e
Table 2. Distribution of Possible Etiologic and Confounding Factors among Study Groups According to Recruitment Sites.*
Variable
Both Sites
Rural Site
Community
and Hospital
Community
Case Patients Control Patients
Case Patients Control Patients
(N = 381)
(N = 757)
P Value
(N = 176)
(N = 177)
P Value
no./total no. (%)
no./total no. (%)
History
Mother did not attend secondary
323/366 (88.3)
554/753 (73.6)
<0.001
162/172 (94.2)
154/176 (87.5)
0.03
school
Death of a parent
25/284 (8.8)
22/554 (4.0)
0.004
13/168 (7.7)
5/163 (3.1)
0.06
Recent hematinic treatment
85/376 (22.6)
61/754 (8.1)
<0.001
40/176 (22.7)
7/177 (4.0)
<0.001
Recent antimalarial treatment
232/375 (61.9)
346/755 (45.8)
<0.001
107/176 (60.8)
79/177 (44.6)
0.002
History of transfusion
57/378 (15.1)
38/756 (5.0)
<0.001
24/176 (13.6)
9/177 (5.1)
0.006
Malnutrition†
Wasting
52/330 (15.8)
43/695 (6.2)
<0.001
24/169 (14.2)
9/174 (5.2)
0.005
Iron deficiency
97/208 (46.6)
288/415 (69.4)
<0.001
71/101 (70.3)
76/95 (80.0)
0.12
Vitamin B12 deficiency
95/312 (30.4)
94/603 (15.6)
<0.001
46/142 (32.4)
20/143 (14.0)
<0.001
Vitamin A deficiency
228/247 (92.3)
172/262 (65.6)
<0.001
113/126 (89.7)
44/83 (53.0)
<0.001
Viral infections
HIV
45/357 (12.6)
41/682 (6.0)
<0.001
7/176 (4.0)
5/176 (2.8)
0.56
Parvovirus B19
5/294 (1.7)
2/609 (0.3)
0.03
2/143 (1.4)
0/147
0.15
Epstein–Barr virus
89/269 (33.1)
102/566 (18.0)
<0.001
43/128 (33.6)
34/133 (25.6)
0.16
Bacteremia
54/359 (15.0)
14/353 (4.0)
<0.001
20/171 (11.7)
ND
Parasitic infections
Plasmodium falciparum
226/380 (59.5)
321/750 (42.8)
<0.001
91/176 (51.7)
93/175 (53.1)
0.79
Hyperparasitemic P. falciparum‡
45/380 (11.8)
24/750 (3.2)
<0.001
17/176 (9.7)
3/175 (1.7)
0.001
Recent or current P. falciparum
243/334 (72.8)
336/696 (48.3)
<0.001
113/169 (66.9)
98/171 (57.3)
0.07
Hookworm
29/296 (9.8)
12/642 (1.9)
<0.001
27/154 (17.5)
4/160 (2.5)
<0.001
Schistosoma mansoni
2/296 (0.7)
8/643 (1.2)
0.43
2/154 (1.3)
4/160 (2.5)
0.44
S. haematobium
4/307 (1.3)
8/669 (1.2)
0.89
4/159 (2.5)
6/168 (3.6)
0.58
Genetic disorders§
G6PD ?202/?376
44/318 (13.8)
54/601 (9.0)
0.02
21/152 (13.8)
11/141 (7.8)
0.10
Sickle cell disease
4/238 (1.7)
4/404 (1.0)
0.45
2/118 (1.7)
2/101 (2.0)
0.88
IL10 ?1117 (C/C+C/T vs. T/T)
196/324 (60.5)
332/607 (54.7)
0.09
98/155 (63.2)
75/141 (53.2)
0.08
IL10 ?3585 (A/A vs. A/T+T/T)
22/308 (7.1)
25/575 (4.3)
0.08
8/148 (5.4)
6/134 (4.5)
0.72
IL10 +4949 (G/G vs. G/A+A/A)
97/322 (30.1)
134/606 (22.1)
0.007
48/155 (31.0)
31/140 (22.1)
0.09
Abnormal ratio of interleukin-10 to
4/276 (1.4)
NA
1/122 (0.8)
NA
TNF-?
* All P values are for the comparisons between case patients and community control patients, hospital control patients, or both. Among vari-
ables not included in the table (because they did not meet the preset cutoff for significance) are parental unemployment, household assets,
folate deficiency, trichuriasis, ascariasis, cytomegalovirus infection, hemoglobin C, and tumor necrosis factor ? (TNF-?) alleles or genotypes
(?238, ?308, and ?1031). Recent is defined as within 8 weeks before recruitment. HIV denotes human immunodeficiency virus, IL10 the
gene encoding interleukin-10, NA not available, and ND not done. G6PD?202/?376 denotes boys who are hemizygous and girls who are ho-
mozygous for both the G6PD202A and the G6PD376G alleles.
† Wasting is defined as a weight-for-height z score of less than ?2. Iron deficiency is defined as a ratio of soluble transferrin receptor to log
ferritin (TfR-F index)10 that is greater than 5.6 (unpublished data). Folate concentrations of less than 0.3 ?g per deciliter (6.8 nmol per liter),
vitamin B12 concentrations of less than 20 ng per deciliter (148 pmol per liter), and vitamin A concentrations of less than 20 ?g per deciliter
(0.7 ?mol per liter) are considered to indicate deficiencies.
‡ Hyperparasitemia is defined as more than 100,000 parasites per microliter of blood.
§ The rs classifications for the genetic markers are as follows: IL10 ?1117, rs1800896; IL10 ?3585, rs1800890; IL10 +4949, rs3024500; TNF ?238,
rs361525; TNF ?308, rs1800629; and TNF ?1031, rs1799964. A ratio of interleukin-10 to TNF-? that is less than 1 is considered abnormal.22
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Severe Anemia in Malawian Children
Rural Site
Urban Site
Hospital Control
Community Control
Hospital Control
Patients
Case Patients
Patients
Patients
(N = 176)
P Value
(N = 205)
(N = 203)
P Value
(N = 201)
P Value
no./total no. (%)
no./total no. (%)
no./total no. (%)
143/176 (81.2)
<0.001
161/194 (83.0)
142/200 (71.0)
0.005
115/201 (57.2)
<0.001
3/164 (1.8)
0.01
12/116 (10.3)
6/109 (5.5)
0.18
8/118 (6.8)
0.33
13/176 (7.4)
<0.001
45/200 (22.5)
19/202 (9.4)
<0.001
22/199 (11.1)
0.002
90/176 (51.1)
0.07
125/199 (62.8)
77/203 (37.9)
<0.001
100/199 (50.3)
0.01
13/176 (7.4)
0.06
33/202 (16.3)
7/203 (3.4)
<0.001
9/200 (4.5)
<0.001
19/175 (10.9)
0.35
28/161 (17.4)
6/182 (3.3)
<0.001
9/164 (5.5)
<0.001
63/92 (68.5)
0.78
26/107 (24.3)
76/113 (67.3)
<0.001
73/115 (63.5)
<0.001
30/149 (20.1)
0.02
49/170 (28.8)
24/157 (15.3)
0.003
20/154 (13.0)
<0.001
60/74 (81.1)
0.09
115/121 (95.0)
32/60 (53.3)
<0.001
36/45 (80.0)
0.003
9/172 (5.2)
0.58
38/181 (21.0)
9/171 (5.3)
<0.001
18/163 (11.0)
0.01
1/146 (0.7)
0.55
3/151 (2.0)
1/157 (0.6)
0.30
0/159
0.07
24/127 (18.9)
0.008
46/141 (32.6)
14/148 (9.5)
<0.001
30/158 (19.0)
0.007
9/166 (5.4)
0.04
34/188 (18.1)
ND
5/187 (2.7)
<0.001
93/176 (52.8)
0.83
135/204 (66.2)
74/199 (37.2)
<0.001
61/200 (30.5)
<0.001
11/176 (6.2)
0.24
28/204 (13.7)
3/199 (1.5)
<0.001
7/200 (3.5)
<0.001
98/175 (56.0)
0.04
130/165 (78.8)
83/180 (46.1)
<0.001
57/170 (33.5)
<0.001
8/156 (5.1)
<0.001
2/142 (1.4)
0/164
0.13
0/162
0.13
4/156 (2.6)
0.42
0/142
0/164
0/163
1/162 (0.6)
0.17
0/148
0/171
1/168 (0.6)
0.35
9/145 (6.2)
0.03
23/166 (13.9)
20/161 (12.4)
0.70
14/154 (9.1)
0.18
0/86
0.23
2/120 (1.7)
0/106
0.18
2/111 (1.8)
0.94
83/147 (56.5)
0.23
98/169 (58.0)
92/162 (56.8)
0.83
82/157 (52.2)
0.30
5/140 (3.6)
0.45
14/160 (8.8)
4/153 (2.6)
0.02
10/148 (6.8)
0.51
34/147 (23.1)
0.13
49/167 (29.3)
34/162 (21.0)
0.08
35/157 (22.3)
0.15
NA
3/154 (1.9)
NA
NA

mon in case patients (P<0.001 and P = 0.03, respec- sion to the hospital, nine (37.5%) before receiving
tively). Severe splenomegaly, which was present in a transfusion. We obtained samples of peripheral
11 case patients (3.0%), was not associated with blood from 1105 subjects (97.1%), stool from 1024
thrombocytopenia or leukopenia. Jaundice was subjects (90.0%), urine from 1042 subjects (91.6%),
more common in case patients (5.0%) but was and bone marrow from 348 case patients (91.3%).
not associated with sickle cell disease (P = 1.00), Table 2 lists the features we investigated in the
G6PD?202/?376 (P = 0.70), or splenomegaly (P = 0.30). three groups and gives the P values for differences
Twenty-four case patients (6.3%) died after admis- among the groups. Factors significantly associated
n engl j med 358;9 www.nejm.org february 28, 2008
893
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T h e n e w e ng l a n d j o u r na l o f m e dic i n e
Case Patients vs. All Control
Case Patients vs. Community
Case Patients vs. Hospital
Variable
Patients in Both Sites
Control Patients
Control Patients
Adjusted Odds Ratio (95% CI)
Limited maternal education
Rural
4.2 (1.2–14.3)
3.9 (1.4–10.6)
2.5 (1.5–4.1)
Urban
1.8 (0.9–3.6)
3.2 (1.6–6.5)
Wasting
3.5 (1.1–11.2)
1.0 (0.4–2.3)
1.7 (0.9–2.9)
6.1 (1.9–19.4)
3.9 (1.3–11.9)
Vitamin B
4.1 (1.8–9.4)
1.9 (0.9–3.9)
12 deficiency
2.2 (1.4–3.6)
1.7 (0.8–3.8)
3.1 (1.4–7.1)
13.9 (5.4–36.4)
Vitamin A deficiency
2.0 (0.7–5.6)
2.8 (1.3–5.8)
9.4 (2.6–33.6)
2.1 (0.5–9.9)
Iron deficiency
0.4 (0.1–0.9)
0.8 (0.4–1.9)
0.4 (0.2–0.6)
0.1 (0.0–0.3)
0.2 (0.1–0.4)
0.5 (0.1–2.7)
HIV
0.3 (0.1–1.2)
2.0 (1.0–3.8)
3.7 (1.3–10.5)
2.2 (0.9–5.3)
1.6 (0.7–3.4)
Epstein–Barr virus
1.6 (0.8–3.5)
1.7 (1.1–2.7)
3.3 (1.3–8.2)
1.7 (0.8–3.7)
4.4 (1.5–13.1)
Bacteremia
5.3 (2.6–10.9)
12.8 (3.4–47.5)
0.8 (0.4–1.4)
1.1 (0.6–1.9)
Malaria
2.3 (1.6–3.3)
3.5 (1.9–6.3)
5.8 (3.1–10.8)
Hookworm
4.8 (2.0–11.8)
10.6 (2.6–43.1)
3.8 (1.4–10.5)
5.4 (1.9–15.7)
4.4 (1.6–11.7)
G6PD?202/?376
2.4 (1.3–4.4)
1.3 (0.5–3.3)
1.8 (0.7–5.0)
1.8 (0.8–3.8)
1.8 (0.9–3.6)
IL10 +4949 (G/G)
1.4 (0.9–2.1)
1.8 (0.8–3.7)
1.0 (0.5–2.2)
0.01
0.10
1.0
10.0
100.0
0.01
0.10
1.0
10.0
100.0
0.01
0.10
1.0
10.0
100.0
Less Common
More Common
Less Common
More Common
Less Common
More Common
Figure 1. Adjusted Odds Ratios and 95% Confidence Intervals for Factors Associated with Severe Anemia, According to Study Group
and Recruitment Site.
The model was corrected for the possible confounders of age, sex, recent antimalarial treatment (within the previous 8 weeks), recent
hematinic treatment (within the previous 8 weeks), a history of transfusion (before the previous 4 weeks), and death of a parent. The
goodness of fit of the model was evaluated by the Hosmer–Lemeshow test (P = 0.65). In the combined model, interaction existed be-
tween malaria and site (P<0.001). Limited maternal education indicates mothers who did not attend secondary school. Wasting is de-
fined as a weight-for-height z score of less than ?2. Concentrations of vitamin B
AUTHOR: Calis
12 of less than 20 ng per deciliter (148 pmol per liter) and
RETAKE
1st
ICM
of vitamin A of less than 20 ?g per deciliter (0.7 ?mol per liter) are considered to indicate deficiency. Iron deficiency is defined as a ratio
FIGURE:
2nd
REG F
1 of 2
of soluble transferrin receptor to log ferritin (TfR-F index)10 of more than 5.6 (unpublished d 3rd
ata). Cultures to detect bacteremia were
CASE
performed only in case patients and hospital controls. Hookworm infection was no Revised
t entered in the urban model because the prevalence
4-C
EMail
Line
was less than 5%. The reference single-nucleotide polymorphism (rs) classification for SIZE
ARTIST: ts
IL10 +4949 is rs3024500.21 Because of the high
H/T
H/T
36p6
correlation among the three interleukin-10 p Enon
olymorphisms, only on Combo
e (most strongly associated with severe anemia) was included in the
multivariate model. HIV denotes human immunodeficiency virus, and G6PD?202/?376 denotes boys who are hemizygous and girls who
AUTHOR, PLEASE NOTE:
are homozygous for both the G6PD202A and th Figure has been redrawn and type has been reset.
e G6PD376G alleles.
Please check carefully.
JOB: 35809
ISSUE: 02-28-08
with severe anemia were further explored in a was 33.5% overall and 47.3% in the urban setting.
multivariate and structural equation model (Fig. 1 In the rural setting, a significant association be-
and 2).
tween malaria and severe anemia was found only
in the subgroup of patients who had hyperparasit-
Malaria
emia (9.7%) (adjusted odds ratio for case patients
P. falciparum was identified in 226 case patients vs. community controls, 7.1; 95% confidence in-
(59.5%) and 321 control patients (42.8%) and was terval [CI], 1.4 to 34.6).
the predominant malarial species overall (97.5%).
P. malariae was found in 1.6% and a mixed infec- HIV
tion in 0.9% of the study patients. The attributable HIV infection was found in 86 children (12.6% of
risk of severe anemia associated with P. falciparum case patients and 6.0% of controls). The attribut-
894
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Severe Anemia in Malawian Children
Severe Anemia
?0.22
+0.18
+0.08
+0.21
+0.10
+0.13
Vitamin A
+0.27
Iron Deficiency
Hookworm
HIV
+0.12
Malaria
deficiency
?0.13
+0.09
+0.16
?0.14
Bacteremia
Figure 2. Structural Equation Model for Severe Anemia, Iron Deficiency, and Malaria.
In this exploratory model of the factors associated with severe anemia,25 the sizes of the associations are indicated
AUTHOR: Calis
RETAKE
1st
ICM
by the standardized regression coefficients (range, ?1.0 to +1.0). Inverse (protective) associations are indicated by
FIGURE:
2nd
REG F
2 of 2
red lines. This model was created containing all possible associations between th 3rd
e displayed variables, after which
CASE
Revised
all nonsignificant arrows (P?0.05) were removed. The model also contained all other variables entered in the multi-
4-C
EMail
Line
SIZE
variate model (omitted for clarity). The d ARTIST:
isplayed ts
variables were all adjusted for age; in addition, malaria was adjust-
H/T
H/T
33p9
Enon
ed for previous use of antimalarial agents, and iron defici Combo
ency was adjusted for a history of transfusions (before the
previous 4 weeks) or use of hematinic agents (within the previous 8 weeks) (both omitted for clarity). Replacement
AUTHOR, PLEASE NOTE:
of severe anemia by continuous hem Figure has been redrawn and type has been reset.
oglobin levels and iron deficiency (ratio of soluble transferrin receptor to log
ferritin, >5.6)10 resulted in a virtually identical m Please check carefully.
odel. The overall model fit was valid (root mean square area of ap-
proximation, 0.043; 95% confidence interval, 0.039 to 0.048).
JOB: 35809
ISSUE: 02-28-08
able risk of severe anemia associated with HIV sent in 36.8% of children with bacteremia. Among
was 6.2% overall and 15.4% in the urban setting. case patients, bacteremia was less common in chil-
Among severely anemic children, significantly dren with malaria than in those without malaria
more HIV-infected than HIV-uninfected children (21 of 208 [10.1%] vs. 32 of 150 [21.3%], P = 0.003).
had Epstein–Barr virus infection (15 of 30 [50.0%] Among control patients, bacteremia was also less
vs. 69 of 226 [30.5%], P = 0.03) or bacteremia (11 of common in children with malaria than in those
42 [26.2%] vs. 38 of 300 [12.7%], P = 0.02), where- without (3 of 146 [2.1%] vs. 11 of 207 [5.3%],
as significantly fewer HIV-infected than HIV-unin- P = 0.12).
fected children had hyperparasitemia (2 of 44
[4.5%] vs. 42 of 312 [13.5%], P = 0.09) or vitamin Nutrition
B12 deficiency (5 of 39 [12.8%] vs. 85 of 254 [33.5%], Fifty-two case patients (15.8%) and 43 control pa-
P = 0.009).
tients (6.2%) had wasting; the attributable risk of
severe anemia associated with wasting was 6.2%.
Bacteremia
Severely anemic children were commonly stunted
Fifty-four case patients (15.0%) and 14 controls (53.2%) or underweight (49.2%), but for both con-
(4.0%) had bacteremia. The attributable risk of se- ditions the unadjusted and adjusted odds ratios
vere anemia associated with bacteremia was 12.2%. were similar to those for wasting (data not
The most common pathogen was nontyphoid sal- shown). Severe wasting occurred in 3.7% of se-
monella, which was present in 38 of the case pa- verely anemic children.
tients (70.4%) and 11 of the controls (78.6%) who
Vitamin B12 deficiency was found in 95 case
had bacteremia (P = 0.54). No mycobacteria were patients (30.4%) and 94 controls (15.6%) and was
isolated from any of the specimens. Fever was ab- severe (<13.6 ng per deciliter [100 pmol per liter])
n engl j med 358;9 www.nejm.org february 28, 2008
895
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T h e n e w e ng l a n d j o u r na l o f m e dic i n e
in 11.2% of case patients and 2.8% of controls with G6PD?202/?376 (78.4%). Boys accounted for
(adjusted odds ratio, 4.3; 95% CI, 1.9 to 9.9). 68.2% of children with G6PD?202/?376, but after
Macrocytosis was more common in children with stratification, G6PD?202/?376 remained significant-
vitamin B12 deficiency than in children with nor- ly associated with severe anemia in girls (adjusted
mal vitamin B12 levels (P = 0.02), although the odds ratio, 4.1; 95% CI, 1.2 to 13.3) and boys (ad-
sensitivity for vitamin B12 deficiency was low justed odds ratio, 2.2; 95% CI, 1.1 to 4.7).
(17.5%). Severely anemic children with vitamin B12
deficiency ate fewer meals with meat than those
Discussion
not deficient (1.9 vs. 2.7 per month, P = 0.02). Fo-
late deficiency was not found in any child en- In many African hospitals, severe anemia is a lead-
rolled in the study. Vitamin B12 and folate levels ing reason for admission and a major contributor
were inversely correlated with each other among to death. The cause of the anemia has not been
severely anemic children (Pearson correlation co- comprehensively investigated, but we found sev-
efficient, ?0.22; P = 0.01).
eral important associations in this study.
Vitamin A deficiency was found in 92.3% of
Malaria is commonly considered to be a prin-
case patients and 65.6% of controls and was cipal cause of severe anemia in Africa.7 In this
considered severe (less than 10 ?g per deciliter) study, P. falciparum parasitemia was strongly as-
in 32.8% of case patients and 14.9% of controls sociated with severe anemia in the area with
(adjusted odds ratio, 1.6; 95% CI, 0.91 to 2.76). seasonal transmission but not in the area with
Vitamin A deficiency was associated with malaria holoendemic transmission. However, the cumu-
and bacteremia in the structural equation model. lative effect of malaria on an individual person is
Iron deficiency was found in 46.6% of case pa- difficult to assess in holoendemic settings where
tients and 69.4% of controls. Further exploration children are repeatedly infected. Our findings
indicated this finding was not affected by the therefore do not exclude malaria as a predispos-
definition used (Table 3). In the structural equa- ing cause of severe anemia in the rural area but
tion model, iron deficiency was found to be in- indicate that additional or alternative diagnoses
versely associated with bacteremia (P = 0.006).
should be considered in severely anemic children
who receive a diagnosis of malaria infection. In
Hookworm
the structural equation model, malaria and bac-
Hookworm was the most common helminth in- teremia were identified as variables that modify
fection. Thirty-one (75.6%) of the hookworm in- the association between vitamin A deficiency and
fections occurred in children less than 2 years severe anemia. This is in line with earlier obser-
old. The attributable risk of severe anemia asso- vations that vitamin A deficiency is associated
ciated with hookworm in the rural site, where with an increased susceptibility to infection.28 A
95.1% of infections were seen, was 15.9%. In this vitamin A supplementation trial showed a reduc-
site, 10.4% of case patients and 0.6% of controls tion in the incidence of malaria,29 although this
had heavy infections (adjuste