http://jid.oxfordjournals.org/content/194/2/146.

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Parvovirus B19 Infection Contributes to

Severe Anemia in Young Children in Papua
New Guinea
1. James Wildig1,a,
2. Pascal Michon2,a,
3. Peter Siba3,
4. Mata Mellombo2,
5. Alice Ura2,
6. Ivo Mueller2,3 and
7. Yvonne Cossart1

+ Author Affiliations
1
1. Department of Infectious Diseases and Immunology, University of Sydney, Australia;
2
2. Papua New Guinea Institute of Medical Research, Madang, and
3
3. Papua New Guinea Institute of Medical Research, Goroka

1. Reprints or correspondence: Dr. Yvonne Cossart, Dept. of Infectious Diseases and

Immunology, University of Sydney, D06, Sydney NSW 2006, Australia
(ycossart@infdis.usyd.edu.au)

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Abstract
BackgroundSevere anemia (hemoglobin level, <50 g/L) is a major cause of death among
young children, and it arises from multiple factors, including malaria and iron deficiency. We
sought to determine whether infection with parvovirus B19 (B19), which causes the cessation
of erythropoiesis for 3–7 days, might precipitate some cases of severe anemia

MethodsArchival blood samples collected in the Wosera District of Papua New Guinea,
from 169 children 6 months–5 years old with severe anemia and from 169 control subjects
matched for age, sex, and time were tested for B19 immunoglobulin M (IgM) by enzyme
immunoassay and for B19 DNA by nested polymerase chain reaction (PCR). A total of 168
separate samples from children in the Wosera District were tested for B19 IgG

ResultsA strong association between acute B19 infection (positive by both IgM and PCR)
and severe anemia was found (adjusted odds ratio, 5.61 [95% confidence interval, 1.93–
16.3]). The prevalence of parvovirus B19 IgG reached >90% in 6-year-olds

ConclusionsB19 infections play a significant role in the etiology of severe anemia in this
area of malarial endemicity. Given the high levels of morbidity and mortality associated with
severe anemia in such regions, the prevention of B19 infection with a vaccine might be a
highly effective public health intervention

Severe anemia (hemoglobin level, <50 g/L) is very common among young children in
regions where malaria is endemic [1], accounting for an estimated 1 million child deaths per
year [2]. The etiology of severe anemia in these children is complex, with a variety of
factors—including malaria and iron deficiency—contributing [3]

Parvovirus B19 (B19) is common worldwide; antibody studies have indicated that >50% of
people are infected during childhood. Higher rates have been reported among children in
some tropical areas [4, 5]. Outbreaks occurring at 3–6-year intervals have been described [5,
6]

B19 is tropic for red blood cell precursors in the bone marrow, with acute infection causing
impaired erythropoiesis for 7–10 days and complete cessation for 3–7 days. The effect of this
on hemoglobin level varies by individual. In healthy adults, a decrease in hemoglobin level of
∼20 g/L will occur [7, 8], whereas larger decreases have been described in persons with iron
deficiency [9] and malaria [10, 11]. In persons with sickle-cell disease [12] and other
hemolytic disorders, a precipitous decrease in hemoglobin levels can be induced through the
combination of a high rate of red blood cell destruction and complete cessation of red blood
cell production caused by B19. This has been termed “transient aplastic crisis.”

After initial contact with B19, viral replication leads to a dense viremia that starts to decline
once specific IgM is produced on day ∼9 [8]. Virus-induced bone-marrow suppression
begins to recover on day ∼16, and the lowest hemoglobin level occurs soon after (figure 1)
[7, 13, 14]. Thus, the simultaneous detection of B19 IgM and DNA is strongly indicative of
acute infection. B19 IgM usually becomes undetectable after 2–4 months, depending on the
initial level of response [15], although persistence for up to 9 months has been reported [16].
The rate of decrease in viremia is less predictable. The half-life of B19 in blood is not known,
but, even though the virus may become undetectable soon after infection, clinical studies
using highly sensitive nested or real-time polymerase chain reaction (PCR) methods have
shown that B19 DNA can often be detected 6 months or even longer after the onset of illness
in patients who have become IgM negative [17]. After infection, IgG persists; thus, a person
usually is infected only once in a lifetime

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Figure 1

Timing of changes in clinical and virological parameters during acute parvovirus B19
infection in the immunocompetent individual. Data are from various sources [7, 12–15].
RBC, red blood cell

In 1990, it was suggested that B19 infection might be a causative factor in some cases of
severe anemia among young children in areas where malaria is endemic [4]. In that study,
which was conducted in the Republic of Niger and apparently coincided with a B19 infection
outbreak, 54% of children with severe anemia (hematocrit level, <20%) showed evidence of
recent B19 infection. No control group was assessed, so rates of B19 infection in children
with and without anemia could not be compared. Two subsequent studies, from Malawi [18]
and Kenya [3], found little evidence of acute B19 infection in any children (anemic or
control) over the course of 1 year of testing. In the present article, we present the results of a
retrospective case-control study linking B19 infection to severe anemia in a highly malarious
area [19] of Papua New Guinea (PNG)

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Patients, Materials, and Methods

Both case patients and control subjects were selected from archival blood samples collected
between 1996 and 2002 from children 6 months–6 years old who presented with presumptive
malaria (measured fever or reported febrile illness with no obvious respiratory cause) at 2
health centers in Wosera District, East Sepik Province, PNG. These samples had been
collected as part of ongoing routine malaria morbidity surveillance at the PNG Institute of
Medical Research malaria vaccine trial site. Hemoglobin was measured at the time of sample
collection using the HemoCue system (HemoCue). Malarial infection was defined as the
detection of malarial parasites by microscopic examination of the blood film; the different
species of malaria parasite were distinguished and examined individually in the statistical
analyses. All children were treated in accordance with PNG national guidelines for malaria
infection and severe anemia (antimalarial treatment and oral or intramuscular iron
supplementation)

After samples lacking hemoglobin readings were excluded, there were 11,441 samples with
age, sex, blood-slide readings, and hemoglobin measurement data available. Children with
hemoglobin levels of ⩽50 g/L (the World Health Organization [WHO]–recommended cutoff
for severe anemia]) were selected as case patients. A control group of children with
hemoglobin levels of >50 g/L was selected and matched for age (83% within 1 month), sex,
and time (most within 3 months and all within 1 year). When spoiled or lost samples were
excluded, 169 matched pairs of samples were available for testing. The mean age of case
patients and control subjects was 2.87 and 2.84 years, respectively, and 47.9% of children in
both groups were male (table 1). These pairs were tested for the presence of both B19 IgM
and DNA, because either can be a unique marker of recent B19 infection, and the finding of
both in the same specimen is strongly indicative of acute infection. A total of 168 separate
consecutive samples from children 6 months–10 years old who presented to the same health
centers in late 2000 with presumptive malaria were subsequently selected for testing for B19
IgG
Stored plasma from the selected samples was tested for B19 IgM or IgG using the Biotrin
Parvovirus B19 IgM or IgG EIA kit (Biotrin International). The reaction wells were washed
with 200–220 μL of wash solution per use, depending on the available equipment. Plasma
samples that were stained with red blood cells during the collection or storage process were
included. Otherwise, specimens were tested in accordance with the manufacturer’s
instructions. The IgM EIA kit has a reported sensitivity of 86% and a specificity of 95% [20],
and the IgG EIA kit has a reported sensitivity and specificity of 100% [21]. These kits have
been shown to have good specificity when they are tested on serum samples from people with
other infections [22] and in comparison with other kits [23]. All case-control samples were
tested for IgM in duplicate (or in triplicate, if the first 2 results differed). Case and control
specimens were tested side by side in 96-well plates. Because the samples were stored at a
remote field location, a portable Axiom M6 Miniphotometer with a 450-nm filter (Axiom)
was used to measure the optical density of the test reactions. To allow for the measured
precision of the reading device, the equivocal range was extended to 0.8–1.2 times the cutoff
optical density

DNA samples were extracted separately from plasma and erythrocyte pellets of the selected
samples (when available) using the QIAmp 96 DNA blood kit (Qiagen). Two oligonucleotide
primer pairs were designed on the basis of the B19 genomic sequence (GenBank accession
number NC_000883) and used in a nested-PCR approach. Briefly, 1 μL of DNA template (or
PCR product from first-round PCR) was amplified by PCR with 0.5 U of Taq DNA
polymerase (Invitrogen) and supplied buffer, 1.5 mmol/L MgCl2, and 200 μmol/L each dNTP
in 2 consecutive 15-μL reactions, using oligonucleotide primers (400 nmol/L each) specific
for human erythrovirus genotype 1 (B19), as follows: first-round forward primer B19-1F (5′-
CTG TGG TTT TAT GGG CCG CC-3′) and reverse primer B19-1R (5′-AGG TGT GTA
GAA GGC TTC TTC CC-3′), followed by nested forward primer B19-2F (5′-GGG AAA
AGC TTG GTG GTC TGG G-3′) and reverse primer B19-2R (5′-GCG CGG GGT TTC AGT
GTT CC-3′). Both reactions consisted of 2 min at 94°C and 35 cycles of 30 s at 94°C, 30 s at
60°C, and 1 min at 72°C. To ensure specificity, PCR products of the expected sizes for both
reactions were originally sequenced and checked with the DNA database, to verify that the
appropriate target was amplified. A sample with a positive PCR result in either plasma or the
erythrocyte pellet was regarded as positive for statistical analysis

Statistical analysesTo account for matching, the case-control data were analyzed with either
McNemar’s χ2 test or with conditional logistic regression. Correspondence between PCR and
EIA results and the prevalence of malarial infections was tested using Pearson’s χ2 test. All
analyses were done using Stata statistical software (version 7.0; StataCorp). Stata software
uses an uncorrected numerator for the McNemar’s test—that is, (n12-n21)2. An age-specific
prevalence curve for IgG data was fitted using a generalized additive model for binomial data
involving fourth-order splines of the S-PLUS package (version 6.0; Insightful)

Ethics approvalEthics approval for the study was given by the PNG Medical Research
Advisory Committee, and that for the IgM testing was given by the University of Sydney
Human Ethics Committee

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Results
For each specimen, 2 independent B19 diagnostic results were obtained: the presence or
absence of B19 IgM or DNA. Positive results for both in the same specimen were regarded as
strongly indicative of an acute B19 infection

Of 169 case patients with hemoglobin levels of <50 g/L, 51 (30.2%) were positive, 95
(56.2%) were negative, and 23 (13.6%) had equivocal results for B19 IgM, whereas, of
control subjects, 21 (12.4%) were positive, 126 (74.6%) were negative, and 22 had equivocal
results (13.0%). All analyses were done once with equivocal readings considered to be
negative and once with all case-control pairs with equivocal IgM values omitted from the
analysis. Because the results of both analyses did not differ significantly (data not shown),
only results from the former analyses (equivocal = negative) will be presented

Acute B19 infection, defined as both IgM EIA and PCR positivity, showed the strongest
association with severe anemia (odds ratio [OR], 5.0). Independently, only IgM positivity
(OR, 3.0)—and not PCR positivity (OR, 1.25)—was significantly associated with severe
anemia (table 2). If only the samples with the highest optical-density values in IgM testing
(i.e., >3 times the cutoff value) were considered, 25 of 26 were from case patients, and 21 of
these were also positive by PCR (figure 2). No clear temporal clustering of either IgM- or
PCR-positive samples was observed

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Figure 2

Evidence of acute parvovirus B19 infection in case patients and control subjects. Optical
density readings for B19 IgM testing (as multiples of the cutoff optical density value) were
plotted against hemoglobin (Hb) levels for case patients and control subjects. Gray-shaded
area, equivocal range (0.8 < OD < 1.2); black points, polymerase chain reaction (PCR)–
positive samples; white points, PCR-negative samples

In the case patients, but not in the control subjects, PCR positivity was significantly
associated with IgM positivity (table 3). Among the case patients, 14.8% were positive for
IgM only, 11.8% were positive for B19 PCR only, and 15.4% were positive for both,
compared with 8.9%, 19.5%, and 3.6%, respectively, in control subjects (χ2=19.5 [df 3];
P<.001). The ORs (vs. double-negative samples) for association with severe anemia were
5.32 for both IgM and PCR–positive results, 1.89 for IgM-positive results only (P=.075), and
0.61 for PCR-positive results only (table 4)
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Figure 3

Age-specific prevalence of parvovirus B19 IgG in 168 children from the study area who were
<10 years old. All children were selected from among patients with presumptive malaria
attending the Kunjingini health center during the second half of 2002. The age-specific mean
(solid line) and binomial SEs (dashed lines) were predicted using fourth-order regression
splines. White circles, IgG-negative children; black circles, IgG-positive children

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Table 1

Comparison of age, hemoglobin (Hb) level, and malarial parasitemia in case patients and
control subjects

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Table 2

Association of parvovirus B19 IgM and polymerase chain reaction (PCR) positivity with
severe anemia

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Table 3

Prevalence of viremia in case patients and control subjects with and without parvovirus B19–
specific IgM antibody

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Table 4

Risk of severe anemia associated with parvovirus B19 and Plasmodium infection status

Malarial infections were significantly more frequent in case patients (150/169 [88.8%]) than
in control subjects (108/169 [63.9%]) (P<.0001). Significantly more of these were P.
falciparum infections (131/150 [87.3%]) than non–P. falciparum infections (76/108 [70.4%])
(P=.001) (table 1). Both P. falciparum and non–P. falciparum infections were associated with
an increase in risk of severe anemia, compared with that in uninfected children (OR, 5.75
[95% confidence interval {CI}, 2.93–11.26]; P<.001 for P. falciparum infection and OR, 2.16
[95% CI, 0.94–5.00]; P=.07 for non–P. falciparum infection)

We then investigated the interaction between B19 and malarial infections in detail (table 4).
Multivariate analyses showed that the risks of severe anemia associated with Plasmodium and
B19 infections were independent of each other (χ2=7.57 [df 6]; P=.27, likelihood-ratio test).
Acute B19 infection (i.e., PCR- and IgM-positive results) was associated with an increase in
the risk of severe anemia comparable to that associated with P. falciparum infections
(multivariate OR, 5.53 vs. 5.84) (table 4)

In the serum samples from 168 children 1–10 years old from the study area, the age-specific
prevalence of B19 IgG was found to increase rapidly during the first years of life (figure 3),
with 60% of children <2 years old already positive for B19 IgG. Levels continued to increase
until age 6 years, when the prevalence of IgG plateaued at ∼90%

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Discussion
The present results demonstrate that acute B19 infection is a major contributor to severe
anemia in young children in the study area, where malaria is endemic. Indeed, the association
found between acute B19 infection (defined as both B19 IgM and PCR positivity) and severe
anemia (multivariate OR, 5.53) is comparable to that found between P. falciparum infection
(a leading cause of anemia in areas of endemicity) and anemia (multivariate OR, 5.84)

This finding contrasts the situation in developed countries, where B19-induced severe anemia
is rare, except in individuals with hemolytic disorders. A possible reason for this
geographical difference is that, in tropical areas of developing countries, an underlying mild-
to-moderate level of anemia is very common in young children [3]. Cross-sectional surveys
in the Wosera and neighboring populations of PNG found that 5%–11% of children had a
hemoglobin levels of <7 g/dL (I.M., unpublished data). This anemia arises from multiple
factors, such as malnutrition (including iron deficiency), current and past malaria, and other
infections, including hookworm [24]. B19 infection causes a 20-g/L decrease in hemoglobin
level, even in previously healthy adult volunteers without hemolytic disease [7]. Thus, when
acute B19 infection strikes children in a population where anemia is common, the resulting
decrease in hemoglobin level, when superimposed on a preexisting moderate level of anemia,
may be the proverbial “last straw” that pushes an already low hemoglobin level below the 50-
g/L level that defines severe anemia, even in the absence of hemolysis. By contrast, children
in developed countries with normal hemoglobin levels are untroubled by a transient 20-g/dL
decrease in hemoglobin level. Clearly, any children in PNG with a hemolytic disorder would
have an even larger decrease in hemoglobin levels if they developed B19 infection, and they
might be even more prone to B19-induced severe anemia. Although hemoglobin S is not
known to occur in PNG, glucose-6-phosphate-dehydrogenase (G6PD) deficiency has been
noted to be present in 8.7% of people in a nearby region [25]. It is thus conceivable that at
least part of the excess risk of severe anemia after B19 infection is concentrated in children
with G6PD deficiency. Other red blood cell polymorphisms that are common in PNG include
the Gerbich blood group and α- or β-thalassemia [26]. However, among 306 children with
genotypic data available, Gerbich blood group (band 3 deletion) and α-thalassemia were not
significantly associated with an increase in the risk of severe anemia associated with B19
infection (P.M., unpublished data)

Data on the prevalence of B19 IgM and DNA in children living in tropical areas is very
limited. Although studies of adult blood donors in the United States have indicated a
prevalence of B19 IgM of ∼1%, we found a much higher prevalence of B19 IgM, 12%, in the
control population. The characteristics of our control group may offer some explanation for
this, in that they were children, they had had a recent febrile illness, and they lived in PNG.
IgG studies have indicated that B19 infection is much more common in children living in
tropical areas. Indeed, our IgG testing showed that 90% of children in the Wosera District
had evidence of previous B19 infection by age 6 years. Furthermore, B19 causes a febrile
illness, so the febrile children used as control subjects were more likely to have had IgM from
an acute infection than were asymptomatic children. These factors help to explain the high
prevalence of IgM and, thereby, the high number of equivocal results in our study. With a
high rate of positive results, numerous samples will be from children who were recently
positive and whose antibody levels are decreasing, passing through the equivocal range as
they fall

Some studies have indicated that IgM testing yields more false-positive results in populations
with other infections or autoantibodies. It is true that a number of the children sampled in the
present study were likely to have had other infections, whereas the rate of autoantibodies in
this population was not known. However, although older IgM assays had problems with
specificity under these circumstances, the Biotrin B19 IgM and IgG EIA kits that we used
have been shown to perform well with regard to specificity in such samples. Moreover,
because case patients and control subjects were selected from the same population, the false-
positive rates should have been similar in both and should not have had a major influence on
the correlation found between B19 infection and severe anemia

The introduction of sensitive PCR tests for B19 in recent years has revealed that many
patients retain detectable DNA for weeks or months after specific IgM becomes undetectable,
at ∼3 months after infection [13, 27]. This may well explain the high prevalence of B19 DNA
that we found in both case patients and control subjects. The average age of the children in
our study was a little less than 3 years, when the prevalence of B19 IgG is already 60%.
Assuming an added PCR persistence of 3 months the after the loss of IgM, protection for the
first 6 months of age by maternal antibody, and an endemic pattern of infection, random
testing of 3-year-olds in this environment would be expected to detect B19 DNA in 10% of
them. We detected a not-dissimilar rate of 15.6%

Although many of the children with severe anemia tested positive for both B19 IgM and
DNA, our study revealed numerous children who tested positive for only 1 of the 2. Given
the complex temporal nature of the association between levels of viremia and IgM (figure 1),
this is not surprising. At least 2 studies have demonstrated similar discrepancies between
PCR and IgM test results in B19 infection, and those researchers recommended that both be
used to diagnose of recent infection [14, 28]. The fact that the double-positive specimens
showed the strongest correlation with severe anemia is expected, because this pattern of
results is most likely to be seen around the time of hemoglobin suppression (figure 1)

Our finding of a significant association between acute B19 infection and severe anemia is
reminiscent of the observations in Republic of Niger [4] but contrasts with the results of 2
other studies, from Malawi [18] and Kenya [3]. The fact that no association was found in
these latter articles is likely to have stemmed from the short duration of the studies, which
seems to have coincided with periods of low B19 transmission in the respective communities.
Indeed, in the Malawi study, only 13% of children were B19 IgG positive, whereas, in the
Wosera District, 60% of children were IgG positive by age 2 years. B19 often occurs in
outbreaks separated by long periods of inactivity in both temperate and tropical areas [5, 29,
30]. The present study, which used archival samples collected over the course of a 6-year
period, was able to achieve sufficient sample size to clearly demonstrate the association
between B19 and severe anemia. However, the use of archival specimens also meant that the
choice of control subjects in this retrospective analysis was limited by the availability of
samples. Both case patients and control subjects were obtained from the same population of
children presenting to health centers with presumptive malaria (i.e., recent fever with no
obvious respiratory cause) between 1996 and 2002. Because acute B19 infection is
accompanied by fever [31], it is likely to be more common among these clinical control
subjects than among asymptomatic children in the community. The presumably higher
prevalence of B19 infection in the available control subjects is likely to affect the estimates of
the association of B19 infection with severe anemia in a conservative way, so the association
is likely to be less pronounced in the present study than would have been the case if
asymptomatic control subjects from the community had been used for comparison

Our study was based in the Wosera District, an area where malaria is highly endemic [19],
and P. falciparum infection is an important risk factor for severe anemia. Interestingly,
analysis of the results showed that the association of B19 infection with severe anemia is not
altered by malarial infections and that the effects of both are additive. However, longitudinal
studies in young African children have shown that hemoglobin levels are more closely related
to average parasitemia during the preceding 90 days than to concurrent infections [32]. In
addition, hematological responses after symptomatic malarial infections are often
characterized by an initial decrease in hemoglobin levels, followed by an eventual recovery
through increased erythropoiesis [33]. The interaction of B19 and acute malaria infections
may thus be strongest when B19-induced erythropoietic suppression coincides with the
period of increased erythropoiesis during the 1–4 weeks after treatment rather than with the
acute malarial episode. The interactions between the 2 infections can thus only be properly
investigated in longitudinal studies. The present results nevertheless indicate that controlling
B19 infection is likely to lower the risk of severe anemia in all children, irrespective of their
malaria status

Severe anemia is a major cause of morbidity and mortality in young children in areas where
malaria is endemic [2]. Treatment with blood transfusion is limited in availability, is costly to
patients and health-care facilities, and carries the risk of transmission of bloodborne
pathogens, including HIV (even when anti-HIV screening is performed) [34]. Clearly, the
prevention of severe anemia should be a major priority [35]. Further studies are needed to
estimate the effects of B19 infection in other regions where malaria is endemic. However, the
strength of the association between B19 and severe anemia observed in the present study
indicates that the prevention of B19 infections is likely to result in a significant reduction in
the burden of severe anemia in young children in these regions. Efforts to develop a safe and
effective vaccine [36] against B19, which are already under way, should be strengthened,
with the view of its possible use in the prevention of severe anemia among young children,
alongside other measures, including malaria control and nutritional supplementation

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Acknowledgments
We thank the staff of the Papua New Guinea Institute of Medical Research in Goroka,
Madang, and Maprik; the staff of the Department of Infectious Diseases, University of
Sydney; Lawrence Rare and the health center surveillance nurses, for the collection of the
samples; Thomas Adiguma, for managing the databases and helping with the selection of
control subjects; and Livingstone Tavul, for helping with DNA extraction

Previous SectionNext Section

Footnotes
 ↵(See the editorial commentary by Pasvol, on pages 141–2.)
 ↵Potential conflicts of interest: none reported

Financial support: Biotrin International, Ireland (parvovirus IgM and IgG test kits);
PanBio Limited (Australia; assistance with shipping); University of Sydney, Faculty
of Medicine Postgraduate Research Support Scheme; and Daphne Goulston
Scholarship (travel assistance to J.W.)

 ↵J.W. and P.M. contributed equally to the study

 Received December 20, 2005.

 Accepted February 16, 2006.

 © 2006 by the Infectious Diseases Society of America

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