MAJOR ARTICLE

Common Surface-Antigen var Genes of

Limited Diversity Expressed by Plasmodium
falciparum Placental Isolates Separated
by Time and Space
Ayman Khattab,1 Peter G. Kremsner,1,2 and Mo-Quen Klinkert1
1
Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany; 2Medical Research Unit, Albert Schweitzer Hospital,
Lambaréné, Gabon

Plasmodium falciparum placental parasites from Cameroon have been shown to express surface variant var
genes encoding Duffy binding–like (DBL)–g domains that bind chondroitin sulfate A. All 5 domains exhibited
sequences with 39%–55% amino acid (aa) identities and appear sufficiently conserved to function in receptor
binding. Transcripts of 2 samples showed complete conservation over 4 kb, demonstrating for the first time
distinct conserved placental var genes. Four placental isolates from Gabon collected 4 years later expressed
DBL-g sequences with 85%–99% aa identities to those from Cameroon, confirming the conserved nature of
placental variants separated by time and location. Five peripheral parasites from children also displayed DBL-
g sequences with 75%–97% homologies. From this, it can be concluded that P. falciparum parasites expressing
unique var DBL-g genes can cause placental malaria, referred to as varPAM genes. This demonstration of
structurally/functionally constrained DBL-g chondroitin sulfate A–binding domains is relevant to understand-
ing pregnancy-associated malaria pathogenesis and to vaccine development.

Pregnancy-associated malaria (PAM) is a major cause of
infant morbidity and mortality and anemia and death
in the mother [1]. Although adults in areas where malaria
is endemic are normally clinically immune to the disease,
women experiencing their first pregnancies are partic-
Received 25 July 2002; revised 3 October 2002; electronically published 24
January 2003.
Study participants gave written informed consent, and, in the case of children,
consent was obtained from their parents or guardians. Ethical clearance was
obtained from the ethics committee of the International Foundation of the Albert
Schweitzer Hospital (Lambaréné, Gabon).
Financial support: European Commission (grants QLK2-CT-1999-01293 and QLK2-
CT-2001-01302).
Nucleotide sequence data are available in the GenBank database (accession
nos. AF547098–AF547163).
Reprints or correspondence: Dr. Mo-Quen Klinkert, Institute for Tropical
Medicine, University of Tübingen, Wilhelmstrasse 27, 72074 Tübingen, Germany
(mo.klinkert@uni-tuebingen.de).
The Journal of Infectious Diseases 2003; 187:477–83
 2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18703-0016$15.00
ularly more vulnerable to infection and can present
clinical manifestations of the disease. Plasmodium falci-
parum–infected erythrocytes (IEs) are preferentially
sequestered in the placenta by interacting with a gly-
cosaminoglycan, chondroitin sulfate A (CSA), on syn-
cytiotrophoblast cells lining the placenta [2]. CSA acts
as a receptor molecule for P. falciparum erythrocyte
membrane protein 1 (PfEMP-1), which is expressed by
the parasites on the surface of IEs [3–5]. Other receptor
molecules such as hyaluronic acid and IgG also have been
implicated in the adhesion of IEs in placentas [6, 7].
Antibodies from secundigravid and multigravid
women living in areas where malaria is endemic can
block the binding of placental parasites to CSA [8] and
are thought to be generated in a strain-independent
manner, with the capacity of recognizing parasites from
different geographic locations. The panreactive anti-
CSA binding antibodies also react with the surface of
IEs in a sex-specific manner, such that serum samples
from malaria-exposed men do not show any reactivities

Common DBL-g Subtypes in P. falciparum • JID 2003:187 (1 February) • 477

toward IE surface [8, 9]. Taken together, the results suggest the
unusual conserved nature of these placental-sequestering var-
iants (varCSA) of the PfEMP-1 gene.
In several independent experiments, a polypeptide domain
of the var-encoded protein PfEMP-1, the Duffy binding–like
(DBL)–g domain, was shown to be associated with the cyto-
adherence phenomenon involving CSA [3–5]. The CSA-bind-
ing DBL-g domains characterized in our laboratory from clin-
ical isolates from malaria-infected placentas exhibited 39%–
55% aa sequence identities with each other, and this unique
region is thought to maintain functional integrity through a
conserved conformational structure [5]. Of interest, we also
found DBL-g sequences from 2 different parasite isolates, 485
and 701, to be nearly identical to each other, as were 2 other
isolates, 720 and 734. In part, these results provide the molec-
ular basis to support the immunoepidemiological data that
suggest that the CSA-binding ligand is antigenically conserved
and that women who are infected with parasites expressing this
varPAM variant during pregnancies can gain lifelong immunity
against PAM.
Recently, a unique regulatory element located at the 5
un-
translated region of the varCSA gene was correlated with the
expression of this gene involved in placental malaria. This find-
ing provided a plausible explanation for the relative conser-
vation of the varCSA gene in genetically diverse parasites [10].
Because one of the central issues of PAM research concerns
structure, function, and amino acid sequence conservation in
DBL-g mediating the cytoadherence of IEs to the placenta, we
have extended the scope of our earlier study of Cameroonian
placental parasites to look specifically at the expression of this
var gene segment in placental parasites collected from a dif-
ferent study site (Gabon). There was a 4-year gap between our
2 studies. The present study also focused on DBL-g domains
amplified from placental, as well as peripheral, parasites, to
examine the occurrence in different parasite populations of the
var gene fragments encoding DBL-g domains that bind CSA
and cause malaria pathogenesis in pregnant women.
SUBJECTS, MATERIALS, AND METHODS
Study site and participants. Study participants were resi-
dents of Lambaréné, which is situated on the equator in a
typical Central African rainforest area in Gabon. The prevalence
of plasmodial infection shows a hyperendemic pattern, and
transmission is intense and perennial [11]. Placental blood sam-
ples that tested positive for P. falciparum in thick blood smears
were collected from women who presented at the maternity
ward of the Albert Schweitzer Hospital for delivery, and 5 sam-
ples were randomly chosen for the study (parasitemia counts
of 1000–140,000 parasites/mL). These individuals were asymp-
tomatic carriers, and parity varied from 1 to 6. Another group
478 • JID 2003:187 (1 February) • Khattab et al.
of participants included schoolchildren who presented at the
Medical Research Unit of the Albert Schweitzer Hospital with
uncomplicated malaria—their parasite counts were 10,000–
30,000 parasites/mL. All patients were treated with quinine.
Collection of parasites. Placental blood was extracted by
squeezing a piece of placenta tissue into 0.9% NaCl that con-
tained 10 U/mL heparin [12] and was examined for the pres-
ence of malaria parasites in thick blood smears using the Lam-
baréné method [13]. The parasites collected in this way were
also shown to have the CSA-binding phenotype. Packed eryth-
rocytes (100-mL aliquots) were collected from infected blood
by centrifugation through ficoll-hypaque to separate the cell
pellet from the plasma, stored at ⫺80C, and subsequently used
for DNA preparation, polymerase chain reaction (PCR) am-
plification, and genotyping experiments. Peripheral blood was
collected in a monovette, parasite densities were determined,
and packed erythrocytes were collected as described above.
PCR cloning of expressed varPAM genes. PCR amplifi-
cations were carried out to amplify longer var gene sequences
that partially corresponded to 2 identical DBL-g sequences pre-
viously isolated by reverse-transcriptase (RT)–PCR on parasite
isolates from Cameroonian patients (nos. 720 and 734) [5].
The var sequences amplified were ∼4 kb in length. Genomic
DNA from patients 720 and 734 were isolated from 100 mL of
packed erythrocytes, using the blood DNA mini kit (Qiagen)
according to the manufacturer’s instructions. DNA was eluted
in 50 mL of 10 mM Tris buffer (pH 8.5), and 5 mL was used
in a PCR in the presence of the expand amplification enzyme
(Roche Molecular Biology). Primer AF served as a forward
primer and has been shown elsewhere [14] to universally am-
plify DBL-a sequences; 720/734-R (5
-ATC ATT ATC TGC TGT
GTG TGT TGC) was used as a reverse primer, designed around
a highly variable region on the 720/734 DBL-g sequences. The
resulting PCR products were cloned into pCR-XL-TOPO plas-
mid from the TOPO XL PCR cloning kit (Invitrogen). Positive
clones were verified by DNA sequencing.
RT-PCR amplification of DBL-g domains. Total RNA was
prepared from placental parasites Gb21 and Gb35 using the
QIAamp RNA blood mini kit (Qiagen), as described elsewhere
[5, 12]. Placental packed erythrocytes (100 mL) were mixed with
500 mL of the RLT buffer provided. The extraction steps thereafter
were conducted according to the manufacturer’s instructions.
The RNA was pretreated on column with RNAse-free DNAse.
After first-strand cDNA synthesis with random primers (Roche
first-strand cDNA kit) and RT-PCR amplification using degen-
erate primers UNIEBP5
and UNIEBP3
[16], the products were
excised from the agarose gels and sequenced directly or cloned
into the TA cloning vector pCR2.1 (Invitrogen).
Preparation and amplification of genomic DNA. Parasite
DNA was extracted from pellets of packed erythrocytes collected
from the peripheral blood of P. falciparum–infected children

Figure 1. Alignment of sequences from Plasmodium falciparum pla-
cental isolates 720/734 with laboratory clones FCR3var chondroitin sulfate
A (CSA), CS2varCSA, and 3D7chr5var. The total length of the varPAM
sequences being compared is ∼4 kb each and spans N-terminal var
domains from Duffy binding–like (DBL)–a to DBL-g. Percentages in the
boxes refer to the degree of amino acid conservation of the laboratory
clones in comparison to sequence 720. Sequence 734 was erroneously
labeled in a previous study [5] as being identical to 732. CIDR, cysteine-
rich interspersed domain region.
(Gb53, Gb54, Gb55, Gb56, and Gb59) or from blood samples
from infected placentas (Gb35, Gb170, Gb172, and Gb174) using
the QIAmp DNA mini blood kit (Qiagen), according to the
manufacturer’s instructions. Universal UNIEBP5
and UNIEBP3

primers were used to randomly amplify genomic DBL-g se-
quences, and PCR products were analyzed by cloning into
pCR2.1 and nucleotide sequencing. Approximately 25 positive
clones from each PCR fragment were examined.
RESULTS
Analysis of expressed placental var genes. In the present study,
we sought to examine the extent to which var genes expressed
by placental isolates are conserved, on the basis of immuno-
epidemiological data that appear to point to a functional and
structural conservation of the CSA-binding ligand. Because nu-
cleotide sequencing provides the best answer for determining the
degree of polymorphism in a gene, we proceeded to clone and
sequence the expressed var genes of interest.
For this purpose, we concentrated on a previously published
DBL-g sequence named 720 (GenBank accession no. AF334806),
which was ∼600 bp in length and was unexpectedly found to be
98% identical to sequence 734 at the nucleotide and deduced
amino acid sequence levels. The analysis of these 2 expressed
var genes was extended into the 5
upstream sequences encoding
the DBL-a/cysteine-rich interspersed domain region (CIDR)
head structure regions. Clones of ∼4 kb each of the 720- and
734-related sequences (GenBank accession nos. AF547158 and
AF547159, respectively) were isolated and sequenced. The de-
duced amino acid sequence analysis demonstrated that the 2
var genes extending from the DBL-a to the DBL-g regions
shared almost complete conservation with each other, on the
order of 96%. The fact that parasites 720 and 734 analyzed in
Common DBL-g Subtypes in P. falciparum • JID 2003:187 (1 February) • 479
the present study have different genotypes (data not shown)
argues against the possibility that the 2 individuals from whom
the parasites have been isolated had been infected with the
same strain. The results presented here provide clear evidence
for the existence of var genes of genetically diverse placental
isolates that are almost identical, at least in their N-termi-
nal–deduced amino acid sequences.
Comparison of varPAM sequences of placental isolates
and laboratory clones. To determine the extent of similari-
ties between well-characterized var genes (FCR3varCSA,
CS2varCSA, and 3D7chr5var) known to bind CSA and those
placental parasites that cause disease during pregnancy, we
aligned each of the sequences in the form of a diagram (figure
1). The alignment of ∼4 kb of nucleotides covers the N-terminal
half of the var domains, namely DBL-a, CIDR-a, DBL-b, and
DBL-g. The levels of amino acid conservation in each of these
blocks are shown as percentages in figure 1. For simplification,
the comparison was made on sequence 720.
Frequency of expressed DBL-g domains of placental isolates
from Gabon. Next, we examined placental parasite isolates
from Lambaréné, with a focus on their expressed DBL-g se-
quences. RT-PCR products amplified on cDNA prepared from
2 different placental parasites (Gb21 and Gb35) were cloned and
Table 1. Frequency of expressed conserved var Duffy bind-
ing–like (DBL)–g domains from 2 Gabonese placental isolates.
Parasite isolate, Total no.
var DBL-g domain Identity sequenced
Gb21
498-like variant 85% to sequence 498 5
701-like variant 42% to sequence 701/485 3
732-like variant 99% to sequence 732 1
Gb35
498-like variant 80% to sequence 498 5
701-like variant 1 97% to sequence 701/485 2
701-like variant 2 43% to sequence 701/485 1
732-like variant 98% to sequence 732 1
NOTE. In the Gb21 sequence analysis, 5 of 9 clones exhibited 85% aa
sequence identity to the previously published Cameroonian sequence 498, 3
clones could be attributed to a novel sequence with 42% homologies to the
701/485 sequence (first hit in the BLAST search), and 1 clone had 99% aa
sequence identity to sequence 732. The 498 sequence of the Cameroonian
and the Gabonese strains differed from each other by a 19-aa deletion in the
latter sequence. The analysis of Gb35 sequences indicated that 5 of 9 DBL-
g isoforms had 80% aa sequence identity to sequence 498 (with the exception
of the 19-aa deletion). Two clones exhibited 97% conservation to sequence
701/485, whereas 1 was 98% identical to sequence 732. Another sequence
in this set was a novel sequence but showed sequence conservation at 43%
with 701/485 (first hit in the BLAST search). Additional sequences of 8 clones
each were obtained from the products of independent reverse-transcriptase–
polymerase chain reaction (PCR) amplification reactions using Gb21 and Gb35
cDNA and were found to be identical to those of the first amplifications. PCR-
based genotyping experiments using merozoite surface antigen (MSA)–1– and
MSA-2–specific primers [12, 15] indicated that 2 different parasite genotypes
were detected in isolate Gb21, whereas 3 different genotypes were deter-
mined in isolate Gb35.
Figure 2. Comparison of Duffy binding–like (DBL)–g genomic sequences from placental and children’s parasites. Sequences identified in the present
study are labeled varX and are preceded by the number of the clinical isolate (e.g., Gb170var5). Percentages given in each box refer to the percentage
of amino acid conservation to known sequences in the database, provided in the form of their accession nos. Names of clinical isolates from Cameroon,
whose DBL-g domains have been demonstrated elsewhere to bind chondroitin sulfate A (CSA) [3–5], are included in some boxes and, for comparison
purposes, also the 2 laboratory strains FCR3varCSA and CS2varCSA. Boxes are shaded in 3 different gray intensities. Dark gray, sequences that have
at least 75% conservation to the clinical isolates; medium gray, sequences with at least 40% conservation; and light gray, sequences with similarities
to the laboratory strains. White boxes, sequences that have not been implicated in CSA binding. Sequences encoding DBL-g domains that are capable
of binding CSA were detected with similar frequencies in parasites from both pregnant and nonpregnant donors. There were 9 of 28 Cameroon-
specific DBL-g sequences in the placental parasite population, vs. 9 of 29 sequences in the peripheral parasite population. Five additional placental
sequences showed sequence similarities with FCR3varCSA and CS2varCSA, whereas 3 FCR3- and CS2-related sequences were found among the
peripheral parasites.

the expressed DBL-g CSA-binding domains were analyzed. The
complete sequences from 9 clones from each isolate were avail-
able for comparison, and the results are summarized in table 1.
We show here for the first time, to our knowledge, that the
placental parasites collected 4 years apart originating from areas
where malaria is endemic in 2 different African countries, Cam-
eroon and Gabon, are constrained in having a limited and
uniquely conserved DBL-g repertoire. It is conceivable that dis-
tinct sets of DBL-g sequences, such as those described here, are
specifically induced and proliferate during placental sequestra-
tion. Because these are similar sequences with 39%–55% aa iden-
tities, these domains can still retain sufficient structural homology
to function as CSA-binding cytoadherence molecules.
Analysis of genomic DBL-g sequences in placental and pe-
ripheral isolates. To determine the array and frequency of
DBL-g–specific sequences in the natural parasite population in
Gabon, their presence in the genome of P. falciparum placental,
as well as peripheral, parasites was further investigated. A com-
parative analysis of a total of 57 sequences taken from parasites
from the placentas of 4 women and from the peripheral blood
of 5 children indicated that DBL-g sequences were found in every
tested parasite sample, with a frequency ranging from 4 to 9
different sequences, independently of the source of the isolate
(figure 2). However, the possibility cannot be entirely ruled out
that additional DBL-g sequences with greater diversity could also
be identified through the use of primer sets distinct from the
ones selected here. Of interest, all samples, with the exception
of parasites taken from one child, harbored at least 1 of 5 DBL-
g sequences previously isolated from infected placentas from
Cameroonian subjects and characterized for CSA binding. The

480 • JID 2003:187 (1 February) • Khattab et al.


Figure 3. Phylogenetic analysis of unique Duffy binding–like (DBL)–g
sequences from placental and peripheral isolates found in the natural
population of Gabon. Alignment was made with the CLUSTAL program
(available at http://inn-prot.weizmann.ac.il/software/ClustalX.html), and
the tree was created by the CLUSTREE program (part of the CLUSTAL
suite). Bootstrap values (1000 resamplings) are shown along the branch
lines. Sequences are labeled as in figure 2. Sequences that show sig-
nificant identities to previously published DBL-g domains are boxed, as
are those of the laboratory-selected clones.
finding of DBL-g–specific sequences in var genes from peripheral
parasites would also explain why some peripheral P. falciparum
isolates have been reported to bind CSA [17]. The presence of
DBL-g sequences in the natural population with the potential
to bind CSA ensures that these tightly controlled varDBL-g genes
do not get lost in the population in the absence of a selection
pressure, namely the placenta.
A phylogenetic analysis of the 57 DBL-g domains together
with the 2 DBL-g sequences of the laboratory strains [3, 4]
was made (figure 3). The comparison showed that sequences
with highest homologies to some of the Cameroonian DBL-g
sequences (701/485 and 720/734) were found in one cluster
(boxed data), whereas others with homologies with sequences
498 and 732 were found in another cluster (boxed data).
Common DBL-g Subtypes in P. falciparum • JID 2003:187 (1 February) • 481
DISCUSSION
In the present study, we have carried out a further analysis of
placental P. falciparum parasites that have been shown elsewhere
[5] to express distinct subtypes of DBL-g domains that were
capable of binding the glycosaminoglycan CSA. In considering
the likelihood that var gene conservation extends beyond the
DBL-g domains, we analyzed the 5
extensions of 2 expressed
var genes of the parasites 720 and 734. The most striking result
was the finding that the entire 4 kb of their deduced amino
acid sequences were 96% indistinguishable from each other.
Thus, contrary to the belief that var genes are extensively di-
verse, evidence is provided in the present study that some
varPAM genes expressed by placental parasites isolated from
different individuals are unique to the extent that they are
almost totally conserved in their amino acid sequences.
Using the previously described RT-PCR methodology, we
analyzed more closely the DBL-g domains of placental parasites
from a different malaria-endemic area. The samples from the
first study were collected in Yaounde in Cameroon, whereas
those of the second study originated from Lambaréné in Gabon,
and the time lapse between sample collections was 4 years. The
novel and significant result of our var gene analysis is the dem-
onstration of a limited number of distinct highly homologous
varDBL-g sequences that are commonly expressed in parasites
from geographically separated study areas and were collected
at different time intervals.
Most interesting, the 720/734 pair of identical varPAM genes
is ∼98% homologous at the amino acid level to a 3D7 chro-
mosome 5 var gene (3D7chr5var). The 3D7 DBL-g domain was
expressed in a heterologous expression system and has been
shown to bind CSA [10]. Furthermore, this 3D7varCSA gene
has been shown to be expressed by many placental isolates from
Malawi [18], despite the fact that the N-terminal half of the gene
(DBL-1a–DBL-4e) exhibits low homology to the FCR3varCSA
gene, which has been implicated to have a role in placental ma-
laria. This allows us to conclude that 3D7chr5var-like (and by
extension, also 720/734-like) sequences belong to a pool of com-
monly expressed DBL-g varPAM genes set among placental iso-
lates capable of infecting pregnant women. It is also noteworthy
that other workers, using a different degenerate set of primers,
were able to detect the Cameroonian-type of varDBL-g sequences
from placental isolates from Kenya [19]. Even though these se-
quences have an overall amino acid sequence homology only on
the order of 39%–55%, it is very likely that this unique set of
sequences is structurally and conformationally conserved in na-
ture, which is crucial for determining an interaction with CSA.
In fact, the earlier study showed that these DBL-g domains that
were expressed on the surface of COS-7L cells and capable of
binding CSA were predicted to exhibit specific structural features,
such as conserved a-helical stretches. Such conservation of the
CSA-binding ligand can clearly explain how antibodies from
multigravid women can block the binding to CSA of placental
parasites collected from different continents [8] and also how
serum samples from multigravid women can react with antigens
on the surface of CSA-binding parasites, whereas serum samples
from adult men cannot.
The next logical question to ask is how CSA-binding var genes
escape frequent recombination and rearrangement events within
the parasite, processes that form the basis of clonal antigenic
variation of var genes known so far. The mechanism controlling
the regulation of expression of varCSA genes recently has been
shown to involve a unique 1.8-kb 5
varCSA element. The hy-
pothesis was advanced that the FCR3varCSA gene implicated in
PAM has a subtelomeric location in the chromosome and is less
likely to undergo recombination and rearrangement, unlike many
other centrally located var genes. Besides gene location, orien-
tation also appeared to play a role, because the FCR3varCSA
gene was found to be transcribed in the opposite sense from the
more “common” var genes [10]. Moreover, the same unique,
single-copy regulatory element upstream of the initiation codon
was also present in the 3D7varCSA gene as well as in var genes
of placental isolates [10].
It is known that the var-encoded PfEMP-1 is surface exposed
and undergoes clonal variation as a result of the stress exerted
on the parasite by the host immune effector mechanism. In
view of the fact that the majority of the peripheral parasites
bind to CD36 but not CSA, it follows that all CSA-binding
variants are rapidly eliminated by the spleen. As a consequence,
the parasites in the natural population that express the CSA
variant are hardly exposed to the immune system, except when
they infect pregnant women and accumulate in the placenta.
Therefore, CSA-binding var genes, in being under less immune
pressure than the CD36-binding var genes, are not as variable
and can afford to be unique, not only in their conservation
pattern but also in their regulatory elements. Gamain et al. [20]
proposed a model that both binding domains are present on
the same var gene and are mutually exclusive of each other—
that is, a CSA-binding domain on a CD36-binding PfEMP-1
var gene is structurally excluded from recognition by the im-
mune system and vice versa.
From our comparative analysis of genomic DBL-g sequences
from peripheral and placental parasites, it appears that periph-
eral parasites can be selected to express a specific var gene
capable of binding to placentas and cause disease. Such a subset
of varPAM genes expressing a limited but distinct repertoire
of DBL-g domains would be a critical property for the selection
process in the placenta and, consequently, successful seques-
tration and infection of the organ. It is now necessary to per-
form a structural analysis of this common conserved varDBL-
g/CSA complex, to understand how these molecules interact
with each other and to see which amino acid residues are crucial
482 • JID 2003:187 (1 February) • Khattab et al.
for the interaction, as first steps in the design and development
of a vaccine against placental infection in pregnant women.
Acknowledgments
We are grateful to the patients of the Albert Schweitzer Hos-
pital for their cooperation. We thank the scientists and funding
agencies comprising the international Malaria Genome Project
for making sequence data from the genome of Plasmodium
falciparum (3D7) public before publication of the completed
sequence. The Sanger Centre (United Kingdom) provided se-
quence for chromosomes 1, 3–9, and 13, with financial support
from the Wellcome Trust. A consortium composed of the In-
stitute for Genome Research, along with the US Naval Medical
Research Center, sequenced chromosomes 2, 10, 11, and 14,
with support from National Institutes of Allergy and Infectious
Diseases/National Institutes of Health, the Burroughs Wellcome
Fund, and the Department of Defense. The Stanford Genome
Technology Center sequenced chromosome 12, with support
from the Burroughs Wellcome Fund. The Plasmodium Genome
Database is a collaborative effort of investigators at the Uni-
versity of Pennsylvania and Monash University and is sup-
ported by the Burroughs Wellcome Fund
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