Am. J. Trop. Med. Hyg., 76(2), 2007, pp. 251–259
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
FATAL MALARIA INFECTION IN TRAVELERS: NOVEL IMMUNOHISTOCHEMICAL
ASSAYS FOR THE DETECTION OF PLASMODIUM FALCIPARUM IN TISSUES AND
IMPLICATIONS FOR PATHOGENESIS
GILLIAN L. GENRICH, JEANNETTE GUARNER, CHRISTOPHER D. PADDOCK, WUN-JU SHIEH,
PATRICIA W. GREER, JOHN W. BARNWELL, AND SHERIF R. ZAKI*
Infectious Disease Pathology and Malaria Branches, Centers for Disease Control and Prevention, Atlanta, Georgia
Abstract. Plasmodium falciparum is a significant cause of morbidity and mortality in travelers to areas where the
parasite is endemic. Non-specific clinical manifestations may result in failure to recognize malaria until autopsy, when
it is often too late to obtain whole blood for microscopic evaluation. The use of immunohistochemical (IHC) assays in
the detection of three P. falciparum antigens, histidine rich protein-2 (HRP-2), aldolase, and Plasmodium lactate
dehydrogenase (pLDH), was evaluated in formalin-fixed paraffin-embedded autopsy tissues from five travelers to
malaria-endemic areas, whose deaths were initially suspected to have been caused by other bacterial or viral hemorrhagic fevers. The HRP-2 assay was specific for P. falciparum, whereas the aldolase and pLDH assays also reacted with
P. vivax. Immunostaining patterns were predominately cytoplasmic and membranous. P. falciparum antigens were
detected in a variety of organs but were most abundant in the blood vessels of brain, heart, and lung tissues.
been characterized. These proteins are currently used in
whole blood rapid detection kits for non-microscopic malaria
diagnosis with high sensitivities and specificities.13 To our
knowledge, immunohistochemical (IHC) detection of these
proteins has not been previously described in the diagnosis of
P. falciparum infection. The following report describes the
development of novel IHC assays targeting P. falciparum histidine rich protein-2 (HRP-2), Plasmodium aldolase, and
Plasmodium lactate dehydrogenase (pLDH). The IHC assays
were used to diagnose and study the pathogenesis of P. falciparum in tissues from five travelers whose deaths were initially suspected to have been caused by other bacterial or viral
hemorrhagic fevers.
INTRODUCTION
The intra-erythrocytic protozoan Plasmodium falciparum is
the etiologic agent of severe malaria, a systemic illness characterized by acute respiratory distress syndrome, renal insufficiency, and central nervous system involvement that can be
confused clinically with viral or bacterial hemorrhagic fevers.1,2 P. falciparum causes the majority of the 300–500 million acute infections and 1–3 million deaths from malaria
worldwide each year.3 Ninety percent of global malaria mortality occurs in sub-Saharan Africa, where P. falciparum and
its primary mosquito vector, Anopheles gambiae, predominates3; however, international travel permits the importation
of P. falciparum into countries where the parasite is not endemic. Of the 25–30 million travelers who visit malariaendemic countries annually, ∼10,000 return with malaria, often caused by P. falciparum.4,5 In the United States, most
cases of imported malaria are caused by failure to take appropriate chemoprophylaxis.6
The lack of clinical suspicion in non-endemic countries may
result in the underdiagnosis and omission of malaria from a
differential diagnosis that includes other viral and bacterial
hemorrhagic fevers.7–9 A review of malaria deaths among US
travelers from 1963 through 2001 found that diagnostic delays
were common among fatal cases.10 When untreated, P. falciparum infection can progress rapidly to severe malaria, often
leading to coma and death.2 Case fatality ratios for imported
malaria range from 1% to 3.6% in industrialized countries.4
Traditional diagnostic techniques, such as the Giemsastained blood smear, rely on the detection of parasites by
trained microscopists; however, blood samples are not often
available for travelers when diagnosis of malaria is not considered before death.11 Other diagnostic techniques, such as
the routine hematoxylin-eosin (H&E) stain, also have limited
use in the diagnosis of fatal malaria because P. falciparum
parasites are difficult to identify with certainty in tissue
samples.12
Several genus- and species-specific proteins produced by P.
falciparum parasites during intra-erythrocytic growth have
MATERIALS AND METHODS
Patient samples. Formalin-fixed autopsy tissue samples
from five cases of suspect hemorrhagic fevers were submitted
to the Centers for Disease Control and Prevention (CDC)
between 1996 and 2005 for diagnostic consultation. The patients had a recent travel history to malaria-endemic regions.
Histopathology was reviewed by routine H&E stain. At the
time of submission, the primary clinical diagnoses made by
submitters, which included Ebola, Lassa fever, dengue, and
leptospirosis, were excluded by IHC assays using a modified
immunoalkaline phosphatase technique.14–17 Representative
tissues from each of the five cases were tested and evaluated
with P. falciparum IHC assays using three antibodies (HRP-2,
aldolase, and pLDH). Interpretation of the IHCs included
identification of antigen localization in the different tissues
and intra-erythrocytic immunostaining patterns.
Plasmodium spp. controls. Plasmodium ovale and Plasmodium vivax were grown in vivo in Aotus and Saimiri sp. monkeys and P. falciparum was cultured in vitro according to the
methods established by Trager and Jensen.18 Infected erythrocytes and uninfected erythrocytes (negative controls) were
fixed in formalin and embedded in paraffin for IHC testing.
Immunohistochemical assays. Formalin-fixed paraffinembedded tissues were cut at 3 ␮m, deparaffinized in xylene,
and rehydrated in a graded alcohol series. Hemozoin pigment
was removed by soaking tissue sections in alcoholic picric acid
solution (Newcomber, Middleton, WI) overnight.19 To ex-
* Address correspondence to Sherif R. Zaki, CDC/IDPA, 1600 Clifton Road, MSG-32, Atlanta, GA 30333. E-mail: [email protected]
251
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GENRICH AND OTHERS
pose malarial antigens, sections were subsequently pretreated
in one of two ways: 1) antigen retrieval was performed by
incubating sections submerged in Antigen Retrieval Citra Solution (BioGenex, San Ramon, CA) at 98°C in a steamer for
10 minutes or 2) sections were digested in 0.1 mg/mL proteinase K (Boehringer Mannheim, Indianapolis, IN) solution for
15 minutes. Sections were blocked with 20% normal sheep
serum in Tris-saline-Triton (NSS/TST) and incubated with
monoclonal antibodies against malaria proteins (below) for
60 minutes on the Dako Autostainer (DakoCytomation,
Carpinteria, CA). Detection of the bound antibody was performed by using the LSAB2 universal alkaline phosphatase
system (DakoCytomation) that uses 15-minute sequential incubations at room temperature in a secondary biotinylated
anti-mouse/rabbit link antibody, followed by alkaline phosphatase–conjugated streptavidin, and then in a 0.2 mg/mL
naphthol phosphatase-fast red chromogen reagent. Slides
were rinsed, counterstained for 3 minutes in Mayer hematoxylin (Biomeda, Foster City, CA), and mounted with aqueous mounting medium.
Monoclonal P. falciparum antibodies. Optimal working dilutions for antibodies were determined by testing P. falciparum–
infected red blood cells (RBCs) with an IgG1, ␬ anti-HRP-2
antibody (cat. no. C03400M; dilution 1:1,000; Biodesign, Saco,
ME), an IgG1 anti-aldolase antibody (dilution 1:50; donated
by Dr. Norm Moore, Binax, Scarborough, ME), and an IgG1
anti-pLDH antibody (dilution 1:200; donated by Dr. Michael
Makler, Flow, Portland, OR). Negative controls for each slide
consisted of sequential tissue sections incubated with normal
mouse serum and an irrelevant, but isotype-identical primary
antibody. The specificity of the primary antibodies was evaluated by testing cross-reactivity with other Plasmodium species
(P. ovale, P. vivax) and human tissues infected with Babesia
microti, Toxoplasma gondii, Trypanosoma cruzi, Rickettsia
rickettsii, Leptospira species, and dengue, Ebola, Lassa, and
lymphocytic choriomeningitis (LCM) viruses.
RESULTS
Clinical and epidemiologic findings. Clinical and epidemiologic data for the five patients are summarized in Table 1. All
five men traveled to Africa, South America, or the Caribbean
region where P. falciparum is endemic20 and had not taken
malaria chemoprophylaxis during their travel. Cases 1 and 2
were Romanian shipmates who made port in South Africa
and several West African cities before becoming sick and
dying in Conakry, Guinea. It was feared that the men had
acquired Ebola in Libreville, Gabon, 1 week earlier because
the stop there coincided with an Ebola outbreak occurring
northeast of the capital. Their ship was quarantined off the
coast of Guinea until a definitive diagnosis could be made. In
2004, CDC received Case 3, a 36-year-old second mate from
Rhode Island who had made port calls in Freetown, Sierra
Leone; Abidjan, Ivory Coast; and Lagos, Nigeria. Ten days
after leaving Nigeria, Case 3 experienced headache, fever,
chills, myalgia, and bloody urine, and died 1 week after onset
of symptoms, before his ship could reach Galveston, TX.
Weeks before this man’s death, a fatal case of Lassa fever was
confirmed in a New Jersey man with a similar travel history.17
As a result, the ship underwent voluntary quarantine, anchored in the Houston channel off the coast of Galveston
until autopsy tissues could be examined and Lassa fever could
be ruled out. Case 4 was a 32-year-old reporter, native to
Guyana, who had traveled to the city of Demerara to report
on flooding in the area. After complaining of headache and
recurrent fever with diarrhea and vomiting, Case 4 deteriorated into hepatorenal failure and coma and died. Because of
an ongoing outbreak of leptospirosis, local health officials
suspected that this patient had acquired leptospirosis during
his assignment.
Case 5, a 43-year-old man from Virginia, traveled to the
Central African Republic and the Ivory Coast during a 17-day
mission trip in December 2005. On his return, he complained
TABLE 1
Epidemiologic and clinical information on five cases sent to the Centers for Disease Control and Prevention (CDC), 1996–2005
Case
no.
Year
submitted
1, 2
Travel history
Clinical and epidemiologic information
1996
South Africa, Gabon,
and Guinea
3
2004
Sierra Leone, Ivory
Coast, and Nigeria
4
2005
Guyana (western
region)
5
2005
Central African
Republic, Ivory
Coast
Ages unknown; male shipmates from
Romania. Limited clinical information
available, but concomitant Ebola outbreak
in north Gabon, 400 km from Libreville.
36-year-old man; shipmate from the United
States. Headache, fever (> 103°F), chills,
myalgia, and sore throat, nausea, vomiting,
jaundice, pain, “bloody” and “rust-tinged”
urine. Malaria chemoprophylaxis was found
among his belongings, but it was not thought
to have been taken.
32-year-old man; reporter from Guyana.
Headache and recurrent fever with diarrhea
and vomiting, hepatorenal failure, coma.
Reported on flooding in western Guyana,
where an outbreak of leptospirosis occurred.
No malaria chemoprophylaxis was taken.
43-year-old man; missionary from the United
States. Unproductive cough, fever and chills,
muscle and joint aches, dark urine. Malaria
chemoprophylaxis was not taken during the
2-week trip to Africa.
Disease(s) initially
suspected by history and
clinical manifestations
IHC testing
Ebola
Dengue virus, Ebola virus,
Lassa virus P.
falciparum
Lassa fever
Dengue virus, Ebola virus,
Lassa virus, Leptospira
spp., Rickettsia rickettsii,
Yellow fever virus, P.
falciparum
Leptospirosis
Leptospira spp., P.
falciparum
Yellow fever, dengue
Yellow fever virus, dengue
virus, P. falciparum
FATAL MALARIA INFECTION IN TRAVELERS
253
FIGURE 1. P. falciparum HRP-2 assay applied to positive and negative control cells. A, Immunostaining of P. falciparum-infected erythrocytes
(pRBCs), (parasitemia 2.8%; original magnification ×100). Immunostaining pattern of positive control cells was similar with aldolase and pLDH
assays. B, P. ovale infected erythrocytes, (parasitemia 1%; original magnification ×100).
of fever, chills, dizziness, joint aches, and dark urine and died
10 days later. Clinicians suspected dengue and yellow fever
because outbreaks of the viruses were occurring in the areas
visited.
Assessment of P. falciparum IHC assays. The HRP-2 antibody was specific for P. falciparum (Figure 1), whereas the
aldolase and pLDH antibodies reacted with both P. falciparum– and P. vivax–infected erythrocytes (pRBCs). Depigmentation dramatically improved visualization of parasites in
H&E-stained sections and increased antigen exposure in IHC
assays; therefore, depigmentation was routinely performed on
tissues (Figure 2A–D). Other IHC pretreatment conditions
FIGURE 2. Immunohistochemical development techniques. A–D, Depigmentation removes hemozoin pigment in P. falciparum-infected spleen
sections (Case 4). A, Hemozoin pigment (arrow) in a routine H&E stain of spleen section without depigmentation (original magnification ×100)
B, HRP 2 immunohistochemical assay on P. falciparum-infected spleen section without depigmentation; diffuse and abundant hemozoin pigment
(original magnification ×50). C, Routine H&E with depigmentation (original magnification ×100). D, HRP-2 immunohistochemical assay on
spleen section after overnight depigmentation (original magnification ×50). E–H, P. falciparum HRP-2 immunohistochemical assay using different
pretreatment, based upon duration of formalin-fixation (Case 1). E, Lung vessel of tissue sample fixed in formalin for 1 month using proteinase
K and F, antigen retrieval (original magnification ×50). G, Liver vessels of tissue stored for 9 years treated with proteinase K and H, antigen
retrieval (original magnification ×50).
254
GENRICH AND OTHERS
were determined for each antibody on a case by case basis;
overall, antigen retrieval was optimal in exposing parasite
antigens on tissues with longer formalin fixation times (Figure
2E–H). Proteinase K could be used in the HRP-2 IHC assay
on tissues that were stored in formalin from days to several
weeks.
Histopathology and IHC detection of P. falciparum antigens. Heart, lung, and liver tissues were available for all
patients; other tissues (no. of patients) included spleen (4),
kidney (3), and brain (2). The following tissues were
available from only one patient: tongue, trachea, thyroid,
adrenal gland, gall bladder, and testes. Viral and/or bacterial
hemorrhagic fever pathogens suspected at death or autopsy
were ruled out by IHC on tissues from the five cases (Table 1).
The histopathologic features of infection were similar in all
cases. No significant inflammation was observed in liver, kidney, and spleen. Abundant hemozoin pigment was observed
as small black discrete granules within erythrocytes or as diffuse deposits throughout the parenchyma (Figure 2A). Parasitized erythrocytes (pRBCs) generally contained one distinct
hemozoin granule or were heme-depleted (ghost erythrocytes). Kupffer cells and other tissue macrophages also contained fine brown hemozoin pigment (Figure 3A).
After depigmentation, parasites were evident inside RBCs
in systemic blood vessels. Blood vessels were generally
packed with pRBCs and non-pRBCs. Congestion in spleen,
liver, and kidney sections was common (Figure 3A, C, and E),
and erythrophagocytosis by macrophages was also observed
in these tissues. In kidney sections, there were varying degrees of cast formation in tubules and glomerular edema (Figure 3E). In the lungs, intra-alveolar edema and hyaline membranes composed of fibrin and proteinaceous material were
observed (Figure 4A and B), typical of lung injury and early
diffuse alveolar damage. In all cases where tissue was available, aggregation of pRBCs was most prominent in the capillaries of heart and brain (Figure 4C–F).
In general, HRP-2 immunostaining was greatest in representative tissues, followed by aldolase and pLDH. All representative tissues from study cases were positive by HRP-2 and
aldolase assays; the pLDH assay, however, did not produce
immunostaining in all tissues from Case 2 and in the renal
tissues of Case 3—perhaps because of severe autolysis.
HRP-2 immunostaining revealed pRBCs in hepatic and
splenic sinusoids (Figure 3B and D); immunostaining was
abundant in the vasculature of lung, heart, and central nervous system tissues (Figure 4B, D, and F). Kupffer cells and
other tissue macrophages also showed granular antigen staining. In spleen sections, there was immunostaining of discrete
extracellular “dots,” representing parasites (Figure 3D). In
the RBCs, there were two predominate immunostaining patterns: punctate and specific for the intraerythrocytic parasite
or membranous (Figure 4D). Staining of the intraerythrocytic
parasite was observed with or without erythrocyte membrane
staining, and peripheral membrane staining highlighted hemoglobin-depleted erythrocytes. HRP-2 and aldolase antigens were detected in the endothelium of various organs, as
well as in renal tubular epithelium and casts (Figure 3F and
G), whereas the pLDH immunostaining pattern was predominately cytoplasmic and specific for intra-erythrocytic parasites
(Figure 4D).
DISCUSSION
In this study, three novel IHC assays targeting HRP-2, aldolase, and pLDH were developed and confirmed severe P.
falciparum infection in five travelers whose death was initially
suspected to be caused by other infectious agents of hemorrhagic fevers. Malaria is the most common cause of fever in
travelers returning to industrialized countries from malariaendemic countries.21 However, the clinical features of disease
are non-specific, and in countries where P. falciparum is not
endemic, fatal malaria is often not suspected.22,23 The pathologic features of malaria resemble many other viral, rickettsial, and bacterial infections2,24; as a result, an unequivocal
diagnosis can be made only by laboratory testing.
IHC assays and histopathologic review confirmed P. falciparum infection in study cases. As previously reported, abundant hemozoin pigment, a byproduct of parasite metabolism,2
was distributed diffusely throughout peripheral tissues and in
blood vessels in the central nervous system, findings consistent with reports of hemozoin localization.25 The density of
hemozoin increases in proportion to the duration of falciparum infection and decreases with appropriate therapy.25,26
In this context, the quantity of hemozoin deposits observed in
each of the study cases strongly suggested that none had
taken malaria chemoprophylaxis during their travel.
The hallmark of P. falciparum infection is sequestration,
characterized by adherence of mature stage falciparum
pRBCs (trophozoites and schizonts) to endothelial cells of
capillaries and venules.27 Studies in humans and animal models have described sequestration of trophozoites and schizonts
in a variety of tissues, including brain, heart, lung, skeletal
muscle, and subcutaneous tissue.2,28,29 The ability of mature
stage falciparum-infected erythrocytes to evade circulation in
the bloodstream30,31 underscores the use of tissue-based diagnosis such as IHC, in fatal cases. As a result of sequestration, peripheral blood parasitemia, traditionally evaluated using a Giemsa-stained blood smear, may not necessarily correlate with the severity of clinical illness; thus, the severity of
infection may be underestimated.32,33
Sequestration in study case tissues, as evidenced by IHC
and histopathologic findings in this report, correlated with the
clinical manifestations of multiple organ failure, which is a
common feature of severe malaria and is associated with high
mortalities.34,35 P. falciparum infection causes respiratory
symptoms that resemble influenza-like illness; in correlation,
the study cases described in this study had pulmonary edema
with intra-alveolar hyaline membranes and proteinaceous debris associated with malarial antigens. Pulmonary edema is
associated with high parasitemias and often leads to acute
respiratory distress syndrome.36,37
Rust-tinged urine, described for several of the patients in
this study, is associated with hyperbilirubinemia caused by
erythrocyte destruction.38 In this study, malarial antigens
were detected in tubular epithelial cells in association with
erythrocyte casts. Progression to renal failure is reported in
30% of patients who are admitted to intensive care units with
falciparum malaria.35 The pathologic findings in the kidneys
of study cases, including congestion and cast formation in
renal tubules, are an important feature of this illness.2,39
Other studies have shown that renal mesangial and proliferative changes, in addition to tubular necrosis, are also associated with P. falciparum infection. Extracellular “dots” of
FATAL MALARIA INFECTION IN TRAVELERS
255
FIGURE 3. Pathologic findings in liver, spleen and kidney (Case 3). A, Hemozoin deposits in Kupffer cells and congestion of sinusoids with
erythrocytes (original magnification ×100). B, P. falciparum HRP-2 IHC assay showing pRBCs in liver vessel and sinusoids (original magnification
×158). C, Congestion of red pulp, decreased white pulp content (H&E, original magnification ×158). D, Abundant HRP-2 immunostaining in
pRBCs, and process of pitting (arrow; original magnification ×158). E, Cast formation in renal tubules (H&E, original magnification ×158). F, P,
falciparum HRP-2 immunostaining throughout renal tubule (original magnification ×158). G, P. falciparum aldolase immunostaining in renal
tubular epithelium (original magnification ×100).
parasites were also observed in spleen sections, showing the
process of pitting, or removal of the parasite from the RBC
(Figure 3D).40 The engorgement of CNS vessels and capillaries with pRBCs and non-pRBCs, which was seen in two study
cases for which tissue was available, is characteristic of severe
malaria and is associated with neurologic impairment.30,41
Histopathologic review was strongly suggestive of P. falciparum infection; however, the IHC assays developed in this
report were essential for definitive diagnosis. In severe malaria infections, such as the study cases presented here, the
accumulation of hemozoin pigment in tissues often obscures
malarial antigens present in erythrocytes. On optimizing the
IHC assays, we found that hemozoin pigment obscured antigens present in erythrocytes and regularly performed the depigmentation step after deparaffinization using an established
method.19 Pretreatment conditions also took into account the
cross-linking property of formalin fixation.42 In general, antigen retrieval pretreatment was more effective in improving
immunostaining than proteinase K; this was especially true
for archival tissues stored in formalin for > 1 month.
The HRP-2 antibody used here was specific for P. falci-
parum, whereas the aldolase and pLDH antibodies reacted
with both P. falciparum and P. vivax. Immunostaining patterns were consistent with what is known about the proteins
from previous studies. HRP-2 immunostaining was observed
within pRBCs, on the pRBC cell membrane, and in endothelial cells. HRP-2 is a 30-kd water-soluble protein localized in
the parasite food vacuole and on the erythrocyte membrane,
exclusively produced by P. falciparum.43–45 Because HRP-2 is
eventually excreted in blood and accumulates in correlation
with disease severity,46 it has been targeted for rapid detection tests, such as ParaSightF.47,48 Immunostaining with P.
falciparum aldolase, a 41-kd tetrameric polypeptide49 and a
key glycolytic enzyme in parasite metabolism, was similar.
Intra-erythrocytic immunostaining is supported by studies of
recombinant P. falciparum aldolase, which suggest that the
enzyme associates with the parasite or host cytoskeleton.50
Aldolase is also detected in the blood and it is another target
in rapid tests like the Now Malaria Test.13,51 HRP-2 and aldolase immunostaining in renal tubules provides further evidence that the proteins are excreted in the urine. Previously,
antigenic proteins ranging from 29,000 to 224,000 Mrs were
256
GENRICH AND OTHERS
FIGURE 4. Pathologic findings in lung, heart and CNS of study cases. A, Intraalveolar edema and intraalveolar macrophages with fine brown
hemozoin pigment (Case 4, H&E, original magnification ×50). B, P. falciparum HRP-2 immunostaining, showing hyaline membranes (arrow) and
pRBCs in vessel (Case 1, original magnification ×100). C, P. falciparum HRP-2 immunostaining throughout cardiac vessels (Case 4; original
magnification ×100). D, pLDH immunostaining in heart vessel; note discrete parasite (arrow) and membraneous (arrowhead) immunostaining of
pRBCs (Case 5, original magnification ×158). E, pRBCs throughout CNS vessel in the absence of inflammation (Case 3, H&E, original
magnification ×153). F, P. falciparum HRP-2 immunohistochemical staining of pRBCs in vessels (Case 3, original magnification ×50; inset, ×158).
isolated in urine samples from adults with P. falciparum infection.52 Furthermore, Genton and others53 showed that
urine samples were adequate specimens for the diagnosis of
P. falciparum using the ParaSight-F, which is usually applied
only to whole blood.
pLDH is another soluble glycolytic enzyme, targeted in the
OptiMal rapid test. Compared with the HRP-2 and aldolase
assays, pLDH immunostaining was less abundant, limited to
discrete staining of the parasite. The difference in relative
abundance may be caused by a rate of clearance that is higher
for pLDH antigens.54 Overall, the most abundant immunostaining was observed using the HRP-2 assay, which has been
shown to clear more slowly from the blood; HRP-2 antigens
may persist for up to 2 weeks even after malaria is cured in
the patient.54,55
The IHC assays described here provide further insight of
reticuloendothelial interactions through the visualization of
pRBCs sequestered in blood vessels and of parasite antigens
localized in endothelial cells. Of particular interest in this
study was the immunostaining of HRP-2 and aldolase antigens in endothelial cells. The mechanisms of endothelial cell
adhesion that allow falciparum parasites to sequester in blood
vessels have been studied extensively. Falciparum-infected
erythrocytes produce erythrocyte membrane protein 1
(PfEMP-1), which binds to endothelial cell receptors intercellular adhesion molecule 1 (ICAM-1),56 vascular cell adhesion
molecule 1 (VECAM-1), platelet/endothelial cell adhesion
molecule 1 (PECAM-1), P-selectin, E-selectin, CD36,57
thrombospondin, and chondroitin sulfate-A, constitutively
expressed on vessel linings.58 Subsequent to adhesion, the
release of soluble parasite proteins and their accumulation in
vasculature may cause cytokine recruitment and endothelial
cell destruction.59 In the CNS, it has been hypothesized that
activation of cerebral endothelial cells causes a disruption of
the blood–brain barrier that could result in the penetration of
brain parenchyma by parasite proteins.60
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FATAL MALARIA INFECTION IN TRAVELERS
As described above, vascular endothelial cell interactions
in peripheral and central nervous system tissues are crucial to
the survival of P. falciparum and contribute to the virulence
of infection. The deposition of fibrin and P. falciparum IgG
and IgM immune complexes along the walls of cerebral vessels has been reported.41,61 Furthermore, ultrastructural studies have found that areas of P. falciparum sequestration are
associated with endothelial cell edema and narrowing of the
capillary lumen.62 The immunostaining of HRP-2 in endothelial cells suggests the protein plays a role in the extracellular
environment, perhaps in cytokine recruitment. In vitro studies
have shown that when the parasitized erythrocyte has reached
its final stage of maturation, the erythrocyte bursts and releases ∼90% of the HRP-2 content.63 In vitro culture and
affinity chromatography studies describe how soluble parasite
antigens from culture supernatants activate neutrophils and
monocytes, resulting in the production of oxygen radicals.64
Both aldolase and HRP-2 are soluble parasite proteins, and
their association with endothelial cells seen in IHC assays
suggests that they play a role in the cytokine response described above; however, endothelial cell interactions with
HRP-2 and aldolase remain to be explored.
While the majority of travelers are aware of the risk of
acquiring malaria during international travel, only ∼50%
comply with an appropriate chemoprophylaxis regimen.65 In
a recent example, five siblings from Chicago, IL, were diagnosed with P. falciparum on their return from Nigeria; three
of the cases were severe, requiring admission to an intensive
care unit.66 The family assumed incorrectly that malaria drugs
were to be taken for treatment only and did not realize that
the drugs were to be used for chemoprophylaxis. Nonetheless,
the timely exclusion of malaria from a differential diagnosis
that includes other viral and hemorrhagic fevers is critical to
infection control. The quarantine of the crew aboard the ships
on which Cases 1, 2, and 4 traveled exemplifies the public
health implications for timely diagnosis and rule-out of common etiologies such as malaria among exotic diseases like
Ebola that are transmissible from person-to-person. The incidence of other travel-associated hemorrhagic fevers continues (imported yellow fever, typhus, Lassa fever, etc.), and
IHC assays described in this report can provide important
information with broad implications for public health. When
the first clues of malaria infection are found at autopsy, the P.
falciparum HRP-2, aldolase, and pLDH IHC assays developed in this report may definitively confirm P. falciparum and
provide insights into the pathogenesis of malaria.
Received July 31, 2006. Accepted for publication August 16, 2006.
Acknowledgments: We thank Dr. Makler, Flow Inc., and Dr. Norm
Moore, Binax Inc., for kindly donating samples of antibodies for use
in this study. We also thank Mitesh Patel for assistance with receiving
and in-processing all tissue specimens.
Financial support: This research was supported in part by an Emerging Infectious Diseases Fellowship from the Association of Public
Health Laboratories and by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention,
National Center for Infectious Diseases, Infectious Disease Pathology Activity, by the Oak Ridge Institute for Science and Education
through an interagency agreement between the US Department of
Energy and the CDC. The findings and conclusions in this report are
those of the authors and do not necessarily represent the views of the
funding agencies.
Authors’ addresses: Gillian L. Genrich, Jeannette Guarner, Christopher Paddock, Wun-Ju Shieh, Patricia Greer, and Sherif R. Zaki,
CDC/NCID/IDPA, 1600 Clifton Rd., MS-G32, Atlanta, GA 303294018, Telephone: 404-639-3889, Fax: 404-639-3043. John W. Barnwell,
CDC/NCID/Malaria Branch, 4770 Buford Hwy., MS-F36, Bldg 109,
Chamblee, GA 30341-3717, Telephone: 770-488-4528, Fax: 770-4884253.
Reprint requests: Sherif R. Zaki, CDC/NCID/IDPA, 1600 Clifton
Rd., MS-G32, Atlanta, GA 30329-4018.
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