Introduction
We report the unique case of a 19-year-old nonimmune patient with Plasmodium vivax
monoinfection, confirmed by PCR in the peripheral blood and in the spleen section,
who was splenectomized due to spleen rupture two days prior to the diagnosis and treatment
of the malarial infection. Microscopic analyses evidenced white pulp expansion and
a diffuse hypercellularity in the splenic red pulp, with intense proliferating plasmablasts
in the subcapsular and perivascular compartments as well as large numbers of intact
P. vivax–infected reticulocytes in the cords, in the absence of other concomitant
infectious diseases. To our knowledge, this is the first full detailed immunohistopathological
characterization of a nontreated P. vivax–infected spleen.
Description of Case
A 19-year-old man was admitted at a secondary-care hospital in the Brazilian Amazon
with acute abdominal pain after jumping from a tree denying to have suffered any direct
abdominal trauma. A diagnosis of spleen rupture and intra-abdominal haemorrhage was
performed. He was submitted to splenectomy, during which a large spleen of 1,300 grams
with a capsule rupture in the colic surface was obtained. The patient lived in an
urban area of Manaus and denied previous episodes of malaria. For the last month before
the traumatic event he had been working in a building project in a rural area where
malaria transmission is endemic and referred that in the two days preceding surgery
he had been feeling feverish and complaining of mild headache and asthenia. After
surgery, he developed high fever, chills, and generalized myalgia. The initial diagnostic
investigation performed by the Surgery Department only revealed anemia (Hb 8.7 g/dL)
and thrombocytopenia (66,000 platelets/mm3). The patient was then transferred to a
tertiary-care infectious diseases reference center.
A thick blood smear disclosed P. vivax infection with a semiquantitative parasitemia
between 10.000 and 100.000 parasites/mm3. Real-time polymerase chain reaction (PCR)
from the peripheral blood confirmed P. vivax monoinfection. Treatment was initiated
with chloroquine (1,500 mg divided in 3 days) and primaquine (30 mg/day during 7 days).
Both fever and parasitemia were cleared within 48 hours. The patient was discharged
in the third day of treatment, and he did not develop any relapse nor had any other
complications through a 1-year follow-up.
The study was approved by the Institutional Review Board of the Fundação de Medicina
Tropical Heitor Vieira Dourado and the National Committee of Ethics in Science and
Technology (CONEP Process No.: 25.001.011.792/2009-15), and the patient consented
his case to be published.
How Frequent Are Splenomegaly and Spleen Rupture in Malaria Infection? Are P. vivax–Infected
Patients at Higher Risk of Complications?
Enlargement of the spleen is a well-known clinical feature of malaria, and it is estimated
to occur in 70%–80% of acute cases, with its size normalizing after successful treatment
[1]. In areas of intense transmission, the spleen is palpable in 50%–80% of individuals,
being directly associated with immunity acquisition, as shown by its higher prevalence
in children and correlation with both antibody levels and host genetics [2], [3].
More reliable and accurate diagnostics techniques have been used to measure malaria
transmission, but for a long time, especially during WHO's Malaria Eradication Campaign
in the 1950–60s, the proportion of palpable spleens in a given population (the spleen
rate) was used to the deployment of control efforts, highlighting its epidemiological
utility [4], [5].
Left-upper-quadrant abdominal pain in a patient with malaria should prompt the physician
to consider the diagnosis of splenic infarction or spleen rupture [6], [7]. The real
incidence of splenic complications is unknown as there is substantial underreporting
of mild, asymptomatic, and nonsevere cases, but some reports point that it can be
as high as 8.8% among patients with abdominal pain [8]. An extensive review of published
cases of splenic rupture demonstrated that it is most commonly observed during the
primary attack in nonimmune individuals [9]. Although there were no differences in
the clinical presentation related to the infecting species, it seems that P. vivax
leads to more marked spleen enlargement than infection with other species [9]. The
pathophysiological events leading to rupture are not completely understood, but it
seems that it is mainly a mechanical event related to the rapid enlargement of the
organ with preceding splenic infarction occurring in a minority of cases [9].
What Are the P. vivax–Induced Changes in the Spleen?
Histologically, hematoxylin-eosin (HE) staining revealed that, compared to a normal
spleen (Figure 1A), the most significant changes in the P. vivax–infected spleen were
a white pulp expansion and a diffuse hypercellularity in the splenic red pulp (Figure
1B). The periarteriolar lymphoid sheets of the white pulp were enlarged, and well-developed
secondary lymphoid follicles were easily found. A prominent infiltration by immunoblasts
and plasma cells was observed in the cords as well as a striking intrasinusoidal histiocytosis.
Although the histological characteristics of the spleen resembled a diagnosis of B-cell
lymphoma, it could be excluded after performing additional tests. A nested PCR performed
in the spleen sections demonstrated the presence of P. vivax and excluded coinfection
with P. falciparum (not shown). Infections by HIV, EBV, CMV, and HHV8 were not detected
through immunohistochemistry and in situ hybridization. Thus, P. vivax infection was
the only condition affecting the patient's spleen after extensive diagnostic investigation.
10.1371/journal.pntd.0001934.g001
Figure 1
Immunohistochemical staining of P. vivax–infected and normal spleen sections.
HE staining of the normal (A) and P. vivax–infected (B) spleen sections evidencing,
respectively, periarteriolar lymphoid cuffs and red pulp cords depleted of lymphoid
cells (inset 20× normal) and a prominent white pulp expansion with hyperplastic germinal
centers in secondary lymphoid follicles highlighting in the inset a striking red cord
infiltration by immunoblasts and reactive plasma cells (20× P. vivax–infected). (C)
The normal spleen reveals a scattered CD138 positive plasma cell distribution in the
subcapsular and perivascular compartments (2×); higher magnification view shown in
the inset (40×). (D) In contrast, a marked increase of CD138 positive plasma cells
is observed in the P. vivax–infected spleen (2×), including the detection of mitotic
activity among several CD138 positive plasma cells shown in the inset (40×). (E) Double
immunostaining with CD138 (brown) and Ki-67 (red) demonstrated the lack of proliferation
in plasma cells in the normal spleen (20×). (F) In contrast, there is a significant
increase in CD138 and Ki67 positive plasmablasts in the P. vivax–infected spleen (20×).
A series of cell markers was used to determine the effects of P. vivax infection in
this organ (Table S1). Noticeably, a mild follicular hyperplasia (CD20, CD10), mild
red cord hyperplasia (CD2, CD3, CD5, CD7), expansion of monocytes-macrophages (CD68),
and plasmablast expansion and proliferation (CD138, MUM-1, Ki 67) in subcapsular and
perivascular areas, as well as large expression of B-cell and antibody markers (IgM,
IgG, IgD, Lambda, and Kappa light chains) were observed when compared to sections
from the spleen of a normal individual who suffered a trauma-forcing splenectomy.
In contrast, none of the other markers used to identify T cells (Granzyme B, CD4,
CD8, CD57, FOXP3, and TCRbeta), dendritic cells (CD123), natural killer cells (CD56),
NK cells and histiocytes (CD16), endothelial cells (CD31 and CD34), myeloid and monocytic
cells (CD33), neutrophil granulocytes/monocytes (myeloperoxidase), normal erythroid
cells at all stages of differentiation (glycophorin A), or megakaryocytes (CD61) showed
a difference in location or expression of these receptors in the spleen from the P.
vivax patient as compared to the normal spleen (Table S1). Worth highlighting, as
compared to the normal spleen (Figure 1C), a marked increase in CD138 positive plasma
cells was observed in the P. vivax–infected spleen (Figure 1D). Moreover, only very
rare CD138 positive cells were in mitosis in the normal spleen (Figure 1C, inset),
whereas abundant mitotic figures were found in the P. vivax–infected spleen (Figure
1D, inset). A double immunostaining with CD138 and the Ki-67 proliferation marker
confirmed the plasmablastic expansion of double Ki-67 and CD138 positive cells in
the P. vivax–infected spleen in the subcapsular and perivascular compartments (Figure
1E and 1F).
Immunofluorescence assays of spleen sections were performed using antibodies raised
against conserved motifs of P. vivax VIR proteins, previously shown to specifically
recognize P. vivax–infected reticulocytes from human patients [10], to determine the
presence of parasites. Results demonstrated the presence of large numbers of parasites
in the red pulp (Figure 2A) and specificity was demonstrated using pre-immune sera
(Figure 2B). Noticeably, confocal images using anti–P. vivax VIR and anti-CD68 (a
marker of macrophages) antibodies revealed intact P. vivax–infected reticulocytes
characterized by dotted pattern of staining mostly outside macrophages (Figure 2C
and inset) and the presence of large amounts of parasite pigment as revealed by reflection
contrast (Figure 2D).
10.1371/journal.pntd.0001934.g002
Figure 2
Immunohistofluorescence images of P. vivax–infected spleen sections.
(A) Spleen section showing P. vivax parasites mostly in the cords of the red pulp
as detected by polyclonal antibodies against Vir proteins. (B) Negative control using
preimmune sera. Nuclei are shown in blue and the bright field image of the tissue
in gray. Scale bar: 20 µm. (C) Double staining showing CD68 macrophages in red and
parasites stained in green. Scale bar is 10 µm. (D) Reflection contrast (magenta)
was used to detect parasite pigment.
Discussion
Here, a 19-year-old man suffered a traumatic spleen rupture in the course of an acute
untreated nonsevere infection with P. vivax and was submitted to splenectomy due to
profuse intra-abdominal hemorrhage. In a malaria-endemic tropical setting such as
the Western Brazilian Amazon region, a thick blood smear must be included in the initial
diagnostic work-up, as it can reliably provide a prompt diagnosis and treatment. This
unique case allowed us to determine the histopathological effects of an active P.
vivax infection in the spleen of a nonimmune individual. Our findings revealed that
in addition to well-described splenomegaly and diffuse cellular hyperplasia associated
with malaria infections, there were intense proliferating plasmablasts in the subcapsular
and perivascular compartments as well as intact P. vivax–infected reticulocytes outside
macrophages in the cords.
The spleen is a complex organ involved in both the removal of damaged and parasitized
red blood cells and in the generation of immunity, consequently having a pivotal role
in malaria [11], [12]. In P. falciparum, several structural and functional changes
have been comprehensively described, and these have been associated with the capacity
of parasitized cells clearance and severe disease manifestations. The essential role
of the spleen in the control of parasite loads is also reinforced by higher parasitemias
and increased clinical severity in patients who suffered splenectomy [13]. Histopathological
analyses of the spleen in natural malaria infections have been limited to snapshots
of postmortem specimens in patients mostly having received antimalarial therapy; our
patient's misfortune provided a rare opportunity to explore extensively an untreated
P. vivax–infected spleen.
In an extensive study of the spleen from Vietnamese patients who died from late complications
of P. falciparum infection, attention was drawn to a marked architectural disorganization
and loss of B cells from the marginal zone [14]. However, as the authors themselves
emphasize, these alterations are very unlikely to reflect what occurs in the majority
of patients who are able to control the infection. The review of spleen rupture from
P. vivax–infected patients published by Lubitz in 1949 remains the most complete description
of the alterations in the acute infection by this parasite so far [15]. In this study,
most cases had acute infection and showed follicle hyperplasia with active germinal
centers and stretching of splenic parenchyma and capsule. Similar observations were
made in two other cases of spleen rupture due to P. vivax
[16]. Our results also showed white pulp expansion and a diffuse hypercellularity.
Moreover, the use of different cellular markers allowed us to identify an increase
in B cells, plasmablasts and plasma cells, all of them implicated in humoral antibody
responses. Although intense proliferation of B cells, resembling splenic commitment
of the spleen had been previously described in two hyperreactive malarial splenomegaly
(HMS) case [17], this is the first report showing these similar histopathological
features in an acute P. vivax infection, highlighting how difficult it can be to distinguish
between malarial infection and malignant disorders in endemic settings.
B cells and antibodies play a prominent role in the development of immunity against
asexual infections in human malaria [18], [19]. Fast production of antibodies is elicited
by B cells, which after antigen encounter migrate to T-cell-rich zones of secondary
lymphoid organs, including the spleen. From there, independently of whether B cells
encounter T-cell-dependent or T-cell-independent antigens, B cells become plasmablasts
for subsequent differentiation of plasma cells [20]. Noticeably, in experimental infections
of C56BL/6 mice with the P. chabaudi chabaudi AS nonlethal strain, remodeling of the
spleen and induction of plasmablasts in extrafollicular compartments have been described
[21]. Similar results have been obtained in experimental infections of Balb/c mice
with the P. yoelii 17× reticulocyte-prone nonlethal strain (unpublished data). Interestingly,
a population of extrafollicular splenic plasmablasts responsible for T-cell-independent
antibody responses has also been described in mice infected with the intracellular
bacterial pathogen Erlichia muris
[22]. In humans, this infection causes a disease known as ehrlichioses, which is characterized
by splenomegaly, lymphopenia, and thrombocytopenia, thus resembling the clinical symptoms
of the P. vivax patient reported here. In striking contrast, no extrafollicular plasmablast
proliferation was observed in histopathological examinations of the spleen of patients
dying of severe falciparum malaria. It is thus tempting to speculate that P. vivax
infections elicit extrafollicular plasmablastic proliferation and a large T-cell-independent
immune response.
In spite of an intense B-cell antibody plasmablastic response and expanded number
of intrasinusoidal macrophages in the cords, immunofluorescence analysis revealed
the presence of large numbers of P. vivax–infected reticulocytes in the cords. Although
it is plausible that some of these parasites were simply detected in their passage
through the spleen, it is difficult to reconcile this sole explanation with the large
numbers detected and the co-localization studies demonstrating that they were mostly
outside macrophages. Noticeably, cytoadherence of the reticulocyte-prone nonlethal
P. yoelii 17× strain to a spleen blood barrier of fibroblastic origin has been shown
[23]. Most important, cytoadherence of P. vivax–infected reticulocytes to different
endothelial receptors have been recently demonstrated [24], [25]. Whether the large
numbers of P. vivax–infected reticulocytes observed in the cords are due to mechanical
trapping [13] as opposed to active adherence remains to be determined.
Conclusion
In endemic areas, malaria is a main cause of spleen rupture and should always be considered
in the initial work-up. Although most cases of spleen rupture can be managed conservatively,
surgery may be necessary when severe hemorrhage occurs. The misfortune of our patient
provided the unique opportunity of performing, to the best of our knowledge, the first
detailed immunohistopathological study of a human spleen from a P. vivax patient having
an active infection with no other clinical confounding effects and that had not been
drug-treated before splenectomy. Similar to P. falciparum, P. vivax infections induced
white pulp expansion and a diffuse hypercellularity in the splenic red pulp. In contrast
to P. falciparum, P. vivax induced a striking proliferation of plasmablasts in extrafollicular
compartments of the spleen. This characteristic of the spleen resembled a diagnosis
of B-cell lymphoma leading to an extensive work-up to clarify the patient's diagnosis.
Last, our observations add to experimental and histopathological evidence [10], [24],
[26], [27], challenging the paradigm that there is no sequestration in deep microvasculature
in P. vivax infections. The presence of large numbers of intact P. vivax–infected
reticulocytes observed in the cords is the matter of further investigation.
Key Learning Points
Plasmodium infection is an important cause of atraumatic spleen rupture in malaria-endemic
areas.
P. vivax infection leads to several macroscopic and microscopic changes in the infected
spleen.
The splenic microscopic changes induced by malarial infection can resemble lymphoma
and an extensive differential workout might be necessary.
The massive proliferation of plasmablasts in the P. vivax–infected spleen and the
large numbers of intact P. vivax–infected reticulocytes observed in the cords may
have important implications on the acquisition of innate immunity in vivax malaria.
Supporting Information
Table S1
Markers, application, and findings used in the phenotypic in situ characterization
and distribution of cells in the spleen of the P. vivax patient.
(DOCX)
Click here for additional data file.