www.elsevier.com/locate/micron

Micron 38 (2007) 651–658

Anionic sites on Toxoplasma gondii tissue cyst wall: Expression,

uptake and characterization

Erick Vaz Guimaraes a, Mariana Acquarone a, Laıs de Carvalho b, Helene Santos Barbosa a,*a Laboratorio de Biologia Estrutural, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz,

Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazilb Laboratorio de Cultura de Celulas, Departamento Histologia e Embriologia, Instituto de Biologia,

Universidade do Estado do Rio de Janeiro, RJ, Brazil

Received 17 July 2006; received in revised form 5 September 2006; accepted 7 September 2006

Abstract

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide, which causes widespread human and animal

diseases. The need for new therapeutic agents along with the biology of these parasites has fueled a keen interest in the understanding of the

nutrients acquisition by these parasites. Studies on the characterization of the T. gondii cyst wall as well as the contribution of the host cell to this

formation have been little explored. The aim of this paper was to investigate the electric surface charge of the T. gondii tissue cysts by ultrastructural

cytochemistry, through polycationic markers, employing ruthenium red (RR) and cationized ferritin (CF). Glycosaminoglycans revealed by RR

were localized on the cyst wall as a homogeneous granular layer electrondense, all over its surface. The incubation of living tissue cysts with CF for

20 min at 4 8C followed by the increase of temperature to 37 8C indicated that T. gondii cyst wall is negatively charged and that occurs an

incorporation of anionic sites by the cyst wall, through vesicles and tubules, and their posterior location in the cyst matrix. So, as to identify which

group of molecules produces negative charge in the cyst wall, we used enzymes for cleavage on different types of molecules, demonstrating that the

negative charge in the cyst wall is mainly produced by phospholipids. Our results, described in this work show, for the first time, the negativities of

the cyst wall, the incorporation and the traffic of intracellular surface molecules by T. gondii cyst wall.

Our model of study can give an important contribution to the knowledge of the biology and the processes involved in nutrients acquisition by

bradyzoites living inside the cysts and, and also be applied as a target for the direct action of drugs against the cyst.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Toxoplasma gondii; Tissue cysts; Bradyzoites; Anionic sites; Enzymatic treatment

1. Introduction

The electric charge on cell surface plays an important role in

some cellular processes, including cell–cell interaction, cellular

differentiation and endocytosis (van Oss, 1978; Spangenberg

and Crawford, 1987; Mutsaers and Papadimitriou, 1988).

Concerning the membranes composition of eukaryotic cells, it

has been demonstrated that the possible candidates for

producing negative charge in the membranes surface are

mainly: the carboxylate and sulphate groups in the mucopo-

lysaccharides acids; phosphate groups in phospholipids, and the

carboxylate groups, widely distributed in glutamic and

neuraminic acid (Weiss, 1969; Mehereshi, 1972). The

* Corresponding author. Tel.: +55 21 2598 4413; fax: +55 21 2260 4434.

E-mail address: helene@ioc.fiocruz.br (H.S. Barbosa).

0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2006.09.002

interaction of surface charges between host cells and protozoa,

together with other specific mechanisms, contributes to the

internalization process of these organisms (Klotz, 1994;

Kleffmann et al., 1998; Maruyama et al., 1998). In the case

of Toxoplasma gondii tachyzoites, in spite of the absence of

lectin bind sites on its surface (Sethi et al., 1977; Handman

et al., 1980; Hoshino-Shimizu et al., 1980; Derouin et al.,

1981), these forms have negative surface charge with an

electrophoretic mobility (Cintra et al., 1986), similar to the

protozoa that present ‘‘coat’’ of glycoconjugate as Trypano-

soma cruzi (Souto-Padron et al., 1984). Proteases and

glycosidases enzymatic studies developed with T. gondii

tachyzoites showed that residues of sialic acid are not present

on the surface of these parasites. The treatment with

phospholipase C reduced the electrophoretic mobility of the

parasites, indicating that phosphate groups must contribute to

their negative surface charge (Cintra et al., 1986). Akaki et al.

E.V. Guimaraes et al. / Micron 38 (2007) 651–658652

(2001), analyzing the surface charge of protozoan under atomic

force microscopy, described that the area of initial contact

between parasites, such as T. gondii, Leishmania amazonensis,

Entamoeba histolytic and T. cruzi, and the host cell presents

positive charge.

The cyst wall is important to the maintenance and integrity

of the parasite inside of the host cell for long periods and it has

been accepted the hypothesis that it is produced by

parasitophorous vacuole membrane modifications, after the

invasion of the tachyzoites, and its interconversion to

bradyzoites (reviewed in Weiss and Kim, 2000). The T. gondii

cyst wall may limit the communication of the parasite with its

host cells and also the antigen presentation to the host,

contributing to the persistence of this intracellular parasite. The

structure and function of the cyst wall components have been

poorly characterized (Sethi et al., 1977; Meingassner et al.,

1977; Derouin et al., 1981; Zhang et al., 2001).

A basic property of eukaryotic cells is the endocytosis,

which represents the capacity to incorporate extracellular

fluids, molecules, solutes and particles to the inside of

intracellular vesicles through different mechanisms. Informa-

tion of endocytosis mechanisms by Apicomplexa parasites has

started to be elucidated (Coppens et al., 2000; reviewed in

Robibaro et al., 2001). The ultrastructural evidences of

endocytosis in T. gondii indicated the micropore as the

structure responsible for the nutrients incorporation, for both

tachyzoites and bradyzoites forms (Nichols et al., 1994).

Recently, receptor mediated endocytosis in T. gondii was

described, through the bond and internalization of sulphated

glycans similar to the heparin, while fluid-phase markers were

incorporated by non-specific pinocytosis (Botero-Kleiven

et al., 2001). Ultrastructural and functional evidences of an

endocytic pathway in T. gondii have been described, in which

acidic compartments were located in the rhoptries, suggesting

that these organelles are related to lysosomes (Shaw et al.,

1998). In addition, it has been demonstrated in tachyzoites, the

existence of tubule-vesicle compartments associated with the

Rab5, a molecular marker for early endosomes (Robibaro

et al., 2001).

The incorporation and traffic of intracellular nutrients in T.

gondii are fields yet to be explored in detail such as in

tachyzoite, bradyzoite and tissue cyst. With all the evidences

above, the aim of this work is to verify ultrastructurally the

tissue cysts surface electric charge, using cationized ferritin

(CF) as a cationic marker, to analyze the dynamics of its

incorporation and fate in the tissue cysts, besides the nature of

anionogenic groups using enzymatic treatment.

2. Materials and methods

2.1. Parasites

T. gondii tissue cysts of ME-49 strain (Type II) were used.

The parasites were maintained in C57BL/6 female mice,

weighing about 12–18 g each, and inoculated intraperitoneally

with about 30 cysts/animal. After 4–8 weeks, the tissue cysts

were isolated from the brain, as described below.

2.2. Cysts isolation and purification

The methodology used was adapted and modified from that

described by Freyre (1995) and Popiel et al. (1996). Mice were

killed in CO2 chamber, and the brains were surgically removed

under aseptic conditions. After immersion at 4 8C in phosphate

buffered saline (PBS), the brains were washed in the same

buffer to remove blood cells, fragmented with the aid of

scissors, and macerated in PBS by successive passages of the

fragments using 18-23 G needles. After that, 20 ml of the total

suspension were placed between slide/coverslip (24 mm �32 mm), and the total number of cysts was determined in the

total area of the coverslip using light microscopy. The

procedures with animals were carried out in accordance with

the guidelines established by the Fundacao Oswaldo Cruz-

FIOCRUZ, Committee of Ethics for the Use of Animals, by

license CEUA 0229-04.

Tissue suspension containing cysts was filtered with cell

dissociation sieve-tissue grinder kit (Sigma Chemical Co.) to

remove small tissue fragments and cellular debris. Then, the

suspension was centrifuge at 400 � g for 10 min and the pellet

was resuspended in Eagle’s medium supplemented with 25%

dextran (about 1 brain per 2.5 ml of final solution). After

centrifugation at 2200 � g for 10 min, the pellet containing the

cysts was recovered and resuspended in PBS, centrifuged at

400 � g for 10 min to remove the dextran solution, and then

diluted in the same buffer. The determination of the cysts

number was carried out as described above.

2.3. Detection and fate of anionic sites

The CF was used to verify the presence of anionic sites on

cysts wall surface and its fate in tissue cyst. After isolation, the

cysts were incubated with 200 mg/ml of CF in PBS, pH 7.2, for

20 min at 4 8C. The intracellular traffic of anionic sites was

monitored by the posterior incubation of the cysts for 1–48 h at

37 8C. Then, the cysts were washed three times in PBS for the

removal of ferritin particles that did not adhere to the surface of

the cysts wall. The material was fixed in 2.5% of glutaraldehyde

(GA) buffered in 0.1 M of sodium cacodylate buffer containing

3.5% of sucrose, pH 7.2, for 1 h at 4 8C. The samples were then

washed in the same buffer three times for 10 min each and post-

fixed for 1 h at 4 8C in 1% of osmium tetroxide (OsO4) in 0.1 M

sodium cacodylate buffer, rinsed, dehydrated and embedded in

Epoxy resin. Thin sections were stained with uranyl acetate and

lead citrate, and then examined under a Zeiss EM10C

transmission electron microscope.

2.4. Ruthenium red (Luft, 1971)

In these assays, the tissue cysts, after isolation and

purification, were fixed in 2.5% GA in 0.1 M sodium

cacodylate buffer containing 0.02% of ruthenium red, pH

7.2, for 1 h at 4 8C. After the fixation, the cysts were washed in

0.1 M of the same buffer two times for 10 min each and post-

fixed in 1% OsO4 containing 0.02% of ruthenium red in 0.1 M

of sodium cacodylate buffer for 30 min at 4 8C. The cysts were

E.V. Guimaraes et al. / Micron 38 (2007) 651–658 653

then washed three times in 0.1 M of sodium cacodylate buffer

containing 0.01% of ruthenium red for 10 min each; dehydrated

and embedded in Epoxy resin as described above.

2.5. Enzymatic treatments of tissue cysts

The cysts were treated with the following enzymes:

phospholipase A2 (PLA2) from Naja mossambica mossambica

(10, 50 and 100 mg/ml); phospholipase C (PLAC) type IX from

Clostridium perfringens (0.2, 0.5, 1 and 2 U/ml) and

Neuraminidase from Vibrio cholerae (0.2 and 0.5 U/ml) diluted

in 0.85% NaCl solution, pH 7.4, during 30 and 60 min at 37 8C;

protease type XIV from Streptomyces griseus and trypsin from

porcine pancreas (1, 10, 100 and 500 mg/ml) diluted in 0.85%

NaCl solution, pH 7.4, during 5 min at 37 8C. After the

treatments, the material was washed in 0.85% NaCl solution

three times and incubated with 200 mg/ml of CF diluted in the

same medium, pH 7.4, for 20 min at 4 8C. The experimental

control was kept by incubating tissue cysts with only 200 mg/ml

of CF for 20 min at 4 8C without any enzymatic treatment.

Figs. 1–3. Anionic sites in T. gondii tissue cysts using cationized ferritin (CF). Fig.

temperature to 37 8C for 1 h, showing formation of clusters of CF on the surface o

tubules (T) from the cyst wall (CW). Particles were found in invaginations of the CW

Tissue cysts showing a vesicle with CF particles near the apical complex (AC) of intr

(V) and tubules (T) containing CF particles in the cyst matrix next to the granular

The cysts were then washed three times in the same buffer and

processed for observation under transmission electron micro-

scopy as described above.

3. Results

3.1. Detection and fate of anionic sites

The cationized ferritin (CF) is a polycationic derivative

electrondense of native ferritin ionized at physiological pH

that allows experiments with living cells. The CF was used in

our studies as a marker of surface anionic sites of tissue

cysts. The incubation of the living tissue cysts with CF for

20 min at 4 8C and subsequent elevation of temperature to

37 8C for 1 h, revealed the distribution of the CF in patches

(Fig. 1) and also as a fine particle layer with uniform

distribution on the cyst wall (Fig. 2), indicating that T. gondii

cyst wall is negatively charged. In these conditions

invaginations of the cyst wall were filled with the tracer

(Figs. 1 and 2). Vesicles of different diameters and tubules

1. Incubation of the tissue cysts for 20 min at 4 8C and subsequent elevation of

f the CW (arrowhead), internalization of CF particles through vesicles (V) and

(arrow), in tubules and vesicles in the cystic matrix (*) below the CW. Fig. 2.

acystic parasites. Fig. 3. High magnification allows to observe details of vesicles

region. Bars: 0.5 mm.

E.V. Guimaraes et al. / Micron 38 (2007) 651–658654

containing ferritin particles were localized right below the

granular region (Fig. 3), in the cystic matrix (Figs. 2 and 3)

and next to or in direct contact with the membrane of

intracystic bradyzoites (Figs. 2 and 4–6).

3.2. Ruthenium red

The ultrastructural analysis of glycosaminoglycans in thin

sections of T. gondii tissue cysts after incubation with the

Figs. 4–6. Anionic sites in T. gondii tissue cysts using CF. Figs. 4 and 5. In detail, ve

the bradyzoityes membrane (Bz). Fig. 6. After 2 h of FC incubation at 37 8C, it w

intracystic parasites. Bars: 0.5 mm.

ruthenium red showed a fine granular electrondense layer all

over surface and in the invagination of the cyst wall (Fig. 7).

Small vesicles filled with the marker, located right below the

internal face of the membrane and also in the granular region,

are transversal cuts of membrane invaginations containing

ruthenium red (Fig. 7). The intracystic matrix and the parasites

did not present marking, since it is a result of the well known

property that ruthenium red do not penetrate in whole

membranes.

sicles (V) of different sizes and tubule (T) containing the label are observed near

as possible to find vesicles containing ferritin very near and also adhered to

E.V. Guimaraes et al. / Micron 38 (2007) 651–658 655

Fig. 7. Detection of glycosaminoglycans in T. gondii tissue cysts incubated

with ruthenium red (RR). Revelation of glycosaminoglycans on the T. gondii

cysts membrane after incubation with RR. This image shows detail of the

membrane of the cyst, the invaginations with particles of ruthenium red (arrow)

and transversal cuts of these invaginations making visible the vesicles contain-

ing the marker (arrowhead). CW: cyst wall. Bar: 0.5 mm.

3.3. Enzymatic treatments of tissue cysts

In order to identify which group of molecules is responsible

for the negative charge of the cyst wall, we cleaved the surface

molecules enzymatically. The cyst control, incubated with CF

(Fig. 8) and after treatment with PLAC, maintained the labeling

on the cyst surface (Fig. 9). The incubation of tissue cysts with

PLA2 (10 and 50 mg/ml) for 30 min at 37 8C revealed that this

was the only enzyme that diminished the expression with CF.

Cysts showed a decrease of the CF distribution when treated

with 10 mg/ml of PLA2 (Figs. 10 and 11), when compared with

the control (Fig. 8) that was incubated only with CF. No

significant decrease in the expression of anionic sites in the cyst

wall was observed when other enzymes (neuraminidase,

protease type XIV, trypsin) were employed (data not showed).

Depending on the concentrations of the enzymes, the cysts

breached, turning impracticable the analysis of the anionic sites

expression. In a same cyst, the surface presented CF followed

by areas without any marking with the CF (Fig. 10). The higher

magnification of Fig. 10 shows the cyst wall area label with CF

(Fig. 11). No label was observed using 50 mg/ml of enzyme

(Fig. 12). Our results showed that the groups of molecules that

are probably producing negative charge in the cyst wall are the

phospholipids.

4. Discussion

Our results with ultrastructural cytochemistry using CF and

ruthenium red clearly demonstrated that the T. gondii cyst wall is

negatively charged. Studies on the plasma membrane of protozoa

have suggested that its components play an essential role in

pathogenic processes, interacting with molecules of the surface

of the host cells, activating the invasion process (Pimenta and De

Souza, 1983; De Carvalho et al., 1985; Cintra et al., 1986;

Kleffmann et al., 1998; Akaki et al., 2001). Thus, the analysis of

the surface molecules of the pathogens and their target cells is

primordial for the better knowledge of this interaction.

There are few studies about the surface charge of the T.

gondii and the molecules which are responsible for the origin of

these charges (De Carvalho and De Souza, 1990; Akaki et al.,

2001; Stumbo et al., 2002). Locksley et al. (1982) described

that the tachyzoite surface has a negative charge and Cintra

et al. (1986) demonstrated that this charge is sensible to

phospholipase C, suggesting that phosphate groups contribute

to its surface charge. The characterization of the surface

components of bradyzoites and tissue cysts has been little

explored, despite the high relevance of both in the course of

chronic toxoplasmosis (reviewed in Dubey et al., 1998; Weiss

and Kim, 2000). The formation of the cyst wall and the

constitution of its matrix are events that follow the

differentiation of bradyzoites and these walls have been

considered as results of the modification of the parasitophorous

vacuole membrane that surrounds the parasite within the host

cell (Gross et al., 1995; Bohne et al., 1996, 1999; Weiss and

Kim, 2000). Our results demonstrated the presence of negative

electric charge on the surface of tissue cysts, using two cationic

markers: (i) CF that presents electrostatic binds to the negative

charge molecules and (ii) ruthenium red, an inorganic marker,

in which atoms of oxygen, ruthenium and amine all together in

known configuration as complex amine, present high affinity

with polyanionic substances, such as the glycosaminoglycans.

The positive reaction to ruthenium red in Plasmodium berghei

merozoites surface and in T. gondii tachyzoites was described

by Seed et al. (1974) and Cintra et al. (1986), respectively.

Considering the absence of binding to lectins on the surface of

P. berghei and T. gondii, Cintra et al. (1986) suggested that

ruthenium red linkage is only an electrostatic interaction of the

polycationic marker with the parasite surface containing

anionic components whose surface electric charge could be

given by anionic phospholipids. Such a conclusion cannot be

inferred from tissue cysts, since the groups of Sethi et al.

(1977), Derouin et al. (1981) and Zhang et al. (2001)

demonstrated, yet with conflicting results, residues of

carbohydrates on tissue cysts surface, although residues of

sialic acid have not been demonstrated, nor has the presence of

glycosaminoglycans in the composition of cyst wall been

investigated. We are demonstrating for the first time the

negative charge of the cyst wall, using two markers that present

different properties to bind the anionic groups on the surface of

tissue cysts of T. gondii. The nature of the membrane

components of these cysts that produce this negativity remains

an open question, but our results with the enzymatic treatment

of tissue cysts denote the participation of phospholipids on the

anionic nature of the cyst wall.

Amongst all the enzymes used in the treatment of the tissue

cysts, only phospholipase A2 was efficient in reducing the

anionic sites of the surface of these cysts. PLA2 is a family of

lipolytic enzymes involved in the phospholipid digestion,

remodeling of cell membranes and host defense. They cleave

the fatty acyl bond at the sn-2 position of membrane

glycerophospholipids to generate unsaturated fatty acids and

lysophospholipids. In mammals, this activity results in the

release of arachidonic acid from the membrane phospholipids,

which is the starting material for the generation of a wide range

of biologically active lipid mediators, including prostaglandins,

leukotrienes, thromboxanes and prostacyclins (reviewed in

Valentin and Lambeau, 2000; East and Isacke, 2002).

There is no literature on studies about the lipids composition

of the cyst wall. Our results suggest that the negative electric

charge of the cyst is given partially or totally by some groups of

the fatty acids tail that bind to carbon 2 of the glycerol structure.

These conclusions are due to the absence or decrease of CF in

E.V. Guimaraes et al. / Micron 38 (2007) 651–658656

Figs. 8–12. Enzymatic treatment in T. gondii tissue cysts. Fig. 8. Experimental control without enzymatic treatment showing the CW labeled with CF over the

surface. Fig. 9. Cysts treated with 1 U/ml phospholipase C maintain the labeling for CF. Fig. 10. Treatment of the cysts with 10 mg/ml phospholipase A2 presenting for

30 min decrease in the labeling of the anionic sites on CW after incubation with CF. Bradyzoites (Bz). Fig. 11. High magnification of cyst of Fig. 10 showing the

region containing CF particles adhered to the cyst wall membrane. Fig. 12. Cyst treated with 50 mg/ml phospholipase A2 showing absence of labeling of the anionic

sites on CW when post-incubated with CF. Bar: 0.5 mm.

E.V. Guimaraes et al. / Micron 38 (2007) 651–658 657

cysts treated with PLA2. The occurrence of cysts with discrete

marker after the enzymatic treatment can be a consequence of a

partial access of the enzyme to some cysts. However, it is little

probable, since the cysts remained under constant agitation

during the treatment. Another hypothesis that can be considered

is that the fatty acids tail of carbon 1 bind to glycerol own

similar cleaved tail for PLA2, also producing negative charge,

that would only be completely eliminated by treating the cysts

with PLA1 and/or B. The presence of cysts with marked and not

marked regions can be in consequence of a heterogeneous

distribution of the glycerolphospholipids after treatment where

not all the negative charge was eliminated, or of the

heterogeneous distribution of other molecules that also give

negative charge to the cyst wall.

We demonstrated ultrastructurally, using CF that negatively

charged molecules present in the cystic wall are incorporated

by tubules and vesicles formed from the membrane that

delimits the cyst wall and its posterior localization in the

matrix. Previous works about the ultrastructure of the cyst wall

had described the presence of these structures in the granular

region and matrix of the cysts, being its origin unknown

(Wanko et al., 1962; Matsubayashi and Akao, 1963; Jacobs,

1967). Mehlhorn and Frenkel (1980), through the ultrastruc-

tural analysis of the cyst wall in the skeletal muscle, suggested

that the vesicles found in the granular region could be derived

from the cyst membrane. We confirmed the origin of these

structures from the membrane, supported by the first

demonstration of the traffic of a marker (CF) of the cyst

membrane and its intracystic fate. Additionally, we verified the

presence of vesicles, neighbor or close to the bradyzoites

plasma membrane. There are no evidences of the fusion of these

vesicles and the parasites membrane, but we can suggest that it

could be one of the pathways of nutrients originating from the

host cell. We also did not find any evidence of the incorporation

of vesicles containing cationized ferritin through the micro-

pore, as described by Nichols et al. (1994), when they observed

vesicles and cellular debris in micropores of intracystic

bradyzoites.

Our observations, showing the cyst wall through its

delimiting membrane with many invaginations which provably

enlarged the cyst surface area, facilitating the exchange of

materials between the intracystic parasites and cytosol of the

host cell, are in accordance with Jones et al. (1986), Ferguson

and Hutchison (1987) and Nichols et al. (1994). The nutrients

required by bradyzoites confined inside the cyst wall may be

necessary for its maintenance, multiplication and the synthesis

of amylopectin granules (Guimaraes et al., 2003; Dzierszinski

et al., 2004). The amylopectin is synthesized in the bradyzoite,

suggesting that the biogenesis of these complex carbohydrate

structures may be of key importance in the bradyzoite

differentiation and persistence of cysts during host infection

by T. gondii (Coppin et al., 2003). Tachyzoites in the interior of

parasitophorous vacuole develop a vesicle-tubular network that

seems to be partly contiguous to the parasite plasma membrane

and to form connections to the parasitophorous vacuole

membrane (Sibley et al., 1986, 1995; Halonen et al., 1996).

This network, due to its large surface area, could play an

important role in the exchange of solutes between the

intracellular parasite and its host (Sibley et al., 1995; Labruyere

et al., 1999; reviewed in Saliba and Kira, 2001). The argument

that the study of the endocytosis in bradyzoites is limited in

virtue of the damages caused to the parasites during the

processes of their isolation from tissue cysts by mechanic or

enzymatic digestion (Nichols et al., 1994) deserves to be

reviewed for two reasons: first, the methods of isolation of

bradyzoites from more recent tissue cysts and the bioassays in

mice have demonstrated ‘‘in vivo’’ infectivity, at least

minimizing the effect of the processes on the viability of the

parasites (Freyre, 1995; Popiel et al., 1996); second, the

endocytosis by bradyzoites can be potentially explored through

experimental protocols, using whole cysts.

Our model can give an important contribution to the

knowledge of the biology and the processes involved in the

nutrients acquisition by cysts and bradyzoites, and be also

applied as a target for the direct action of drugs against of T.

gondii cyst.

Acknowledgements

Support was provided by Conselho Nacional de Desenvol-

vimento Cientıfico e Tecnologico (CNPq), Fundacao Carlos

Chagas Filho de Amparo a Pesquisa do Estado do Rio de

Janeiro (FAPERJ), Programa Estrategico de Apoio a Pesquisa

em Saude—PAPES IV and Instituto Oswaldo Cruz-Fiocruz.

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