CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 4,489-499 (197%

Immunologic and lmmunopathologic Studies in Congenital Chagas’ Disease1

ANA SZARFMAN,~

Institute de Patologia Regional, Resistencia, Chaco

PATRICIO M. COSSIO, ROBERTO M. ARANA,~

Laboratory of Rheumatology and Immunology, Centro de Education Medica e lnvestigaciones Clinicas (CEMIC), Buenos Aires

JORGE URMAN

Hospital Municipal R. Sarda, Buenos Aires

EDUARDO KREUTZER

Escuela Municipal de Cirugia Cardiovascular, Hospital Durand, Buenos Aires

RUBEN P. LAGUENS,AMADA SEGAL,~

Ctitedra de Patologia II, Fact&ad de Medicina, La Plata

AND

LEPOPOLDO COARASA

Hospital para la Madre y el Niho, Resistencia, Chaco

Received November 5, 1974

Six children with congenital Chagas’ infection were studied. Four of the six children died; the main cause of death was encephalitis in two cases, cardiac involvement with heart fail- ure and pulmonary hyperinfection in one case, and pulmonary hyperinfection in the re- maining child. Three cases were positive for IgM circulating antibodies, which reacted in the indirect immunofluorescence technique with endocardium, vascular structures, and striated muscle (EVI antibody). The mothers of these babies did not show EVI reactivity in this immunoglobulin class. In two cases, EVI antibodies were demonstrated to react with self-tissue. Skeletal muscle biopsies performed in three children showed deposits of im- munoglobuhns when they were studied by the direct immunofluorescence technique. In two of these cases, ultrastructural immunohistochemical methods revealed that the gamma

I Supported in part by grants from Comision para el Estudio Integral de la Enfermedad de Chagas, grant No. 24241/72 of the University of Buenos Aires, and by the World Health Organization and Fundacion Cossio.

p Fellow of the Research Worker Career, Consejo National de lnvestigaciones Cientificas y TCc- nicas, Buenos Aires, Argentina.

:I Member of the Research Worker Career, Consejo National de Investigaciones Cientificas y Ttc- nicas, Buenos Aires, Argentina. Send correspondence to Roberto M. Arana, M.D., CEMIC, San- chez de Bustamante 2560, Buenos Aires, Argentina.

’ Member of the Research Worker Career, Comision de lnvestigaciones Cientificas y Ttcnicas de la Provincia de Buenos Aires, Argentina.

489 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

490 SZARFMAN ET AL.

globulins were bound to the plasma membrane of skeletal muscle and endothelial cells. In these two cases, a severe myositis was also observed.

These findings suggest the possible pathogenic role of the EVI antibody in some of the lesions observed in congenital Chagas’ disease.

INTRODUCTION

Although as early as 19 11, Carlos Chagas suspected the possibility of trans- placental transmission of T. cruzi in a 2-month old baby (l), it was not until 1949 that this phenomenon was demonstrated in a human newborn by detection of trypanosomes in peripheral blood (2). Since then a number of investigators have confirmed this observation (3-6), and the clinical characteristics of congenital Chagas’ disease, as well as its high rate of mortality, were described (3, 5). The transmission of T. cruzi from infected mothers to their babies is not very frequent, and it has been shown to be between 2 to 4% in babies born alive (3, 6).

The reason why T. cruzi crosses the placenta in such a small proportion of in- fected mothers is unknown; the observation that many mothers who have a new- born with congenital Chagas’ disease have healthy children born before and after the birth of a sick child is of interest (3).

The pathogenesis of congenital Chagas’ disease is not completely clear. Although some lesions appear to be related to the direct invasion of the parasite (7, S), in other lesions, consisting of inflammatory reactions that involve vascular structures of the fetus and placenta (4, 7-8), the presence of the parasite cannot be demonstrated.

In a previous study (9), a circulating antibody with complement fixing ability reacting with endocardium, blood vessels, and striated muscle (EVI antibody) was observed in 95% of individuals with Chagas’ heart disease and in 45% of asymptomatic people infected with T. cruzi. The EVI antigenic system cross- reacts with T. crud, and we have also demonstrated the autorreactivity of the EVI antibody (9). Recent studies, employing ultrastructural immunochemical methods, have shown that the specificity of the EVI antibody is directed against the plasma membrane of striated muscle and endothelial cells (10).

In the present study, the possible induction of EVI antibody synthesis in the infected newborn and its participation in the pathogenetic mechanism of some of the lesions of congenital Chagas’ disease were investigated.

MATERIAL AND METHODS

Patients

A total of six newborns (one pair of twins) and their five mothers were stud- ied. Four cases of congenital Chagas’ infection (Cases l-4) were from a nonen- demic area (Buenos Aires city). The mothers in the study were from an endemic area, had a positive serology for T. cruzi infection, and had the diagnosis of Chagas’ disease made during pregnancy. Serological tests for T. cruzi infection were performed during 1971 and 1972 for 5275 pregnant women studied in ob- stetrical centers of Buenos Aires. In this group, 566 pregnant women had a posi-

CONGENITAL CHAGAS’ DISEASE 491

tive serology. In these cases, each newborn was investigated for the presence of congenital Chagas’ disease with the following methods: (a) clinical and routine laboratory examination of the newborn daily during the first 3-5 days of life and monthly up to 6 months of life; (b) peripheral blood examination, by the Pizzi et al. method (1 l), for the detection of parasites in blood samples; (c) when a posi- tive parasitemia was found, isolation of T. cruzi by mouse inoculation; (d) deter- mination of specific anti-T. cruzi antibodies as described in the following. In this manner, four of the six reported cases of congenital Chagas’ disease were de- tected early. In the remaining newborns, in whom signs or symptoms of T. cruzi infection were not observed, and with no parasitemia, specific anti-T. cruzi an- tibodies were positive in the IgG fraction at delivery (mothers presented IgG- anti-T. cruzi antibodies). In these newborns, a progressive decrease in the titer of the IgG-anti-T. cruzi antibodies was observed, and all were negative at 6 months of life.

Neither clinical nor electrocardiographic changes compatible with myocar- diopathy were observed in the four mothers of the congenital cases, and the clinical study as well as the routine blood and urine analyses was also normal. Toxoplasmosis and syphilis were ruled out by serological procedures (12), and obvious infections during pregnancy due to other etiologies were not apparent in their clinical histories. The other two newborns with congenital Chagas’ disease (Cases 5 and 6) were from an endemic area (Resistencia city, Chaco, Argentina) and were nonidentical twins (different blood groups). The mother was also diag- nosed during pregnancy and presented the same clinical characteristics of the other four mothers from Buenos Aires. The study of these two cases was planned with emphasis on conditions to keep the newborns in the postpartum period free of the possibility of an “acquired” infection. Although the five mothers had lived in primitive houses with the insect vector, in their infancy and adolescence, they had in the last few years improved their socioeconomic condi- tion. Serum from postpartum mothers was obtained simultaneously with the first serum specimens obtained from each child.

Case Reports

Case 1. This was a male born in the 33rd week of pregnancy with a birth weight of 1,600 g. He was not severely infected. The initial finding was hyper- bilirubinemia (9 mg% at 30 hr of life), which was treated with phototherapy and remitted at the 5th day of life. Hepatosplenomegaly was noticed on the 7th day. Splenomegaly was no longer present 2 months later, but the liver was still enlarged at 3 months. Myocarditis was not present. At 6 months he was released from the hospital in apparently good health. Blood samples of this case were ob- tained at 1, 13, 3 1, 80, and 160 days of life. Parasitemia was positive in the first four samples and negative in the last one.

Case 2. This was a male born after an uneventful full term pregnancy with a birth weight of 1,600 g. Hepatosplenomegaly, anemia, and high levels of serum bilirubin were present at delivery. A few days later he developed generalized hypotonia, poor suckling, and lymphocytosis in the cerebrospinal fluid (38 cells/mm3), dying on the 30th day of life with marked signs of encephalitis, which

492 SZARFMAN ET AL.

appeared as the main cause of death, weighing 190 g below the birth weight. Myocarditis was not present. Blood samples of this case were obtained at 3, 12. and 30 days of life. Parasitemia was always positive. At necropsy, nests of amas- tigotes were found in the urinary bladder. In the central nervous system (CNS). encephalitic alterations were observed, constituted mainly of large perivascular lymphoplasmocytic infiltrates. In the gastrointestival tract, necrosis of many neurons of the Auberbach plexus with lymphocytic infiltrates were observed.

Case 3. This was a male born after an uneventful full term pregnancy with a birth weight of 1,900 g. Hepatosplenomegaly, high levels of serum bilirubin, ane- mia, and severe metabolic acidosis were detected. At birth the infant showed signs of CNS involvement with abnormal findings in the cerebrospinal fluid (protein concentration: 140 mg%, cells: 90 lymphocytes/mm3), and T. cruzi was isolated from this fluid. Myocarditis was not present. In spite of therapy, signs of CNS involvement became more severe, and the infant died on the 33rd day of life weighing 235 g below his birth weight. Apparently, encephalitis was the main clinical cause of death. Blood samples were obtained at 1 and 30 days of life. Parasitemia was positive in both samples. At necropsy, nests of amastigotes were found in the esophagus, stomach, and urinary bladder. The CNS presented significant alterations of encephalitis, with large perivascular lymphoplasmocytic infiltrates and thrombotic vessels.

Case 4. This was a male born in the 36th week of pregnancy with a birth weight of 2,800 g. Slight hepatosplenomegaly was observed at delivery, and it increased later. Cardiac enlargement (cardiac : thoracic ratio 65%) and elec- trocardiographic signs of left ventricular hypertrophy were also seen. At the 45th day, severe alterations of ventricular repolarization and heart failure appeared. Good response was obtained with digitalis and diuretics, and he was released from the hospital at 4 months of age. One month later, in spite of treatment, heart failure was again observed, and the child developed a superimposed bron- chopulmonar infection and died at home. Necropsy was not available, but a skeletal muscle biopsy was performed at the 3 1st day of life. Blood samples were obtained at Days 28, 43, and 85 of life. Parasitemia was positive in the first two samples.

Case 5. This was a female born in the 36th week of pregnancy with a birth weight of 1,920 g. Jaundice, coluria, and hepatosplenomegaly were observed early. After 30 days, the child improved; 7 days later an urinary infection ap- peared (Klebsiella sp.) that remitted with gentamicine. Progressive anemia and hypotonia developed with deterioration of her general condition. At the 59th day, a bronchopulmonar infection with vomiting, convulsions, and muscular tremor appeared, and she died 4 days later. Clinical cause of death was at- tributed to the confluence of these multiple factors, which facilitated a terminal pulmonary hyperinfection. Necropsy was not available. Cardiac involvement was not observed during life. A skeletal muscle biopsy was obtained at the 16th day of life. Blood samples were obtained at 5 and 43 days, and parasitemia was always present.

Case 6. This was a female born in the 36th week of pregnancy, with a less severe form than her heterozygous twin sister (Case 5), with a birth weight of 1,990 g. She presented with anemia and hepatosplenomegaly and improved after

CONGENITAL CHAGAS’ DISEASE 493

treatment. At the 50th day she was released to her home in good condition. Car- diac involvement was not observed. A skeletal muscle biopsy was performed at the 16th day of life. Blood samples were obtained at 5 and 43 days of life, and parasitemia was positive in both samples.

All cases were treated with Nifurtimox (a nitrofuran derivative at 10 mg kg-’ day-‘) starting on the 2nd day after detection of parasitemia. The longest treat- ment was done in Case 1 (5 months) because parasitemia was still positive in the 3rd month of life.

In all the congenital cases, T. cruzi was isolated by inoculation of blood samples in mice on the 28th day of life in Case 4 and within the first 5 days of life in the other five cases.

Serological Determinations for Chagas’ Disease

The following techniques were used: (a) complement fixation test (13); (b) direct agglutination by the microtiter method, employing epimastigotes treated with trypsin and form01 (14); and (c) indirect immunofluorescent technique, using epimastigotes as antigen (15).

The distribution of specific anti-T. cruzi antibodies in the serum IgG and IgM fraction was studied by employing a modification of the agglutination test (16) and by the use of fluorescein-labeled monospecific anti-IgG and anti-IgM an- tisera mentioned in the following.

Immunojluorescence Technique

For the investigation of the EVI antibody (9), the sera were studied by the indirect immunofluorescence technique as described elsewhere (17) using 2 pm cryostat sections of mice, bovine, and human skeletal and heart muscle. Goat antihuman gamma globulins (IgG, IgA, and IgM) and rabbit monospecific anti- human IgG, IgM, and IgA were prepared in our laboratory (9) and labeled with fluorescein isothiocyanate (12.5 pgfmg protein) as described by Nairn (18). Pro- tein : fluorescein ratio was 1 : 5 for the antigamma globulin and the anti-IgM an- tisera and 1 : 3 for the anti-IgG and anti-IgA antisera. Fluorescein-labeled an- tiserum to human C3 was commercially obtained (Hyland Division Travenol, Los Angeles, California). All antisera were absorbed with mouse liver and bovine skeletal and heart muscle powder before being used. Bovine skeletal and heart muscle powder was used since in the experience of the authors, it is com- pletely adequate for removing nonspecific fluorescence of the antisera when they are employed with murine or human striated muscle cryostat sections. Human striated muscle powder was not used since it was observed that on the one hand, it did not present major advantages in relation to bovine powder in order to abol- ish nonspecific reactivity and on the other hand, it diminished the titer of the anti-human gamma globulin antiserum, probably because of the immunoglobulins that the tissue powder retains. After absorption with tissue powder, labeled an- tisera were used for direct treatment of normal mouse and human striated muscle cryostat sections (selecting blood group A+ and B+ individuals); although satisfactory negative results were obtained, antisera were further absorbed with a pool of AB+ washed red blood cells: 2 ml of packed cells/ml of serum were used. Mixtures were incubated at 37°C for 1 hr, left overnight at 4”C, and cen-

494 SZARFMAN ET AL.

trifuged at 10,000 g in the cold. Direct immunofluorescence on skeletal muscle biopsies (biceps), obtained by a minimal surgical procedure, was performed on tissue from Cases 4, 5, and 6. Tissue was immediately frozen at -20°C and processed as in a previous report (9). The sections were studied with the antisera already described and with labeled rabbit antihuman fibrinogen serum (9). The antifibrinogen antiserum was prepared by immunizing with plasma and absorbing with serum. The specificity was controlled by immunoelectrophoresis. The pro- tein: fluorescein ratio was 1 : 3. and it was used at a 1 : 10 dilution.

Blocking experiments were performed by applying the same unlabeled goat antihuman gamma globulin antiserum before applying the labeled antiserum.

For evaluating EVI autorreactivity. the skeletal muscle sections of the biopsies were treated with citrate buffer saline pH 3.2 to remove the bound gamma globulin (9). The eluted sections were employed for the indirect im- munofluorescence technique, with the patients’ own sera, in an autologous reac- tion. The autologous reactions were also performed after absorbing the chil- dren’s sera with lyophilized epimastigotes of T. cruzi and with homogenates of murine heart and skeletal muscle: absorptions were performed as in a previous report (9). As a control, children sera were absorbed as previously described (9) with human red blood cells, which did not remove the EVI antibody (9). FOI studying the action of the citrate acid buffer on the EVI antigen, mouse skeletal and heart muscle sections were treated for 2 hr in Coplin jars with the buffer and then employed as substrate for the indirect immunofluorescence technique. In this manner, we observed that the acid buffer treatment did not affect the EVI antigen.

A Leitz Ortholux microscope with a dark field condenser and an Osram lamp HBO 200 with UG 1 filter were used, and an ultraviolet excluding filter was placed in the eyepiece of the microscope.

Ultrastructural lmmunohistochemical Studies

The same batch of goat antihuman gamma globulin antiserum employed for the immunofluorescence technique was utilized. The globulin fraction of the an- tiserum was separated and labeled with horseradish peroxidase as reported else- where ( 10). Skeletal muscle biopsy samples were fixed 4 hr at 4°C in 4% for- maldehyde in 0.1 M phosphate buffer pH 7.3. After fixation, the samples were washed overnight in the same buffer and sectioned at 100 Frn with a freezing microtome. The sections were rinsed in PBS and incubated at 4°C in one of the following ways: (a) incubation for 3 days with goat gamma globulin antihuman gamma globulin, labeled with peroxidase; (b) incubation with rabbit gamma globulin antirat IgG (absorbed with normal human serum), labeled with perox- idase; (c) incubation with free peroxidase ( 1.2 mglml in PBS); (d) same as (a) but “blocking” previously with unlabeled goat gamma globulin antihuman im- munoglobulins. In every case, peroxidase-labeled antibodies were diluted in PBS to contain 0.5 mg globulin/ml. After treatment, sections were rinsed for 2 hr in several changes of PBS, and peroxidase activity was investigated according to the histochemical method of Graham and Karnovsky (19) with diaminobenzidine chlorhidrate. The sections were then rinsed in PBS, cut into small pieces, post- fixed in 1% osmium tetroxide in phosphate buffer pH 7.5, and embedded in

CONGENITAL CHAGAS’ DISEASE 495

Araldite (20). Thin sections were mounted on copper grids and examined un- stained with a Philips electronmicroscope operated at 60 kV.

Thick sections (1 pm) of the epoxy resin embedded material were examined under the light microscope.

Pathological Studies

In the skeletal muscle biopsies, routine histological procedures were per- formed.

Electron Microscopic Studies

In Cases 5 and 6, routine transmission electron-microscopic studies were also done with skeletal muscle biopsies. The tissue samples were fixed for 24 hr in cold (4°C) 5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, rinsed in the same buffer, postfixed, and embedded in Araldite (20). Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips 200 electron microscope.

RESULTS

Presence of Circulating EVI and Specific T. cruzi Antibodies in Mothers and Newborns (Table I)

Five of the six newborns with congenital Chagas’ disease and three out of the five mothers had circulating EVI antibodies as demonstrated by the indirect im- munofluorescence technique, employing the antihuman gamma globulin serum and an anti-C3 serum (9).

Fluorescein-labeled monospecific antisera were used for studying the im- munoglobulin class with EVI and anti-T. cruzi activities, both in mothers and newborns.

In the mother of Case 1, EVI and anti-T. cruzi antibodies were present both in the IgM and IgG classes. In the newborn, EVI antibodies were in the IgG class until the 3 1st day of life; the latest examinations were done at the 80th and 160th days, and IgG-EVI antibody was absent. Anti-T. cruzi antibodies shifted from IgM to IgG.

EVI antibodies were not detected in Case 2 nor in his mother’s serum, whereas anti-T. cruzi antibodies were of IgG and IgM class in both instances.

In the mother of Case 3, EVI reactivity was observed only in the IgG class; in her child, it was present in IgM and IgG up to the 4th week, when the infant died. Anti-T. cruzi antibodies were IgG in the mother and both IgM and IgG in the infant.

The mother of Case 4 was EVI negative; the child had EVI activity in the 1gM antibody at the 28th day of life. After 85 days, EVI antibodies were of the IgG class. Anti-T. cruzi antibodies also changed from IgM to IgG.

The mother of Cases 5 and 6 (heterozygous twins) had circulating IgG-EVI antibodies: Case 5 also had EVI activity in the IgG class, whereas in Case 6 it was present in both IgM and IgG classes. T. cruzi specific antibodies were of IgG class in the mother and in Case 5, whereas in Case 6 they were of both IgM and IgG classes.

496 SZARFMAN ET AL..

TABLE I TMMUNOGLOBULIN CLASS OF ANTI-T. cn4;i SPECIFK ANTIBODIES AND UF EVI

ANTIBODIES IN SERA OF NEWBORN INFANTS WITH CONGENITAI

CHAGAS’ DISEASE AND THEIR MOTHERS

Infants (days after delivery)

Case Antibodies MotheP ~-

kM Pas w pos Specific

1 anti-T. crrczi IgG POS pos’ pas

EVI W Pas neg neg W Pas Pas Pas

kM Specific

2 anti-T. cnrii IgG

EVI kM I@

W Specific

3 anti-T. cvuzi IgG

EVI IgM

IgG

POS

Pas neg neg

neg

POS

neg

POS

Pas Pas

pos’ POS”

w3 w neg neg

-____-

Pas

POS’

Pas Pas

I&f neg Specific

4 anti-T. uu;i IgG Pas

EVI kM w kG w

5a

I&f Specific anti-T. u44;i IgG

EVI kM 16

and

6”

W Specific anti-T. cruzi IgG

EVI W I&

neg w

Pas neg Pas

pos’

neg Pas

Pas

pos’

Pas Pas

pos

pas

neg pas

pos

POS

POS’

net4 neg

neg

pas’

neg w

pos”

neg neg

PO5

ti (encephalitis)

___---

POS’ tl” (encephalitis) pos POS

.-.. __ - .___..--__---- .~-~~-~

pos pos w

pas” pas’ pos’ ci t cardiopathy . pos pos neg hyper- neg pos pos infection)

w

pus

w pos

d (hyperinfection)

pos

pos’ pos POS

a Allogenic twins. a Serum samples were obtained simultaneously with the first sample of each child, c Positive parasitemia. d Died. e Nests of amastigotes at autopsy. f Negative parasitemia.

CONGENITAL CHAGAS’ DISEASE 497

Studies on Skeletal Muscle Biopsies

Skeletal muscle biopsies of three EVI positive newborn infants (Cases 4, 5, and 6) were studied by the direct immunofluorescence technique. All biopsies showed the presence of bound immunoglobulins, and the positive reaction was observed near the sarcolemma, as it has been described in adult cases (9). In Case 4, deposits were identified as IgM, in Case 5 as IgG, and in Case 6 as both IgM and IgG classes. Blocking experiments performed with unlabeled antisera resulted in negative reactions, and acid buffer treatment of the muscle sections removed the bound gamma globulin, whereas control washes had no effect.

Staining procedures with the fluorescein-labeled antifibrinogen antiserum gave negative results.

The acid eluted skeletal muscle sections of Cases 4 and 6, both with circu- lating IgM-EVI antibodies, were used in the indirect immunofluorescence tech- nique, with the patients’ own sera. Positive results were observed with the human gamma globulin-labeled antiserum and the IgM-labeled antiserum, and the pattern was similar to an EVI positive test. The autologous reactivity disap- peared after absorbing the patients’ sera with epimastigotes of T. cruzi, or with murine skeletal muscle or heart homogenates or both (9). Absorption with human red blood cells did not affect the autologous reactivity.

The treatment of the sections with normal human sera, or with sera of individ- uals infected with T. cruzi but without circulating EVI antibodies, gave negative results.

The fine structures to which the gamma globulin was bound in vivo were stud- ied in the skeletal muscle biopsies of Cases 5 and 6 by direct ultrastructural im- munohistochemical methods. Positive staining for gamma globulin was observed in the plasma membrane of the muscle fibers and the endothelial cells.

Light microscopy and transmission electron microscopy were used for study- ing the skeletal muscle biopsies of Cases 5 and 6. Both showed the presence of important myositic lesions, which were more marked in the former. No parasites were observed in the lesions of both cases. The light microscope study of Case 5 showed a heavy mononuclear exudate present among the muscle fibers and severe sclerosis replacing the muscle bundles. In the periphery of the inflamma- tory and scarred areas, muscle cell destruction was observed with the appear- ance of multinucleated myoblasts. In Case 6, similar lesions of less severity were observed.

The electron microscope study of those muscle biopsies confirmed and ex- tended the observations made with the light microscope. In the periphery of the inflammatory areas, the muscle fibers presented morphological alterations such as an increase of the sarcoplasm and a decrease of the number of myofibrils and cell organelles. At places there were cell stumps containing numerous nuclei, suggesting regeneration attempts.

DISCUSSION

The present report shows that an intrauterine infection with T. cruzi induces the synthesis of EVI antibodies by the fetus during the fetal and perinatal period in some instances. This is demonstrated by the presence of IgM-EVI antibodies

498 SZARFMAN ET AL.

in the sera of three newborn infants in the absence of IgM-EVI antibodies in the serum sample obtained simultaneously from their mothers. This observation and the persistence of IgM-EVI antibodies in the same children through a much longer period of time than the half life of IgM (21) ruled out the possibility of transfer of maternal IgM-EVI antibodies through the placenta by altered perme- ability due to an inflammatory process (22).

In two of the cases reported here. the autorreactivity of the IgM-EVl an- tibody was demonstrated. This can be explained by the presence of cross- reacting antigens between T. cruzi and host tissues (9, 10). In the case of con- genital Chagas’ disease. this mechanism appears to operate during an intra- uterine infection.

In three congenital cases with circulating EVl antibodies in \ivo, gamma globulin bound to skeletal muscle structures was demonstrated. Ultrastructural immunochemical methods showed that it was located in the plasma membrane of the fibers and endothelial cells. In a recent report (lo), it has been shown that the EVI antibodies react with those structures, which poses the possibility that the deposited gamma globulin represents the in vivo fixation of that antibody. The comparison between the immunoglobulin class (or classes) of the deposits and the immunoglobulin class with EVI reactivity in the infants and their mothers suggests that the source of tissue-bound immunoglobulin is. with high probability, EVI antibody provided by the fetus in some cases or from the mother in other cases or from both.

Lesions of myositis had been described with some frequency in congenital Chagas’ disease (3). Severe myositic lesions were observed in two of our cases that were not related to the presence of the parasite. It is of interest that vasculitic lesions of the esophagus in two reported cases of congenital Chagas’ disease were not related to the histological distribution of the T. cruzi in that organ (8). Since in our two cases with myositic lesions in vivo immunoglobulins bound to plasma membranes were found, it would be reasonable to associate both observations and to suppose that EVI antibodies are possibly related to the pathogenic mechanism. This is supported by the presence of circulating EVI an- tibodies in five out of the six newborns studied. Unfortunately, in Case 3. in which EVI antibodies were absent, no immunopathological studies could be carried out. Therefore, in this case the possibility of a limited synthesis of EVI antibodies cleared from the circulation by deposition on tissues cannot be ruled out (23).

The reasons why the parasite crosses through the placenta in such a small proportion of infected mothers is unknown. In some cases, a placentitis with vascular involvement not related to the presence of parasites has been described (6, 7). Since EVI antibody reacts with endothelial cells and the placenta is an extremely vascularized tissue, it is of interest to speculate on the possibility of a pathogenic role of the antibody on this organ. In this respect. im- munopathological studies of the placenta would be of great help.

ACKNOWLEDGMENTS We are grateful to Dr. Irving G. Kagan for his encouragement throughout our studies and for the

revision of the English manuscript, to Dr. Maxime Seligmann for his valuable advice, and to Miss M. B. Casanova for her expert technical assistance.

CONGENITAL CHAGAS’ DISEASE 499

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