HIGH LEVELS OF IgG4 TO SCHISTOSOMA MANSONI EGG ANTIGENS IN
INDIVIDUALS WITH PERIPORTAL FIBROSIS
ALDA M. S. SILVEIRA, JEFFREY BETHONY, ANDREA GAZZINELLI, HELMUT KLOOS,
LU´CIA ALVES DE OLIVEIRA FRAGA, MARIA CAROLINA B. ÁLVARES, ALUIZIO PRATA, HENRIQUE L. GUERRA,
PHILIP T. LOVERDE, RODRIGO CORREA-OLIVEIRA, AND GIOVANNI GAZZINELLI
Departamento de Bioquı ´mica e Imunologia, e Escola de Enfermagem, Universidade Federal Minas Gerais, Belo Horizonte, Minas
Gerais, Brazil; Laborato ´rio de Imunologia da Universidade Vale do Rio Doce, Governador Valadares, Minais Gerais, Brazil; Centro
de Pesquisas Rene ´ Rachou-FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil; University of California, San Francisco Medical
Center, San Francisco, California; Santa Casa de Miseraco ´rdia de Belo Horizonte, Belo Horizonte, Minas Gerais, Brazil; Faculdade
de Medicina do Tria ˆngulo Mineiro, Uberaba, Minas Gerais, Brazil; State University of New York at Buffalo, Buffalo, New York
267 individuals from an area in which schistosomiasis mansoni is endemic. Based on information obtained from clinical
and sonographic examinations of this sample, the individuals were divided in three groups: 1) 204 individuals without
periportal fibrosis, and liver and spleen enlargements; 2) 41 individuals without periportal fibrosis, but presenting with
organopathy, with or without organomegaly; and 3) 22 individuals with periportal fibrosis, regardless of their status as
having hepatomegaly and/or splenomegaly. Levels of IgG4 to SEA were significantly higher in sera from patients with
fibrosis compared with the patients from the other two groups. We also found significantly higher levels of IgG4 against
SEA in egg-negative patients with fibrosis compared with egg-negative patients from the other two groups. This report
demonstrates a specific humoral response in patients presenting with initial fibrosis, a form of schistosomiasis transient
between intestinal and severe hepatosplenic.
Specific IgG4 and IgE responses to adult worm antigen and soluble egg antigen (SEA) were examined in
Schistosoma mansoni has a wide geographic distribution in
Africa, South America, and the Caribbean. The disease is due
mainly to eggs deposited in host tissue by the adult female.
Egg antigens induce granuloma formation and fibrosis,
mostly in the intestine and liver (portal system). Most infected
individuals living in endemic areas are asymptomatic, but a
few develop Symmers’ periportal fibrosis of the liver and its
sequelae of portal hypertension and esophageal varices.
Many investigators attribute the intensity of infection as the
main cause of morbidity in schistosomiasis infection.1,2How-
ever, the intensity of infection may also be related to behav-
ioral routines (water contact),3,4age,5and sex.6,7Individual
susceptibility that is associated with the immunologic8,9and
genetic profiles10–12of each individual living in the endemic
area is also important.
The contribution of each of these factors to the sequential
development of pathology in S. mansoni infections leading to
hepatosplenic disease is not completely understood. Studies
of human immunity to schistosomiasis in endemic populations
have indicated that parasite-specific antibody isotypes vary in
their association with either susceptibility or resistance to in-
fection/reinfection.13–16High levels of IgE and IgG4 are pro-
duced during helminth infection. Evidence for a significant
correlation between susceptibility to reinfection to S. mansoni
in humans and increased production of IgG4 antibody was
first reported by Auriault and others.17In fact, elevated pro-
duction of IgG4 was consistently associated with increased
susceptibility to reinfection,18and low production of this iso-
type was found in patients resistant to infection (endemic
normal individuals)19and to reinfection.13The association
between high levels of IgG4 and susceptibility was thought to
result from the blocking effect of this isotype on the protec-
tive effect of IgE.18More recently, a positive correlation be-
tween IgG4 to soluble egg antigen (SEA) and intensity of
infection has been reported.20Previous reports also associ-
ated susceptibility with heavy infection as a high risk for the
development of periportal fibrosis.1,21However, there are
few studies linking humoral immune response with morbid-
With the introduction of ultrasound examinations in schis-
tosomiasis field surveys, it has become possible to identify
individuals with different grades of periportal thickness due
to fibrosis, a typical lesion of hepatic schistosomiasis corrobo-
rated by histopathologic studies of liver biopsies.25–27
In the present study, we used ultrasonography to grade
periportal fibrosis in patients from an area in which S. man-
soni is endemic in an attempt to correlate the development of
hepatosplenic disease with aspects of the humoral response
associated with susceptibility to S. mansoni infection. This is
part of an extensive study to elucidate the contributions of
demographic, behavioral, immunologic, and genetic factors
on the development of susceptibility and resistance to S. man-
soni infection and pathology.
MATERIALS AND METHODS
Study design. This investigation was conducted in an area
endemic for schistosomiasis, Co ´rrego dos Melquı ´ades, in the
Minas Gerais State of Brazil. It was reviewed and approved
by the Ethical Committee of Fundac ¸a ˜o Oswaldo Cruz, Min-
istry of Health. Local health care personnel explained the
study to the village population. It was stressed that all indi-
viduals would receive treatment irrespective of whether or
not they participated in the study. Participants were enrolled
only with their explicit approval, or with that of their parents
in the case of children less than 15 years of age. A cross-
sectional survey was then conducted, which enumerated in-
dividuals and assigned them personal identity numbers and a
household identity number. The clinical history of partici-
pants was collected, along with their quantitative stool egg
counts as determined by duplicate Kato-Katz thick smears.
Fecal examinations were completed in May 1998. In Septem-
ber, 340 participants were given physical examinations. Indi-
viduals with symptoms of the disease (abdominal pain and
diarrhea) or those having liver and/or spleen enlargement
detected by abdominal palpation even during deep breaths or
Am. J. Trop. Med. Hyg., 66(5), 2002, pp. 542–549
Copyright © 2002 by The American Society of Tropical Medicine and Hygiene
whose abdominal palpation was difficult (expontaneous
muscle contraction or an excess of fat) were scheduled for an
ultrasound examination. A total of 267 individuals were ex-
amined by ultrasonography. The physical examination and
ultrasonography were done independently by different phy-
sicians. From September to December, 10-ml blood samples
were collected in sterile vacutainer tubes (Becton Dickinson,
Rutherford, NJ). At the same time infected patients received
treatment with oxamniquine at the Brazilian standard dose of
15 mg/kg of body weight for adults and 20 mg/kg of body
weight for children. One year after treatment, feces were col-
lected for evaluation of reinfection. Egg-negative individuals
and those that did not return a feces sample were not included
in the calculation of the reinfection rate. Active infection with
S. mansoni was defined as the presence of eggs in the stools.
Malaria and leishmaniasis, confounders for hepatosplenic dis-
ease, are not endemic in the study area.
Detailed methods and the results of the water contact sur-
vey undertaken in the area have been previously re-
ported.28,29Questions concerning the frequency of water con-
tact were derived from a direct water contact observation
study in the same area.28Briefly, participants were inter-
viewed individually as to their water contact behavior.29
Questions consisted of the frequency per week for each water
contact behavior and the source of the water (e.g., well, bam-
boo conduit). Parents were asked about their children’s water
contact behavior only when the children were less than six
years old; no proxy responses for water contact behavior for
participants more than six years old were accepted. All water
contacts were divided into potentially infected (streams, ca-
nals, fish ponds, “bicas”) or potentially safe sites (springs,
wells, water hoses, household faucets, wash basins, and show-
ers sites). The determination of an infected or safe site was
based on the presence (infective) or absence (safe) of snail
intermediate hosts determined by malacologic surveys under-
taken at each water contact site. Water contact frequencies
that occurred at potentially infective sites were then multi-
plied by standardized duration and body immersion values
and defined as total body minutes (TBM).29
Ultrasound evaluation. Two hundred sixty-seven subjects
were subjected to abdominal ultrasonography using a por-
table Hitachi EUB-200 machine (Tokyo, Japan). Patients less
than five years old were excluded. Liver size, portal-vein di-
ameter, thickness of the walls of peripheral portal branches,
spleen size, and splenic vein diameter were assessed as de-
scribed elsewhere.25–27Liver span was measured both in the
midclavicular line and the midline. The liver was also exam-
ined for smoothness of surface. The ultrasound criteria for
schistosomal hepatic fibrosis are multiple diffuse echogenic
areas scattered throughout the liver. Portal vein diameter was
measured at its entrance into the porta hepatis and its bifur-
cation inside the liver. The spleen intercostal spaces were
evaluated using oblique and longitudinal scanning of the left
upper quadrant. The splenic size and texture were noted and
the gallbladder was examined for wall thickness and stones.
The following classification was adopted to assess the mor-
bidity of S. mansoni infection:25grade 0 ? periportal fibrosis
thickness less < 3 mm; grade I ? thickness of 3–5 mm. There
were no significant differences between the control, pathol-
ogy, and fibrosis groups in relation to portal and splenic veins
diameters (cm, mean ± SD) (portal vein: 1.1 ± 0.2, 1.2 ± 0.2,
and 1.1 ± 0.2; splenic vein: 0.7 ± 0.1, 0.8 ± 0.2, and 0.7 ± 0.2,
respectively). The term organopathy was used for individuals
with alterations in size, surface or parenchyma (liver or
spleen) without periportal echogenicity (periportal fibrosis).
Although most of these patients were infected, the liver al-
terations may not be due to schistosomiasis because they did
not have the characteristic periportal thickening limited to the
portal vein and its tributaries. The physicians performing ul-
trasound examinations were not aware of the infection status,
results of the clinical examination, and water contact results
of the patients undergoing ultrasonography.
Parasitologic examination. Infection intensities were mea-
sured by individual fecal egg counts, expressed in eggs per
gram of feces (epg) performed by the Kato-Katz30method on
stool samples obtained on three consecutive days. An indi-
vidual’s fecal egg count was the arithmetic mean of these
three determinations. Due to the important role of diagnostic
sensitivity in causing a truncation in the frequency distribu-
tion of egg output,31every attempt was made to standardize
the reading of slides, including having the same technicians
prepare and read all slides. Quality control was ensured by
blinded cross-checking of slides between technicians.
Preparation of crude S. mansoni antigens. Sera were ex-
amined by enzyme-linked immunosorbent assay (ELISA) for
antibody isotypes reactive to a soluble worm antigen prepa-
ration (SWAP) and SEA. These antigens were prepared ac-
cording to methods previously described.32,33The protein
concentration was determined with a bicinchoninic (BCA) kit
(Pierce, Rockford, IL).
Indirect ELISA. The ELISA was performed using SWAP
and SEA. Briefly, plates (Maxisorb; Nunc, Roskilde, Den-
mark) were coated with 100 ?l of SWAP or SEA at a con-
centration of 5 ?g/ml in carbonate-bicarbonate buffer (pH
9.6). The plates were then sealed and incubated overnight at
4°C. The next day the plates were washed with 0.15 M phos-
phate-buffered saline (PBS, pH 7.2). The plates were blocked
with 0.15 M PBS, 0.05% Tween 20 (PBST), and 10% fetal
bovine serum for 1 hr. The plates were then decanted and 100
?l of sample sera was added in duplicate two-fold dilutions
(from 1:100 to 1:800). The plates were covered and kept over-
night at 4°C. The plates were then washed five times in 0.15
M PBST and 100 ?l of biotin-conjugated anti-human IgG4
(Zymed, San Francisco, CA) was added to each well at a
dilution of 1:1,000 for IgG4. The plates were incubated for 90
min at room temperature and then washed five times with
PBST. One hundred microliters of 1:1,000 streptavidin horse-
radish peroxidase (Amersham, Piscataway, NJ) was then
added to each well and incubated for 90 min at room tem-
perature. One hundred microliters of o-phenylenediamine
(OPD) (Sigma, St. Louis, MO) containing 0.03% hydrogen
peroxide was then added to each well. The reaction was
stopped with 50 ?l/well of 12.5% H2SO4and the optical den-
sity (OD) was measured at 450 nm using an automated
ELISA reader (Molecular Devices, Sunnyvale, CA). The con-
ditions for the measurement of IgE were the same as de-
scribed for IgG4 except that 100 ?l of a 1:500 dilution of
alkaline phosphate-conjugated IgE (Pharmingen, San Diego,
CA) was added to each well and incubated for 90 min at room
temperature. The plates were washed five times in PBST and
100 ?l of 10% diethanolamine in 0.01% MgCl2(pH 9.8) con-
taining 1 mg/ml of p-nitrophenylphosphate was then added.
The plates were then covered and stored in the dark for 2 hr
and the absorbance was measured at 405 nm.
IgG4 TO EGG ANTIGENS AND S. MANSONI MORBIDITY
Quality control of indirect ELISA. Each plate contained
three controls to check for non-specific binding of conjugate,
chromogen, or streptavidin to the plate. A plate was repeated
if one of these controls had an OD > 0.050 nm. In addition,
each plate contained duplicates of a 1:100 dilution of sera
from three S. mansoni-negative individuals from Brazil. A
plate was repeated if the OD for one of these three controls
was greater than 0.050 nm. Pearson product moment corre-
lations were used to determine if wells on the edge of the
plate had higher or lower OD values (e.g., edge effect) than
wells in the center of the plate. Sample duplicates with a
coefficient of variation greater than 10% were retested.
Variation between plates during a single run (interplate varia-
tion) was measured by the coefficient of variation between
the three positive controls on each plate. If the positive con-
trols between plates on a single run had a coefficient of varia-
tion greater than 10%, the entire run was repeated.
Statistical analysis. Data were analyzed by least square
analysis of variance technique using general linear models
procedures. Scheffe’s test was used to determine if a signifi-
cant difference (P < 0.05) could be found between groups
related with intensity of infection, TBM, and IgE and IgG4
levels to SWAP and SEA. The levels of antibody isotypes
were examined after root square transformation of OD +1 to
approximate a normal distribution. For the same reason, we
used natural log transformed TBM values. To allow log trans-
formation of zeros, a value of 1 was added to egg counts.
Logistic regression analysis was used to examine the relation-
ship between age and sex with status of infection and mor-
bidity (periportal fibrosis). The goal of modeling was not to
build a predictive model, but to quantify the association be-
tween study variables. For this analysis, age was categorized
into two groups (? 20 and > 20 years) because of the small
number subjects presenting with periportal fibrosis. All analy-
ses were performed using the Statistical Analysis System, ver-
sion 6.12 (SAS Institute, Inc., Cary, NC).
Characterization of the sample by age, sex, intensity of in-
fection, prevalence, and water contact. Clinical and ultra-
sound examinations were carried out on 267 individuals. The
distribution of patients by age, intensity of infection, preva-
lence, and water contact before treatment is shown in Figure
1. Individuals less than 20 years of age showed the highest
intensity of infection (mean ? 72.4 epg, 95% confidence in-
terval [CI] ? 28.8–182.1) and prevalence (93%, 95% CI ?
87–99). In individuals more than 20 years of age, the intensity
of infection varied from 3.5 epg (95% CI ? 2.6–6.6) to 17.8
epg (95% CI ? 7.9–40.3) and the prevalence varied from
36% (95% CI ? 19–54) to 69% (95% CI ? 52–86). Water
contact ranged from a mean TBM of 128 (95% CI ? 113.9–
142.2) in the 11–20-year-old age group to a mean TBM of 86
(95% CI ? 71.0–104.9) in those more than 60 years old. The
TBM was significantly higher in the 11–20-year-old age group
only in comparison with the group > 60 years old (P < 0.05).
Interestingly, although individuals in the 41–50-year-old
age group showed similar TBM levels compared with those in
the 11–20-year-old age group, the intensity of infection of this
group was 6.3 times less (P < 0.05). Males had a higher in-
tensity of infection than females (26.56 epg, 95% CI ? 18.1–
38.9 versus 14.67 epg, 95% CI ? 10.0–21.5) (P < 0.05), but
water contact values were not significantly different between
genders. Furthermore, water contact values were not signifi-
cantly different in egg-positive and egg-negative individuals.
Characterization of the study sample based on clinical and
sonographic examinations. The association between age and
sex with infection or periportal fibrosis was assessed by logis-
tic regression analysis (Table 1). The results show an associa-
tion between infection and age with the odds of infection 8.16
times greater for individuals ? 20 years old compared with
older (> 20 years old) individuals. No association was found
with gender. Conversely, the presence of periportal fibrosis
was associated with gender, since the odds of morbidity were
3.46 times greater for males than for females, with no asso-
ciation with age.
For further analysis, the study sample was divided into
three groups based on individual clinical and sonographic
records (Table 2). The first group included 204 individuals
with no periportal fibrosis, liver or spleen enlargement (con-
trol group). The second group included 41 individuals without
thickening of the portal vein wall, but presenting with orga-
nopathy (cholecystitis, steatosis, hepatic cysts, or renal cysts),
with or without organomegaly (pathology group). The third
group included 22 individuals with periportal fibrosis
(echogenic thickening ? 0.3–0.5 cm) with or without orga-
nomegaly (fibrosis group).
Our data demonstrate that individuals in the pathology
group were significantly older (41.8 years [95% CI ? 35.5–
48.0]) than individuals in the control group (P < 0.05). Al-
though the fibrosis group showed an intensity of infection at
least 2.5 times greater than the other two groups, it was not
significantly different. There were no significant differences
for TBM between the three groups. Moreover, no significant
differences in TBM were observed between egg-negative and
egg-positive individuals (Table 2), as well as between genders
(Figure 2). However, despite no statistical significance, both
male and female subjects in the fibrosis group showed a mean
intensity of infection at least twice as high as that of the other
two groups (Figure 2).
Analysis of antigen-specific IgG4 and IgE in sera from pa-
tients with/without periportal fibrosis. To investigate whether
humoral immune response would differ between patients with
and without periportal fibrosis, the levels of IgG4 (Figure 3)
and IgE (Figure 4) antibodies to SEA and SWAP were com-
(total body minutes [TBM]) data for Schistosoma mansoni by age
group (n ? 267). *Geometric mean TBM; **Geometric mean eggs
per gram of feces (epg) [ln(epg + 1)]. Values in parentheses are the
number of individuals in each age group.
Intensity of infection, prevalence, and water contact
SILVEIRA AND OTHERS
pared between individuals from the control, pathology, and
fibrosis groups. Our results show that the sera levels of IgG4
against SEA were significantly higher in individuals in the
fibrosis group compared with both the control group and the
pathology group (P < 0.05). No statistic differences were ob-
served for the levels of IgE against SEA or IgG4 or IgE
against SWAP between all three groups. When the patients
were stratified by gender, the levels of IgG4 to SEA were
significantly higher in sera from males than from females in
the control and pathology groups (P < 0.05). Although the
level of IgG4 to SEA was higher in males than in females in
the fibrosis group, the difference was not statistically signifi-
cant. The levels of IgE against SEA were similar between
genders in all three groups. For isotypes against SWAP, the
levels of IgG4 were substantially higher in the sera from
males than from females only in the pathology group (P <
0.05) and for IgE in the control group (P < 0.05).
Increased levels of IgG4 against SEA in egg-negative pa-
tients with periportal fibrosis. To further characterize the hu-
moral immune response in individuals with and without peri-
portal fibrosis, the sera levels of IgG4 and IgE were analyzed
after stratifying the patients based on the presence or absence
of eggs in their feces (Figure 5). Higher sera levels of IgG4
against SEA and SWAP were found in egg-positive individu-
als compared with egg-negative individuals in both the con-
trol (P < 0.05) and the pathology groups (P < 0.05), but not in
the fibrosis group. However, analysis of sera IgG4 against
SEA within the egg-negative subgroup demonstrated higher
levels of this antibody only in the fibrosis group compared
with the control (P < 0.05).
Using the same approach to analyze levels of IgE against
SEA and SWAP, we found higher levels of this isotype in the
egg-positive individuals only in the control group for SEA (P
< 0.05). When serum levels of IgE against SEA and SWAP
within the egg-negative subgroup were analyzed, we found no
differences in these levels between groups. No differences
were observed when the levels of IgG4 and IgE against SEA
and SWAP were analyzed in the egg-positive subgroup.
In this cross-sectional study, we have performed a detailed
descriptive analysis of individuals living in an area where
schistosomiasis is endemic to evaluate the relationship be-
tween demographic features, parasitologic status, behavior
routines, and the immune response in patients with periportal
By characterizing the sample by age, sex, intensity of infec-
tion, prevalence, and water contact, we have found a typical
relationship between age and either prevalence or intensity of
infection. Individuals less than 20 years old showed the high-
est prevalence and intensity of infection compared with other
age groups. The decrease in prevalence and intensity of in-
fection with age has been explained as a multifactorial phe-
nomena that, in part, results from changes in exposure to
cercariae due to decreased contact with infected water,3,34
development of acquired immunity,35,36and differences in
hormones levels or physiologic features, such as thickening of
cutaneous fat observed with aging.37Our data are consistent
with this multifactorial hypothesis, since the decrease in
prevalence and intensity of infection in older subjects could
not be explained by the lower exposure to infected water in
all age groups. The lower TBM (water contact) observed in
the 31–40-, 51–60-, and < 60-year-old age groups could ac-
count for their lower prevalence and intensity of infection.
However, individuals in the 21–30- and 41–50-year-old age
groups, regardless of similar TBM, showed decreased preva-
lence and less intensity of infection compared with individuals
less than 20 years of age (Figure 1). Thus, a difference in TBM
does not explain an age effect in prevalence and intensity of
infection in the Melquı ´ades study sample. As has been sug-
gested by other studies in areas where schistosomiasis is en-
Odds ratios (ORs) and 95% confidence intervals (CIs) for age and sex in relation to morbidity or infection with Schistosoma mansoni
No. (%) OR (95% CI)No. (%)OR (95% CI)
Age group (years)
80 (92.0)1.20 (0.48–2.98)8.16 (3.56–18.68)
Descriptive analysis for individuals infected with Schistosoma mansoni stratified by periportal fibrosis, age, intensity of infection, and
Group Echogenicity† Organomegaly (%)No. Age (years) epg‡
Total Egg +Egg −
* Geometric mean of total body minutes (ln [TBM]). n ? number of individuals.
† Periportal fibrosis (cm).
‡ epg ? geometric mean eggs per gram of feces (ln [epg + 1]).
§ Difference between pathology and control groups (P < 0.05).
IgG4 TO EGG ANTIGENS AND S. MANSONI MORBIDITY
demic, age-acquired immunity could also play a role in the
prevalence and intensity of infection.8,38–42
Periportal fibrosis detected by ultrasonography has been
considered the most important indicator for morbidity in hu-
man schistosomiasis.43,44In this study, data from clinical and
ultrasonographic examinations allowed us to identify in a
population of 267 a group of 22 individuals with echogenic
lesions indicating severe chronic schistosomiasis and another
group of 41 individuals with organopathy but without peri-
portal fibrosis. Using a logistic regression model, we did not
find any association of age with morbidity, but we observed
an association between age and infection as previously de-
scribed for human schistosomiasis.45However, 77% of the
individuals with periportal fibrosis were male, with an odds
ratio of 3.46, compared with females (Table 1). Interestingly,
TBM was similar for both genders (Figure 2). Similar asso-
ciations between gender and morbidity have been reported
for S. mansoni and S. haematobium infections.45–47
In addition to gender, it has also been proposed that severe
schistosomiasis is more often observed in subjects with a
higher intensity of infection.1,21In this regard, our results
show that patients in the fibrosis group have increased inten-
sity of infection in comparison with the pathology and the
control groups (Table 2). Moreover, we observed that one
egg antigen (SEA) (A) and an adult worm antigen preparation
(SWAP) (B) in sera from patients from the control, pathology, and
fibrosis groups. The levels of IgG4 against SEA from the fibrosis
group were significantly higher than those in the control and pathol-
ogy groups (P < 0.05). Data points are optical density (OD) values at
450 nm. The horizontal lines indicate the arithmetic mean.
Levels of IgG4 against Schistosoma mansoni solubleFIGURE 5.
soluble egg antigen (SEA) and an adult worm antigen preparation
(SWAP) in sera from patients from control (I), pathology (II), and
fibrosis (III) groups after subgrouping the patients based on the pres-
ence (open bars) and absence (solid bars) of eggs. Bars show the
mean ± 95% confidence intervals of optical density values read at 450
and 405 nm.
Levels of IgG4 and IgE against Schistosoma mansoni
water contact (total body minutes [TBM]) data for Schistosoma man-
soni in individuals from the control, pathology, and fibrosis groups
separated by sex. *geometric mean [ln(TBM)]; **geometric mean
epg [ln(epg + 1)]. Data points and vertical lines show the mean ±
Intensity of infection (eggs per gram of feces [epg]) and
antigen (SEA) (A) and an adult worm antigen preparation (SWAP)
(B) in sera from patients from the control, pathology, and fibrosis
groups. Data points are optical density (OD) values at 405 nm. There
was no difference between the groups. The horizontal lines indicate
the arithmetic mean.
Levels of IgE against Schistosoma mansoni soluble egg
SILVEIRA AND OTHERS
year after treatment, 23.1% of the patients in the fibrosis
group had become reinfected in contrast with 9.8% and 6.9%
of patients from control and pathology groups, respectively.
In general, it has been difficult to demonstrate a relation-
ship between intensity of infection and morbidity. Both posi-
tive and negative associations have been reported.48–51Inter-
pretation of this discrepancy has been extensively discussed
and includes the reduction in egg excretion leading to an
underestimation of parasite load in patients with periportal
fibrosis, the chronic evolution of the disease that does not
synchronize morbidity and the peak of infection intensity, and
differences on the clinical phenotypes counted as morbidity
markers, such as hepatosplenomegaly or fibrosis. We suggest
that the intensity of infection, although important in disease
progression, is not the only critical factor in the development
of fibrosis and other components such as the immune re-
sponse and host genetic factors may play critical roles.
Most immunologic studies on S. mansoni have focused on
the relationship between the immune response and develop-
ment/maintenance of different clinical forms of the disease or
the mechanisms of resistance and susceptibility to infection or
reinfection.13,15,16The current study adds to these studies by
examining the association between humoral immune re-
sponse and the development of periportal fibrosis.
Early studies comparing anti-SEA IgG4 before and after
chemotherapy demonstrated that the level of this isotype de-
creased following a decrease in egg output.52In this paper, we
are dealing with two supposedly causal variables associated
with the intensity of infection (Figure 3): fibrosis and the
levels anti-SEA IgG4. To address this confounding question,
the anti-SEA IgG4 levels in our study was examined after
stratifying the population into two groups: egg-negative and
egg-positive. As shown in Figure 5, the association between
anti-SEA IgG4 and fibrosis still remained significant among
individuals without eggs in their stool. The current literature
links levels of anti-SEA IgG4 with susceptibility,13,18,19but
our data suggest that the levels of IgG4 may also be involved
in the development of fibrosis. These patients, although egg-
negative, live in same endemic area, have similar water con-
tact as the infected individuals, and presented with high pe-
ripheral blood mononuclear cell proliferative responses to
adult worm and eggs antigens, as well as with cytokine syn-
thesis in their cell culture supernatants (A. M. S. Silveira, G.
Gazzinelli, L. F. Álvares-Oliveira, J. Bethony, A. Gazzinelli,
C. Carvalho-Queiroz, M. C. B. A´lveres, F. C. L. Silva, A.
Prata, P. T. LoVerde, and R. Correa-Oliveira, unpublished
data). Furthermore, the coefficient of variability of the Kato
quantitative method for patients with less than 20 epg is very
high (89.55);53thus, patients with a low intensity of infection
may be not detected. These data suggest that these egg-
negative individuals are infected or previous sensitized by S.
mansoni. In relation to fibrotic, egg-negative patients, the
possibility that the abnormal echogenicity reported is caused
by S. mansoni infection is very high because 8–12 months
after chemotherapy, the lesions detected by ultrasonography
disappeared (T. V. B. Magalha ˜es, A. M. S. Silveira, M. C. B.
A´lvares, and G. Gazzinelli, unpublished data).
In conclusion, IgG4 specific for SEA was associated with
the initial stages of fibrosis, which was graded by the
echogenic pattern of the periportal tree by ultrasound. This
allowed the identification of the critical step in the progres-
sion of disease from mild to severe fibrosis.
Acknowledgments: We thank Maria de Fa ´tima da Silva, Marlucy
Rodrigues Lima, and Lilian Cardoso Moreira for field and laboratory
expert technical assistance, and Dr. Olindo Assis Martins Filho for
valuable comments on the manuscript.
Financial support: This work was supported by grants from the Pro-
grama de Apoio a Grupos de Excele ˆncia-PRONEX/CNPq/FINEP
(Brazil), the National Institutes of Health-NIH-ICIDR (AI-45451-
01), the Fundac ¸a ˜o de Amparo a Pesquisa do Estado de Minas Gerais-
FAPEMIG (Brazil), the Conselho Nacional de Desenvolvimento Ci-
entı ´fico e Tecnolo ´gico-CNPq (Brazil), and the UNDP World Bank/
WHO Special Program for Research and Training in Tropical
Diseases. Jeffrey Bethony was supported by an International Re-
search Scientist Development Award (IRSDA) (1K01 TW00009-01)
from the John C. Fogarty International Center of the National Insti-
tutes of Health.
Authors’ addresses: Alda M. S. Silveira, Departamento de Bio-
quı ´mica e Imunologia, Instituto de Cie ˆncias Biolo ´gicas, Universidade
Federal de Minas Gerais, Avenida Antonio Carlos 6627, 31270-901,
Belo Horizonte, Minas Gerais, Brazil. Jeffrey Bethony, Henrique L.
Guerra, Rodrigo Correa-Oliveira, and Giovanni Gazzinelli, Centro
de Pesquisas Rene ´ Rachou, Fundac ¸a ˜o Oswaldo Cruz, Avenida Au-
gusto de Lima 1715, Barro Preto, 30190-002, Belo Horizonte, Minas
Gerais Brazil. Telephone: 55-31-3295-3566, Fax: 55-31-3295-3115.
Andrea Gazzinelli and Helmut Kloos, Escola de Enfermagem, Uni-
versidade Federal de Minas Gerais, Avenida Alfredo Balena 190,
CEP 30130-100, Belo Horizonte, Minas Gerais Brazil. Lu ´cia Alves de
Oliveira Fraga, Laborato ´rio de Imunologia, Centro de Cie ˆncias Bio-
lo ´gicas e da Sau ´de, Universidade Vale do Rio Doce, Campus II, BR
259, Km 3, S/N˚, Bairro Capim, Caixa Postal 295, 35024-820, Gover-
nador Valadares, Minas Gerais, Brazil. Maria Carolina B. Alves,
Centro de Diagno ´stico, Santa Casa de Miserico ´dia de Belo Hori-
zonte, Avenida Francisco Sales 1111, Santa Efige ˆnia, 30150-221, Belo
Horizonte, Minas Gerais Brazil. Aluizio Prata, Medicina Tropical,
Faculdade de Medicina do Tria ˆngulo Mineiro, Caixa Postal 118,
38001-970, Uberaba, Minas Gerais, Brazil. Philip T. LoVerde, De-
partment of Microbiology, School of Medicine and Biomedical Sci-
ences, State University of New York, 138 Farber Hall, 3435 Main
Street, Buffalo NY 14214-3000.
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