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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 1, 1996, pp. 87-94
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1),
Jan/Feb. 1996
Trypanosama cruzi Defined Antigens in the Serological
Evaluation of an Outbreak of Acute Chagas Disease in Brazil
(Catole do Rocha, Paraiba)
Eufrosina S Umezawa, Maria Aparecida Shikanai-Yasuda*, Arthur
Gruber**, Vera L Pereira-Chioccola***, Bianca Zingales**
Instituto de Medicina Tropical, Universidade de Sao Paulo, Av.
Dr. Eneas de Carvalho Aguiar 470, 05403-000 Sao Paulo, SP, Brasil
*Departamento de Doencas Infecciosas e Parasitárias, Faculdade
de Medicina, Universidade de Sao Paulo, Sao Paulo, Brasil
**Departamento de Bioquimica, Instituto de Quimica, Universidade
de Sao Paulo, Sao Paulo, Brasil
***Instituto Dante Pazzanese de Cardiologia, Sao Paulo, Brasil
Code Number: OC96014
Sizes of Files:
Text: 32K
No associated graphics
This investigation was supported by grants from FAPESP, CNPq,
Fundacao Nacional da Saúde, LIM-49-FMUSP and UNDP/World Bank/WHO
Special Programme for Research and Training in Tropical
Diseases
Immunoglobulin G and M humoral response to recombinant protein
B13 and glycoconjugate LPPG Trypanosama cruzi defined
antigens was evaluated by ELISA in 18 patients in the acute phase
of Chagas disease, who were contaminated on the same occasion.
LPPG showed 100% positivity detecting both IgM and IgG
antibodies, while positivity of 55-65% was observed for B13. An
epimastigote alkaline extract (EPI) also showed high sensitivity
for acute IgM (100%) and IgG (90%) antibodies. However LPPG had
better discriminatory reactivity since with EPI two patients
showed negative IgG and several other sera presented OD values
for IgG and IgM antibodies very close to the cutoff. Thus, it is
suggested that detection of IgM antibodies by LPPG may be used
for diagnosis of the acute phase of Chagas disease. An intense
decline of IgG and IgM antibodies to the three antigens was
observed in response to anti-T. cruzi chemotherapy in all
acute phase patients. After treatment, six (30%) individuals
maintained IgG positivity to EPI, LPPG, and B13 with lower
reactivity than that measured at the acute phase. For comparison,
serology of a group of 22 patients in the chronic phase of Chagas
disease and also submitted to chemotherapy was determined.
Positive IgM antibodies to EPI, LPPG and B13 were detected in
only 5-9% cases. In all chronic-phase patients IgG antibodies
highly reactive to the three antigens were present and no
significant decrease resulted after benznidazole administration.
These observations reinforce previous reports that treatment in
the acute phase may reduce or eliminate the parasite.
Key words: Chagas disease - acute phase - serology -LPPG - B13
recombinant protein - Trypanosama cruzi
Chagas disease, caused by Trypanosama cruzi, is a
widespread illness in Latin America where 16-18 million people
(Moncayo 1993) are affected. The most important T. cruzi
transmission mechanism to humans is throughout infected
triatominesÆ feces. The second most important route is
blood transfusion. Human migration from endemic areas to urban
centers is providing a rising risk of transfusional Chagas
disease in all Latin America and in non-endemic countries.
Congenital transmission occurs most frequently by colonization
of placenta by the parasite. The incidence of congenital
transmission varies according to different circumstances and
geographical situations. At present, this problem is deserving
the attention of governmental health programs in few Latin
America countries. Less important mechanisms of transmission are
accidental laboratory contamination and organ transplantation
from infective donors. In this case, the problem is exacerbated
by the immunosuppressive therapy generally administered to the
organ recipient.
In Brazil at least three reports suggest T. cruzi
transmission by oral route. The first describes an epidemic
outbreak in Teutonia with 17 persons infected simultaneously,
probably due to the ingestion of contaminated meal with urine
from infected marsupials (Dias 1979). Another outbreak was
observed in the State of Para where 4 persons seem to have been
infected through the ingestion of food contaminated with
triatomine excreta (Dias 1979). A third outbreak was reported in
the State of Paraiba in the municipality of Catole do Rocha in
October 1986, when 26 individuals acquired acute Chagas disease
most probably by drinking sugar-cane juice contaminated with
feces of triatomines or marsupial secretion (Shikanai-Yasuda et
al. 1991).
Acute phase in Chagas disease is characterized by patent
parasitemia which is followed by a life-long chronic phase with
subpatent parasitemia (Brener & Camargo 1982). The small number
of detected human acute cases and the short duration of this
phase are obstacles for accurate clinical investigations at this
stage of the disease. It has been shown that specific
chemotherapy applied in the acute phase, leads in many cases to
successful cure (Rassi & Luquetti 1992).
Diagnosis of acute phase is mainly accomplished by detection of
the parasite either by direct blood examination or indirectly by
xenodiagnosis, hemoculture and inoculation in experimental
animals. Most of theses tests are time-consuming, show low and
variable sensitivity and can only be done in specialized
centers.
Antibodies, both IgG and IgM, can be detected in serum usually
following the first clinical manifestation of the acute phase of
T. cruzi infection. In the succeeding chronic phase, IgG
antibodies are to be regularly found in serum (Camargo & Amato
Neto 1974, Schmuñis et al.1978, Matsumoto et al.1993).
The antigens commonly used in serodiagnosis of Chagas disease are
fixed epimastigote forms or a complex mixture of proteins and
glycoconjugates extracted from whole parasites (Camargo 1992).
In the last years, efforts have been made for development of new
diagnostic tests employing purified antigens, recombinant
proteins or synthetic peptides in order to make a more
reproducible and specific diagnosis of Chagas disease (Franco da
Silveira 1992).
In this study we present the evaluation of IgM antibodies
significance for the serological diagnosis of the acute phase of
Chagas disease. We investigated the IgM and IgG appearance in a
group of individuals contaminated with T. cruzi in a
festive reunion in Catole do Rocha and those who developed the
acute phase. All individuals were submitted to specific treatment
with benznidazole, as described in the original report which also
presents the clinical characteristics of these patients
(Shikanai-Yasuda et al. 1991). This group shows extremely
interesting and unique features since it constitutes a
homogeneous sample in reference to the date and route of
contamination (possibly with the same strain(s) of T.
cruzi); chronology of appearance of the acute phase and
application of the same chemotherapeutic protocol. For the
serological evaluation we used an alkaline epimastigote extract
(Lissaldo et al. 1994) and two purified antigens: a recombinant
protein, denominated B13 (Gruber & Zingales 1993) and a T.
cruzi glycoconjugate LPPG (lipopeptidophos-phoglycan)
(Lederkremer et al. 1990, Zingales et al. 1993). Data are
compared with serology in a group of chronic chagasic patients
before and after chemotherapy.
MATERIALS AND METHODS
Human sera - Group I: human sera were obtained from 18
individuals who acquired Chagas disease on the same occasion in
Catole do Rocha (Paraiba, Brazil) (Shikanai-Yasuda et al. 1991).
Details of these patients are shown in Table I. All individuals
received benznidazole in a 7 mg/kg/day schedule for children and
5 mg/kg/day for adults during 60 days (Shikanai-Yasuda et al.
1991). After chemotherapy, all patients showed negative
xenodiagnosis as determined by a single application test. Sera
were collected on the 54th day after contamination and three
years after the end of chemotherapy. Two additional individuals
(one male and one female) who acquired Chagas disease by
accidental laboratory contamination were also submitted to
chemotherapy and included in the analysis. Group II: human sera
were obtained from 22 patients in the chronic phase of Chagas
disease from Instituto Dante Pazzanese de Cardiologia (Sao
Paulo). The patients (15 males and 7 females), originary from
different regions of Brazil, but presently living in non-endemic
areas, presented positive serology for Chagas disease and 50% of
them had positive xenodiagnosis. Sera were collected before
treatment with benznidazole (same protocol as above) and 2-7
years afterwards (Pereira et al. 1989). All patients had negative
xenodiagnosis after this period. Group III: the control group
included 20 sera from the blood bank of Hospital das Clinicas
(Sao Paulo) with negative serological reactions to Chagas
disease. Sera were inactivated at 56 C for 30 min and stored in
50% glycerol at -20 C .
Antigens- Epimastigote forms of T. cruzi (Y strain)
grown in liver infusion tryptose (LIT) medium were lysed with 0.3
N NaOH overnight at 4 C with stirring. After neutralization (pH
7.2) with 0.3 N HCl, 2 mM phenylmethylsulfonylfluoride was added
and the homogenate was centrifuged at 12,000 g for 1 min. The
supernatant, denominated EPI (Lissaldo et al. 1994), was stored
at -70 C. The recombinant B13 T. cruzi protein fused to
b-galactosidase was obtained and purified as previously described
(Gruber & Zingales 1993). Lipopeptidophosphoglycan (LPPG) was
purified from epimastigote forms by the butanol method
(Lederkremer et al. 1990). Protein was measured by a Lowry
modified method (Hartree 1972). Neutral sugars were determined
by the phenol-sulfuric acid method (Dubois et al. 1956).
Enzyme-linked immunosorbent assay (ELISA) - Plates (Nunc,
PolySorp, Roskilde, Denmark) were alternatively coated with: 300
ng protein/well of EPI; 50 ng protein/well of B13 recombinant
protein or 100 ng hexose equivalent/well of LPPG. B13 and LPPG
were dissolved in PBS, pH 7.2. EPI was solubilized in
carbonate-bicarbonate buffer 0.05M, pH 9.6. The ideal
concentration of each antigen at different serum dilutions was
established from saturation curves. After antigen incubation for
2 hr at 37 C and for 18 hr at 4oC, plates were blocked with 5%
defatted milk (Nestle) in PBS-Tween 20 (0.05%) for 1 hr at room
temperature. Reactivity was measured incubating 50 ml of coded
sera diluted in 1% defatted milk-PBS-Tween 20 for 1 hr at 37 C.
Serum dilutions are specified in Results. After six washes with
PBS-Tween 20, peroxidase-conjugated goat anti-human IgG or IgM
(Sigma) was added at dilutions of 1:6,000 and 1:5,000,
respectively, for 1 hr at 37 C. The substrates 0.05% H2O2 and
o-phenyle-nediamine (0.4 mg/ml) were added and the reaction was
allowed to develop in the dark for 30 min at room temperature.
The reaction was stopped by the addition of 4 N HCl. The optical
density (OD) was measured at 492 nm in a Multiskan plate reader
(Titertek, Flow). In all tests non-chagasic sera (Group III) were
included. Experiments were performed at least twice and on
different days in duplicates for each serum dilution.
Statistical analysis - Samples recorded OD were analyzed
by StatgraphicsTM 5.1 (Statistical Graphic Corp. 1991) Software.
The cutoff of each reaction was calculated as the mean OD of
twenty non-chagasic sera plus 3SD. Comparison between tests and
samples was performed analyzing all groups by multifactor
analysis of variance (ANOVA), with Bonferroni test. Data are
expressed as the mean OD and the significance of the difference
was considered when the probability of equality was lower than
0.05 (Gardner & Altman 1986).
Complement-mediated lysis (CML) - the assay was done as
described (Umezawa et al. 1993).
RESULTS
Table I shows the individual characteristics of the 18 acute
phase chagasic patients from Catole do Rocha as well as data on
the time of manifestation of acute phase symptoms and period of
fever. The electrocardiographic study in 15 patients performed
from the 33rd to the 55th day of illness gave normal results for
10 individuals. Fourteen individuals were submitted once to
xenodiagnosis and ten (71.4%) were positive. Two of the four
xenodiagnosis negative patients were already under specific
treatment when the test was applied (Table I). Serological
evaluation of these patients was initially done by indirect
immunofluorescence (IIF) tests with sera collected 54 days after
contamination. IgM antibodies were negative in two individuals
while IgG antibodies were detected in all patients (data not
shown). Lytic antibodies were detected in only three patients and
in very low levels. Inconclusive results were obtained for the
other individuals since their sera promoted direct trypomastigote
lysis in the absence of complement or in the presence of
heat-inactivated complement (Table I).
Table 1
Sensitivity of QBC test in a field study of patients infected in
transmission areas of southeastern (State of Bolivar) and south
(State of Amazonas) Venezuela
-------------------------------------------------------
Group^a Number of subjects Number of patients
studied QBC+/GTS+^b
-------------------------------------------------------
2 141 69/70 (99%)^c
3 37 11/11 (100%)
-------------------------------------------------------
a: Group 2, 141 miners of State of Bolivar; Group 3, 37
indigenous children of State of Amazonas
b: QBC = quantitative buffy coat (QBC^r) analysis and GTS =
Giemsa stained thick blood smears
c: number in bracets indicate sensitivity for the QBC - test
relation to GTS.
Table II
Comparison of QBC^a test and GTS^b to determine prevalence (%)
of malaria in a field study of individuals living in endemic
areas of Venuzuela
---------------------------------------------
QBC+ QBC- QBC+ QBC-
Group^c GTS+ GTS- GTS- GTS+
---------------------------------------------
2 49 32 18 1
3 30 59 11 -
---------------------------------------------
a,b: QBC=quantitative buffy coat (QBC^r) analysis and GTS=Giemsa
stained thick blood smears
c: Group 2: 141 miners (State of Bolivar); Group 3: 37 indigenous
children living in Sate of Amazonas.
TABLE III
Sensitivity of the QBC^a test as compared to GTS^b in patients
with Plasmodium falciparum or P.vivax malaria
---------------------------------------------------------------
Patients Sensitivity^d (%) of Range of parasitaemia
infected with^c the QCB text (parasites/ul)
---------------------------------------------------------------
P. vivax (n=63) 100 200-1500
p. falciparum (n=32) 97 100-200
---------------------------------------------------------------
a,b: QBC=quantitative buffy coat (QBC^r) analysis and GTS=Giemsa
stained thick blood smears
c: identification of parasite species was done by microscopic
examination of Giemsa stained thin smears
d: the test sensitivity defined as the percentage of positive GTS
that were positive bt the QBC test.
Levels of IgM antibodies to B13 recombinant protein, to LPPG
glycoconjugate and to an epimastigote alkaline extract (EPI) were
determined by ELISA in 20 acute phase chagasic (APC) patients:
18 from Catole do Rocha (Table I) and two individuals
accidentally contaminated with the Y strain of T. cruzi.
The serum dilutions used for each target antigen were previously
established by cross-titration in order to give the best
discriminatory results. These dilutions are: 1:200 for EPI; 1:100
for LPPG and 1:50 for B13 protein.
IgM reactivity data in Fig. and Tables I and II indicate
positivity of 100% for the APC sera (Group I, n=20) with EPI and
LPPG and of 55% with recombinant B13 protein. Analysis of the
mean OD, based on 95% confidence intervals, was significant for
the three antigens (Fig.). The highest reactivity was displayed
by LPPG (Table II). Sera at 1:100 dilution gave a mean OD for EPI
of .65+/-.20 (data not shown).
Analysis of IgG reactivity in APC sera (Group I, n=20) is
summarized in Table II. The positivity of 20 pre-therapeutic
patients was 90% for EPI, 100% for LPPG and 65% for B13. In this
case also, reactivity to LPPG was the highest. Analysis of
variance was significant between LPPG and EPI or LPPG and B13
(data not shown).
IgM reactivity was assessed three years after chemotherapy of
Group I patients and results are shown in Fig. where individual
serum samples are linked by transversal lines. Data for the two
accidentally contaminated patients are indicated with asterisks.
A clear decline of IgM antibodies was observed in all patients,
but positivity in low titers was observed in 20% of the
individuals for EPI and 30% for LPPG (Table II).
IgG analysis of Group I-treated patients (Table II) showed a
positivity of 45% to EPI (.36+/-.13) and LPPG (.48+/-.20) and 30%
to B13 (.36+/-.10). Six patients (No. 1, 3, 7, 9, 12, 14 in Table
I) remained positive to the three antigens.
For comparison, a group of 22 chronic phase chagasic (CPC)
patients from Instituto Dante Pazzanese de Cardiologia (Sao
Paulo) was analyzed before and after specific chemotherapy (Group
II). Serological data are summarized in Table II. IgM positivity
for the three antigens was detected in 5-9% pre-therapeutic
individuals and 100% of IgG antibodies was observed in samples
collected before and after chemotherapy, with high reactivity to
the three antigens. It was verified that CPC patients submitted
to chemotherapy present IgG mean OD values approximately three
times higher than those displayed by the six patients of Group
I who maintained positive serology after chemotherapy. The
confidence intervals showed no statistical difference between
pre- and pos-therapy IgG reactivity to the three antigens (data
not shown).
DISCUSSION
An intense and early humoral response against LPPG of both IgM
and IgG antibodies is observed in all acute phase patients from
Catole do Rocha, as well as in the two individuals who acquired
Chagas disease by accidental laboratory contamination. The high
antigenicity of LPPG can be ascribed to the parasite membrane
location of this glycoconjugate and to the presence of
galactofuranose in this molecule (Lederkremer et al. 1980,
Golgher et al. 1993). In fact, galactose in the furanoic
configuration is a rare carbohydrate in nature, not represented
in glycoconjugates from vertebrates (Colli 1992). Previous
reports showed IgG antibodies to galactofuranose in sera of
chronic chagasic patients (Schnaidman et al. 1986, Golgher et al.
1993, Zingales et al. 1993). Data presented in this study suggest
that screening of IgM antibodies directed to the
galactofuranose-containing LPPG could be of use to diagnose
Chagas acute phase. For definitive conclusion, a more
representative sample of acute sera has to be analyzed. If the
above observation is confirmed, then measurement of IgM
antibodies to LPPG could be applied for diagnosis of acute phase
in conjunction or substitution of parasitological detection,
obtained by procedures which last 30-60 days. As a clear
advantage, the early diagnosis of acute phase would determine the
immediate application of specific chemotherapy with elevated
indices of cure (Rassi & Luquetti 1992).
Comparison of acute phase antibody reactivity to EPI and LPPG
(Tables I, II) indicates that the alkaline extract of
epimastigotes is also a good target antigen for both IgM and IgG.
However, analysis of data in Fig. shows that IgM antibodies from
some sera gave OD values for ELISA with EPI close to the cutoff
value. In contrast, for LPPG the sera with lowest reactivity have
OD values at least two times the value of the cutoff for this
antigen. This indicates that LPPG has a better discriminatory
reactivity for IgM than EPI. As an additional advantage of LPPG
over EPI, the use of a purified antigen would allow more
reproducible results in serological assays. Furthermore, LPPG can
be easily and reproducibly purified with high yield from
epimastigote forms of several strains of T. cruzi (Golgher
et al. 1993).
A great individual variation in the IgM and IgG reactivity to
LPPG (and EPI) was observed in acute phase patients from Catole
do Rocha (Table I, Fig.). Such behavior does not correlate with
the sex or age of the patient or with clinical data (Table I).
Assuming that most probably the 18 individuals of this group were
infected by the same T. cruzi strain(s), the results favor
the hypothesis that differences in antibody reactivity are
dependent on host genetic factors. Alternatively, it could be
conceived that patients were contaminated with different doses
of parasites.
Recombinant protein B13 shows high specificity and sensitivity
for IgG antibodies in chronic chagasic patients (Table II),
confirming previous observations (Gruber & Zingales 1993,
Zingales et al. 1993). Data in the present study indicate that
B13 protein should not be used for acute-phase serologic
diagnosis since a sensitivity of only 55-65% for both IgM and IgG
antibodies was observed (Table II).
All patients of Group I submitted to chemotherapy show an intense
decline of IgM and IgG antibodies to the target antigens when
assayed three years after treatment (Table II). A natural
disappearance of IgM reactivity to total epimastigotes has been
observed when most chagasic patients enter the chronic phase
(Primavera et al. 1990). In the present study it is observed that
30% (6/20) of the ACP individuals show persistent IgG reactivity
to EPI, LPPG and B13 after chemotherapy. However this reactivity
is lower than that measured before treatment (Table II) or when
compared with data of CPC patients of Group II (Table II).
Analysis of individual parameters of these six ACP patients
indicates no common characteristic of sex, age or particular
serological reactivity in the acute phase (Table I). The only
interesting feature is that three of these patients had
electrocardiographic alterations in the acute phase (Table I).
The decline in IgG antibodies to the three antigens observed in
the majority of Group I patients three years after benznidazole
administration, in contrast to what is observed in the chronic
Group II, could be indicative of the efficacy of treatment
reducing or eliminating the parasite in ACP groups. This could
favor previous suggestions that chemotherapy applied at early
stages of infection can be successful (Rassi & Luquetti 1992).
It should be emphasized that we do not intend to propose that
reduction of specific antibodies should be taken per se as
criterion of cure or drug efficacy. Definitive conclusions can
only be achieved by direct and sensitive parasitological
diagnosis by the use of specific T. cruzi DNA probes, as
shown (Wincker et al. 1994).
It has been reported that a specific class of antibodies ('lytic
antibodies') that lyse trypomas-tigotes by a complement-mediated
reaction (CML) show a progressive decrease in chagasic patients
submitted to successful therapy (Krettli et al. 1982, Galvao et
al. 1993). As a consequence it has been proposed that
negativation of lytic antibodies could be considered a criterion
of cure. In the present study, CML reaction was performed in sera
from Group I patients before and after chemotherapy. Table I
shows that only three ACP patients from Catole do Rocha have
lytic antibodies which promote very low percentage of parasite
lysis. In the remaining sera direct lysis of trypomastigotes in
the absence of complement was observed. These results are
coincident with previous report indicating that antibodies from
acute chagasic patients lyse parasite blood forms independently
from the classic or alternative complement pathways (Gazzinelli
et al. 1990). Measurement of lytic antibodies in Group I patients
three years after chemotherapy gave inconclusive results since
a large variation in the percentage of trypomastigote lysis was
observed in the patientsÆ sera and even in the same serum
sample when the reaction was performed on different days (data
not shown). Similar observations were reported by one of us for
chronic chagasic patients (Pereira et al. 1989). Therefore, in
our hands, lytic antibodies could not be used in assessing
treatment efficacy in chagasic patients from Catole do Rocha.
This reinforces the need of sensitive and direct parasitological
diagnosis with DNA probes.
ACKNOWLEDGEMENTS
To Professor Mario Camargo for encouragement and for critically
reading the manuscript. To AR Ferreira for drawing assistance and
MS Nascimento for technical help.
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Received 4 April
Accepted 22 June 1995
Copyright 1995 Fundacao Oswaldo Cruz
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