Impact of aetiological treatment on conventional and multiplex serology in chronic Chagas disease.

Abstract

The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion, achieved many years post-treatment. One of the main limitations in evaluating treatment for chronic Chagas disease is the lack of reliable tests to ensure parasite clearance and to examine the effects of treatment. However, declines in conventional serological titers and a new multiplex assay can be useful tools to monitor early the treatment impact.
Changes in antibody levels, including seronegative conversion as well as declines in titers, were serially measured in 53 benznidazole-treated and 89 untreated chronic patients in Buenos Aires, Argentina with a median follow-up of 36 months. Decrease of titers (34/53 [64%] treated vs. 19/89 [21%] untreated, p<0.001) and seronegative conversion (21/53, [40%] treated vs. 6/89, [7%] untreated, p<0.001) in at least one conventional serological test were significantly higher in the benznidazole-treated group compare with the untreated group. When not only complete seronegative conversion but also seronegative conversion on 2 tests and the decreases of titers on 2 or 3 tests were considered, the impact of treatment on conventional serology increased from 21% (11/53 subjects) to 45% (24/53 subjects). A strong concordance was found between the combination of conventional serologic tests and multiplex assay (kappa index 0.60) to detect a decrease in antibody levels pos-treatment.
Treatment with benznidazole in subjects with chronic Chagas disease has a major impact on the serology specific for T. cruzi infection in a shorter follow-up period than previously considered, reflected either by a complete or partial seronegative conversion or by a significant decrease in the levels of T. cruzi antibodies, consistent with a possible elimination or reduction of parasite load.

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Impact of Aetiological Treatment on Conventional and
Multiplex Serology in Chronic Chagas Disease
Rodolfo Viotti1*, Carlos Vigliano1, Marı ´a Gabriela A´lvarez1, Bruno Lococo1, Marcos Petti1, Graciela
Bertocchi1, Alejandro Armenti1, Ana Marı ´a De Rissio2, Gretchen Cooley3, Rick Tarleton3, Susana
Laucella2
1Chagas Disease and Heart Failure Section, Cardiology Department, Hospital Eva Pero ´n, San Martı ´n, Buenos Aires, Argentina, 2Instituto Nacional de Parasitologı ´a ‘‘Dr.
Mario Fatala Chaben’’, Buenos Aires, Argentina, 3Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens,
Georgia, United States of America
Abstract
Background: The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion,
achieved many years post-treatment. One of the main limitations in evaluating treatment for chronic Chagas disease is the
lack of reliable tests to ensure parasite clearance and to examine the effects of treatment. However, declines in conventional
serological titers and a new multiplex assay can be useful tools to monitor early the treatment impact.
Methodology/Principal Findings: Changes in antibody levels, including seronegative conversion as well as declines in
titers, were serially measured in 53 benznidazole-treated and 89 untreated chronic patients in Buenos Aires, Argentina with
a median follow-up of 36 months. Decrease of titers (34/53 [64%] treated vs. 19/89 [21%] untreated, p,0.001) and
seronegative conversion (21/53, [40%] treated vs. 6/89, [7%] untreated, p,0.001) in at least one conventional serological
test were significantly higher in the benznidazole-treated group compare with the untreated group. When not only
complete seronegative conversion but also seronegative conversion on 2 tests and the decreases of titers on 2 or 3 tests
were considered, the impact of treatment on conventional serology increased from 21% (11/53 subjects) to 45% (24/53
subjects). A strong concordance was found between the combination of conventional serologic tests and multiplex assay
(kappa index 0.60) to detect a decrease in antibody levels pos-treatment.
Conclusions/Significance: Treatment with benznidazole in subjects with chronic Chagas disease has a major impact on the
serology specific for T. cruzi infection in a shorter follow-up period than previously considered, reflected either by a
complete or partial seronegative conversion or by a significant decrease in the levels of T. cruzi antibodies, consistent with a
possible elimination or reduction of parasite load.
Citation: Viotti R, Vigliano C, A´lvarez MG, Lococo B, Petti M, et al. (2011) Impact of Aetiological Treatment on Conventional and Multiplex Serology in Chronic
Chagas Disease. PLoS Negl Trop Dis 5(9): e1314. doi:10.1371/journal.pntd.0001314
Editor: Helton da Costa Santiago, National Institutes of Health, United States of America
Received March 24, 2011; Accepted July 27, 2011; Published September 6, 2011
Copyright: ? 2011 Viotti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (grant P01AI044979 to RLT); National Fund for Science and Technology of Argentina
(FONCYT PICT 05–38188 to SAL) and Bunge and Born Foundation (to SAL). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: rviotti@arnet.com.ar
Introduction
The chronic form of Chagas disease, now considered to be
globalized is prevalent in Latin America as well as in countries
where T. cruzi-infection is not endemic [1–3]. In Argentina, T.
cruzi infection is diagnosed by the conventional serologic tests
indirect immunofluorescence assay (IFI), indirect hemagglutina-
tion (IHA) and ELISA that measure the level of circulating
antibodies against Trypanosoma cruzi antigenic components (gener-
ally intact or whole parasite lysates), with 2 positive tests out of the
3 performed being required to confirm T. cruzi-infection [4]. This
‘best 2 out of 3’ approach is standard for diagnosis of T. cruzi
infection in most endemic countries.
Even though long term follow-up of chronic Chagas disease
patients treated with benznidazole showed that specific chemo-
therapy can prevent the progression of the heart-related pathology
of Chagas disease in several [5–7], but not all studies [8], a wider
use of the available drugs has not been achieved. One of the main
limitations in evaluating treatment for chronic Chagas disease is
the lack of reliable tests to ensure parasite clearance and to
evaluate the impact of treatment on the evolution of lesions in
target tissues. The main criterion for ‘‘cure’’ has been the
conversion to negative serology on all tests performed [9].
However, this result is often not observed until 8 to10 years
post-treatment and then only in approximately 15% of treated
adult subjects [5–7,10]. Clearly, the identification of better
surrogate markers of parasite load decrease or complete parasite
elimination is needed.
We have recently shown that changes in T. cruzi–specific T cell
responses and in antibody responses to individual parasite proteins
as determined using a multiplex assay can be used as early and
effective predictors of the impact of drug treatment in human
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Chagas disease [11]. In addition, several studies have shown that a
fall in conventional serological titers might be also useful to indicate
treatment impact [5,7,9,12]. However, these protocols have not
been exhaustively explored in short post-treatment follow-up
periods. In this study, the changes in antibody levels specific for
T. cruzi were measured by conventional serologic assays and by a
new multiplex assay during a relatively short follow-up period after
treatmentwithbenznidazoleinchronicallyT.cruziinfected subjects.
Methods
Subjects
Three hundred and twenty eight patients assisted in the first
consultation at the Health Care Section for Chagas disease at the
Hospital Eva Pero ´n, Buenos Aires, Argentina were prospectively
evaluated between 2004 and 2009. For inclusion in the study, a
new serological evaluation by IFI, IHA and ELISA assays was
conducted at the "Instituto Nacional de Parasitologı ´a ‘‘Dr. Mario
Fatala Chaben’’, the National Center of Reference for diagnosis of
T. cruzi infection in Argentina. Of these 328 subjects who had a
previous positive serology for T. cruzi in primary health care
centers, 37 were excluded based upon negative tests at the
Instituto Nacional de Parasitologı ´a ‘‘Dr. Mario Fatala Chaben’’.
Fifteen patients with discordant serology, 2 patients with
associated coronary artery disease, 1 patient with HIV co-
infection, and 1 patient living in an endemic area without vector
control were also excluded from the present study. Adult
individuals older than 21 years of age with 3 positive serological
tests for T. cruzi infection, without heart disease (Group 0) or with
mild heart disease (Group I), and serological follow-up of at least
24 months were included in this study. One hundred and thirty
patients did not achieve the minimum follow-up period and were
excluded for further analysis.
Intervention
Aetiological treatment with benznidazole was offered to all
patients, following the recommendations of the national and
international guidelines for Chagas disease [13–15]. Benznidazole
was administered at a dose of 5 mg/kg/day for 30 days. According
to the principle of intention to treat, all patients, including those
who did not complete the 30 day-treatment period (17%), were
considered in the treated group.
Measurements and study variables
A baseline electrocardiogram (ECG) and a 2-D echocardiogram
were performed to stratify the patients according to a modified
Kuschnir classification as follow: Group 0 has positive serology on
3 conventional serological tests, normal ECG and normal
echocardiogram; and Group I includes subjects with positive
serology on 3 tests, abnormal ECG (arrhythmias or conduction
disturbances) and echocardiogram without left ventricular dilation
or dysfunction [16]. Patients in group II (with dilation and left
ventricular dysfunction) and in group III (with heart failure) were
not included due to the low number of subjects with these
conditions.
Conventional serology.
Serology for T. cruzi infection was
performed on admission and repeated yearly. The serological tests
were standardized at the "Instituto Nacional de Parasitologı ´a ‘‘Dr.
Mario Fatala Chaben" where sera samples were received blinded
for treatment and for the clinical stage of the patients. IHA and IFI
assays were reported as reactive from sequential K titer dilutions
between 1/32 and 1/256. ELISA test was considered positive
when mean absorbance at 490 nm was higher than the cut-off
value of 0.200.
The magnitude in serological titer reduction at different time
points post treatment respect to titers at baseline was calculated for
each subject. Decreases in at least 1 titer dilution on IHA and IFI
(reduction of 50%) and at least a 30% reduction on ELISA assays
compared with baseline values were considered as significant
changes. Serological variations were evaluated annually, while the
latest serological data available was considered for global analysis.
The ELISA cut-off of 30% was defined according to the analysis of
sensitivity/specificity and positive likelihood ratios (sensitivity/1-
specificity) to achieve titer decreases in benznidazole-treated
subjects that were significantly different compared with those in
the untreated group. The minimum follow-up period to detect a
decrease of titers or seronegative conversion respective to baseline
serology was also determined.
Conversion to negative serology was confirmed when titers were
lower than 1/32 for IHA and IFI and the mean absorbance at
490 nm in ELISA assays was under 0.200. Because the standard
for determining a diagnosis of chronic Chagas disease as a positive
result is based on at least 2 tests out of 3 conventional serological
tests, we considered the conversion to negative serology on 2 or 3
tests as an indicator of cure.
Multiplex Serologic Assay.
(32 treated and 12 untreated patients) out of the 142 patients
evaluated by conventional serology were screened for antibodies
reactive to a panel of 14 recombinant T. cruzi proteins in a
Luminex-based format, as previously described [17]. Sampling
was performed prior to treatment and then, 2 and 6 months, and
annually after treatment. Serological responses to each individual
T. cruzi protein were considered to have decreased during the
study period if the mean fluorescence intensity for at least one
recombinant protein decreased$40% relative to that of the time 0
(pretreatment) sample assayed concurrently. The multiplex cut-off
of 40% was defined according to the analysis of sensitivity/
specificity and positive likelihood ratios (sensitivity/1-specificity) to
achieve titer decreases in benznidazole-treated subjects that were
significantly different compared with those recorded in untreated
subjects.
Serum specimens of 44 patients
Author Summary
The main criterion for treatment effectiveness in Chagas
Disease has been the seronegative conversion of previ-
ously reactive serology, generally achieved many years
post-treatment. The lack of reliable tests to ensure parasite
clearance and to examine the effect of treatment is the
main difficulty in evaluating treatment for chronic Chagas
disease. Decreases of conventional and non-conventional
serological titers can be useful tools to monitor the early
impact of treatment. We serially measured changes in
antibody levels, including seronegative conversion as well
as declines in titers in 53 benznidazole-treated and 89
untreated chronically T. cruzi-infected subjects. Seronega-
tive conversion as well as decreases of titers was
significantly higher in treated compared with untreated
patients. A strong concordance was found between
decreases of titers of conventional and non-conventional
serologic tests post-treatment, reaffirming the findings.
When seronegative conversion plus decreases of titers
were considered altogether, the impact of treatment was
higher, in a shorter follow-up period than previously
considered. New tools for monitoring the effectiveness of
treatment of chronic Chagas disease are necessary, and the
results showed in this study is a contribution to
researchers and physicians who assist patients suffering
from this disease.
Impact of Treatment in Chronic Chagas Disease
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Statistical analysis
Continuous measures were expressed as mean and standard
deviation or median and interquartile range of 25–75%, whereas
dichotomous variables were expressed as a result/total and
percentage. Chi square test or Fischer’s exact test was performed
to evaluate differences between discrete variables, whereas Student
t test was applied in the study of continuous numerical variables. A
Spearman rank correlation test and kappa index were applied to
compare the decreases in antibody levels specific for Trypanosoma
cruzi after treatment with benznidazole by the Multiplex and
conventional serological tests.
Sensitivity, specificity and positive likelihood ratios (sensitivity/
1-specificity) for detecting decreases of titers were determined for
the Multiplex assay, and for the 3 conventional serological tests
taken together or each serological taken separately. For the
multivariate analysis, the logistic regression model was used,
including all variables with significance (p,0.05) in the Chi square
test and Student t test, as well as gender and years of residence in
endemic areas for being considered clinically relevant.
The relative risk with a 95% confidence interval and the
number needed to treat to achieve a change in post treatment
serology was determined. All statistical analysis was performed
using Analytical Software Statistix 8.0. The protocol was approved
by the review Boards of the Eva Pero ´n Hospital. Signed informed
consent was obtained from all individuals before inclusion in the
study.
Results
Variations in conventional serological tests
We included 142 patients, 85 female and 57 male, mean ages
42.268.4 years. Fifty three patients (37%) were treated with
benznidazole while 89 remained untreated (63%). Patients who
agreed to receive treatment were marginally younger (mean age
treated
group=43.768.1, p=0.006) and had a higher likelihood of
ECG abnormalities compared with untreated subjects (14/53,
26% of treated patients vs. 4/89, 4% of untreated patients,
p,0.001). The median time and interquartile range (25–75%) of
follow-up was 36 months (32–48) for the entire study population
[48 months (36–60) for treated and 36 months (27.5–48) for
untreated patients, p,0.001]; sex (male in treated group=23/53,
43% vs. male untreated group=34/89, 38%, p=0.54) and years
of residence in endemic areas (treated=14.469.9 vs. untreat-
ed=14.569.1, p=0.94) were not significantly different between
treated and untreated patients.
In order to evaluate treatment success, seronegative conversion
and also decreases of titers in at least one test were determined in
the present study. The frequency of decrease in titers as well as the
conversion to negative results on one or more conventional
serologic tests was significantly greater in the treated patient group
compared with untreated individuals (Table 1 and Figure 1).
Complete seroconversion was found in 4 out of 53 (8%) while
seronegative conversion on 2 tests was observed in 7 out of 53
(13%) treated subjects. Because the standard for determining a
diagnosis of T. cruzi infection as a positive result is based on at least
2 out of 3 conventional serological tests, we extended the criteria of
cure to the seronegative conversion on 2 tests, as well. Thus,
conversion to negative serology on 2 or 3 tests was observed in 11
out of the 53 treated subjects (21%).
IHA titers decreased in 22 out of 53 (41%) treated patients and
in 8 out of 89 (9%) untreated subjects, p,0.001. Mean absorbance
by ELISA decreased in 18 out of 53 (34%) treated and in 6 out of
89 (7%) untreated subjects, p,0.001. Finally, a decline in titers by
IFI assays was observed in 18 out of 53 (34%) treated and in 7 out
of 89 (8%) untreated subjects, p,0.001. In treated subjects, the
median follow-up period to detect a decline in antibody levels was
27 months (interquartile range 25 to 75% 16.5–38.5), while a
group=39.768.4 vs.mean ageuntreated
Table 1. Impact of treatment with benznidazole on conventional serology in chronic Chagas disease.
Serological changes (a)Treated(b) (N=53) Untreated (b) (N=89)p Relative risk (CI 95%)
Number needed
to treat
Decrease of titers on at
least 1 serological test
34 (64) 19 (21)
,0.0013.00 (1.92–4.69)2
Decrease of titers on 1
serological test
21 (40) 17 (19)0.007
Decrease of titers on 2
serological tests
12 (23) 2 (2)
,0.001
Decrease of titers on 3
serological tests
1 (2)0 (0)0.19
Seronegative conversion
on at least 1 test
21 (40) 6 (7)
,0.0018.91 (3.81–20.85)3
Seronegative conversion
on 1 test
10 (19)6 (7)0.03
Seronegative conversion
on 2 tests
7 (13)0 (0%)
,0.001
Seronegative conversion
on 3 tests
4 (8) 0 (0%) 0.009
Seronegative conversion on 2
and 3 tests (Indicator of cure)
11 (21)0 (0%)
,0.00118.47 (2.45–130.06)5
Impact of treatment (c) 24 (45) 2 (2)
,0.00120.1 (4.96–81.87)2
(a) The latest available serological data at the end of the study was considered for analysis.
(b) The figures represent the number of subjects with the indicated changes out of the total evaluated (%).
(c) Includes subjects with decrease of titers or seronegative conversion on 2 or 3 tests.
doi:10.1371/journal.pntd.0001314.t001
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median of 24 months (interquartile range 25 to 75% 21–27.7) was
required to identify seroconversion on 2 or 3 tests.
The aetiological treatment with benznidazole was the only
variable that correlated with the decline of titers or seronegative
conversion as determined by multivariate analysis (Table 2). None
of the treated or untreated patients showed progression of the
heart disease (new ECG changes or evolution to more severe
clinical stages) during the follow-up period of this study.
Altogether, these findings show that taking into account either
conversion to negative serology on 2 or 3 tests and decreases of
titers on 2 or 3 tests, the impact of treatment on conventional
serology might increase to 45%.
Comparison of conventional serology and multiplex
analysis
We have previously shown that treatment with benznidazole
decreased antibody responses specific for a group of T. cruzi
recombinant proteins as detected by a multiplex assay [11].
Decreases of antibody titers by conventional serology, as defined
in Material and Methods, and by multiplex analysis were compared
in a subset of individuals, 32 benznidazole-treated and 12 untreated
subjects (50% male/female with a mean age of 40.767.7) during
the follow-up period of the study. Seventy five percent of the
subjectsassessed showedsimilar trendswith respect tothe combined
3 conventional serologic tests and the multiplex assay (simple
concordance=75%; Kappa index=0.60). In addition, a positive
correlation was found between decreases in antibody titers
measured by conventional serological tests and the multiplex
(r=0.59,p,0.001).Overall,26/32treatedsubjects(81%)and1/12
untreated subjects (8%) showed a decline in antibody responses
considering both conventional serological tests and the multiplex
assay (Table 3). Although the overall results in this set of subjects
were similar using the multiplex and combined conventional
serological tests, the multiplex test was superior in detecting
serological changes when compared to each conventional serologic
test separately (i.e. 7 subjects showed titer decreases by multiplex
while no alterations were recorded by ELISA, Table 3).
Discussion
This study shows the unquestionable impact of aetiological
treatment with benznidazole on conventional and multiplex
serology for T cruzi-infection by three years postreatment. A
Figure 1. Percentages of titers decrease and seronegative conversion in treated and untreated chronically T. cruzi-infected
subjects(a). (a) The latest available serological data at the end of the study was considered for analysis. Q=decrease of titers on at least 1 test;
1Q = titer decrease on 1 test; 2 Q = titer decrease on 2 tests; 3 Q = titer decrease on 3 tests; (2) = seronegative conversion on at least 1 test; 1
(2) = seronegative conversion on 1 test; 2 (2) = seronegative conversion on 2 tests; 3 (2) = seronegative conversion on 3 tests; 2+3 (2) =
seronegative conversion on 2 or 3 tests.
doi:10.1371/journal.pntd.0001314.g001
Table 2. Logistic regression for decrease of titers and seronegative conversion on conventional serological assays in T.cruzi-
infected subjects.
Decrease of titers Seronegative conversion
Coefficient b
PCoefficient b
P
Age0.03 0.260.019 0.55
Gender 0.200.64 0.340.48
Group 0 (without heart disease) 1.110.0940.220.73
Years of residence in endemic area0.008 0.700.04 0.08
Treatment with benznidazole2.87
,0.0012.34
,0.001
doi:10.1371/journal.pntd.0001314.t002
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significant proportion of treated patients reached the extended
criteria of cure (conversion to seronegative on 2 or 3 tests)
presented herein, while an even larger number of subjects showed
declines in the titers on one or more tests compared with untreated
adult subjects in this relatively short follow-up span. The strong
correlation between the combined 3 conventional serologic tests
and the multiplex assay, that measures antibody levels to
individual recombinant proteins, also provides support for the
impact of benznidazole treatment on chronic T. cruzi infection.
Cure rates of 8–40% in a 10–20 year-average of follow-up have
been reported in the late chronic phase of adult patients who
received etiological treatment for T. cruzi infection [18]. Work
from our group showed a conversion to negative serology in only
15% of benznidazole-treated subjects after a 10-year follow-up
study with serological tests performed every 3-year interval of time
[6]. It is noteworthy that in the present study we were able to
detect a 7% rate of seronegative conversion in less than half the
time of our former studies. The more frequent sampling might
likely improve the detection of both seronegative conversion and
decreases in serological titers. In agreement with our findings, a
recent publication has also described complete seroconvertion of
5% in a 3-year follow-up study after treatment with benznidazole
[19]. Because the standard for determining a diagnosis of T. cruzi
infection is based on at least 2 tests out of 3 conventional
Table 3. Reduction of antibody levels by Multiplex and conventional serology after treatment with benznidazole.
Reduction of titers
ID
Multiplex ELISAIHAIFI
%a
N6b
Time of titer
decrease (mo)c
%a
Time of titer
decrease (mo)c
%a
Time of titer
decrease (mo)c
%a
Time of titer
decrease (mo)c
PP 0154–714/6 36-- 506
PP 0341–58 5/6 24------
PP 06-0/3-------
PP 16-0/8-4048--5048
PP 1841–607/1112--7525--
PP 21-0/3-------
PP 2441–68 3/56 60 24-- 507
PP 26- 0/2-------
PP 3140–47 2/312------
PP 44411/531 40 37----
PP 55 62–66 3/72430 24----
PP 62- 0/2-------
PP 67-0/1- 3024 50 63--
PP 83- NRd
----- 50 48
PP 8541–63 4/614 3414-- 7514
PP 93 47–53 3/7246036-- 5052
PP 100 641/112-- 752450 60
PP 113 57–694/5 12-- 75 245036
PP 114 56 1/8363024----
PP 11566–85 7/8 24 3036----
PP 117- NRd
--- 5012 50 12
PP 118 401/3 36---- 50 48
PP 120 41 1/32650 27 50 27--
PP 137-0/9-------
PP 14941–43 2/6 36-- 50 60 5072
PP 16448–96 7/72------
PP 17845–46 3/348-- 50 12--
PP 17951–59 4/636-- 876--
PP 18662–64 3/36------
PP 198401/424----5048
PP 384---------
PP 38557–92 6/82 3024----
aRange percentage of reduction in antibody levels compared with baseline at the end of follow-up.
bNumber of proteins with$40% decreases in reactivity after treatment out of the total reactive proteins prior to treatment.
cTime of follow-up where reactivity decrease was observed.
dNR No reactive prior to treatment. (2) Changes in serological titers were not observed during follow-up.
doi:10.1371/journal.pntd.0001314.t003
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