Annals of Tropical Medicine & Parasitology, Vol. 101, No.

2, 123–132 (2007)

Correlation of the serum concentrations of tumour

necrosis factor and nitric oxide with disease severity in
chronic Chagas disease (American trypanosomiasis)
R. PÉREZ-FUENTES*,{, A. LÓPEZ-COLOMBO{, G. ORDÓÑEZ-TOQUERO1,
I. GOMEZ-ALBINO", J. RAMOS*, E. TORRES-RASGADO*, H. SALGADO-ROSAS*,
M. ROMERO-DÍAZ*, P. PULIDO-PÉREZ* and M. C. SÁNCHEZ-GUILLÉN*
*
Laboratorio de Investigación en Fisiopatologı́a de Enfermedades Crónicas, Centro de Investigación
Biomédica de Oriente, Instituto Mexicano del Seguro Social, km 4.5 Carretera Federal Atlixco–
Metepec, Puebla, C.P. 62340, Puebla, Mexico
{
Facultad de Medicina, Benemérita Universidad Autónoma de Puebla, 13 Sur 2901 Colonia
Volcanes, Puebla, C.P. 72000, Puebla, Mexico
{
Gastro-enterology Service, Hospital de Especialidades, Centro Médico Nacional Manuel Avila
Camacho, Instituto Mexicano del Seguro Social, 2 Norte 2004, Colonia Centro, Puebla, C.P.
72000, Puebla, Mexico
1
Cardiology Service, Hospital de Especialidades, Centro Médico Nacional Manuel Avila
Camacho, Instituto Mexicano del Seguro Social, 2 Norte 2004, Colonia Centro, Puebla, C.P.
72000, Puebla, Mexico
"
Radiology Service, Hospital de Especialidades, Centro Médico Nacional Manuel Avila Camacho,
Instituto Mexicano del Seguro Social, 2 Norte 2004, Colonia Centro, Puebla, C.P. 72000, Puebla,
Mexico
Received 2 February 2006, Revised 5 June 2006,
Accepted 5 June 2006

Pro-inflammatory cytokines such as tumour necrosis factor (TNF) and nitric oxide (NO) are believed to play an
important role in the severity of chronic disease. When evaluated in 71 patients who were seropositive for
Trypanosoma cruzi and 50 apparently healthy controls, the mean (S.D.) serum concentrations of both TNF [7.65
(1.32) v. 4.24 (1.53) ng/ml; P,0.001] and NO [114 (40) v. 74 (21) mM; P,0.0001] were found to be significantly
higher in the patients than in the controls. In addition, patients with chronic, symptomatic disease affecting their
hearts — eight with dilated cardiomyopathy [8.82 (1.47) ng TNF/ml; 142 (45) mM NO] and 17 others with
electrocardiographic alterations [8.37 (1.26) ng TNF/ml; 134 (53) mM NO] — had significantly higher serum
concentrations of these cytokines than 34 patients who were in the asymptomatic, indeterminate phase of the
disease [6.38 (1.35) ng TNF/ml; 99 (28) mM NO]. In those infected with T. cruzi, it therefore appears that serum
concentrations of TNF and NO correlate with disease severity, indicating that these cytokines play some role in the
pathogenesis of chronic Chagas disease.

In the chronic stages of Chagas disease
(human American trypanosomiasis) — a
major cause of morbidity and mortality in
many areas of Latin America — up to 30%
of infected subjects develop at least one of
Reprint requests to: M. C. Sánchez-Guillén.
E-mail: csguillen2002@yahoo.com.mx; fax: z52 244
44 40 122
# 2007 The Liverpool School of Tropical Medicine
DOI: 10.1179/136485907X154593
the main clinical manifestations: cardiomyo-
pathy, neuropathy and/or digestive disease
(Prata, 2001). Although much of the patho-
genesis remains poorly understood, the
normal sequence of the infection, from an
acute phase to an indeterminate and then
chronic phase, indicates that the human
host’s immune system plays some role in the
124 PÉREZ-FUENTES ET AL.
pathology observed (Andrade, 1999;
Moncayo and Ortiz Yanine, 2006).
Pro-inflammatory host cytokines such as
tumour necrosis factor (TNF) have protec-
tive antiparasitic effects in human infections
with Trypanosoma cruzi (Abrahamsohn and
Coffman, 1996), the parasite responsible for
Chagas disease, but TNF is also a direct
mediator of toxic shock (Tracey et al., 1986)
and has also been shown to be deleterious in
some severe, systemic, inflammatory
responses (Tracey and Cerami, 1993).
Excessive production of TNF may contri-
bute to the pathogenesis of some infectious
disease (Beutler and Grau, 1993).
Inhibition of TNF, for example, prevents
mortality in experimental endotoxaemia
(Gerard et al., 1993). Maintenance of
granulomas is an active process in which
cells (both macrophages and T-cells) are
continually recruited from the blood, and
TNF participates in this process at several
levels (Havell, 1989; Flynn et al., 1995;
Saunders and Cooper, 2000; Roach et al.,
2002). In Tr. cruzi-infected mice, deaths
tend to occur when the systemic concentra-
tion of TNF is relatively high (Holscher
et al., 2000).
The central role of TNF in vivo seems to
be the activation of the inducible nitric oxide
synthase (iNOS) of macrophages and the
production of nitric oxide (NO; Nussler and
Billiar, 1993). Production of NO by TNF-
activated macrophages has been shown to
be an effective defence against a wide
range of intracellular pathogens, such as
Toxoplasma gondii (Adams et al., 1987),
Leishmania spp. (Bogdan et al., 1991) and
Mycobacterium bovis (Flesh and Kaufmann,
1991), and TNF is also an important
molecule in the human immune response
to Tr. cruzi (Gazzinelli et al., 1992; Vespa
et al., 1994).
Although NO may itself be cytotoxic, it
reacts with superoxide (O22) to yield
peroxynitrite (ONOO). Peroxynitrite is a
more cytotoxic molecule than its precursor,
causing lipid and thiol oxidation and nitro-
sylation of amino acids on target proteins
(Moncada, 1992). It is highly toxic for
Tr. cruzi (Rottenberg et al., 1996), as well
as for the tissue cells of infected hosts.
The evidence that excessive NO produc-
tion may cause pathology, associated with
the cytokine’s cytotoxic effects, is particu-
larly strong for a variety of immunopatho-
logical conditions, including rheumatic
disease (Farrell et al., 1992) and septic
shock (Wright et al., 1992). Garcia et al.
(1999) found that NO was involved in the
damage to peripheral autonomic neurons
observed in the acute phase of experimental
Tr. cruzi infection. Little is known about the
factors that influence the clinical variability
of Chagas disease (Macedo and Pena,
1998). The aim of the present study was
to explore the serum concentrations of TNF
and NO in cases of Chagas disease and
healthy controls, and see whether they could
be related to the degree of disease severity.
SUBJECTS AND METHODS
Subjects
Patients found seropositive for anti-Tr. cruzi
antibodies and the apparently healthy indi-
viduals used as controls were prospectively
recruited from the blood-bank, cardiology
and gastro-enterology services at the Centro
Medico Nacional Manuel Avila Camacho
(CMN) at the Instituto Mexicano del Seguro
Social in the Mexican city of Puebla.
Seropositivity was determined by indirect
haemagglutination tests (IHAT) and ELISA
(WHO, 1991) based on autochthonous
antigen (see below).
Clinical Studies
Patients and controls were each examined
clinically and a standard questionnaire was
used to record information about previous
illnesses and any general signs and symp-
toms of infections. The physical examina-
tion included a 12-lead electrocardiogram
(ECG), an echocardiogram, a chest X-ray
(at 2-m distance) for the measurement of
 TNF, NITRIC OXIDE AND SEVERITY OF CHAGAS DISEASE
the cardiothoracic index and radiological
imaging of the gastro-intestinal tract, with
barium swallow and oesophageal manome-
try. Patients with any acute disorder or with
a chronic disease other than Chagas were
excluded. Each subject gave full informed
consent and the study protocol was
approved by the research committee at the
CMN.
Clinical Groups
The subjects were divided into groups I–V,
according to their clinical characteristics.
Group I was formed of the healthy controls,
all of whom had been found seronegative for
Tr. cruzi, HIV and Brucella abortus, and
negative in standard tests for syphilis
(VDRL), hepatitis B virus (core and surface
antigens) and hepatitis C virus, with no
evidence of any clinical abnormalities or
alterations and with normal results in renal-
function tests. The other four groups con-
tained patients who had each been found
seropositive for Tr. cruzi, both by IHAT and
ELISA. Group II was formed of patients
who, because they were seropositive for
Tr. cruzi but asymptomatic, with no ECG
abnormalities consistent with Chagas dis-
ease and no abnormalities of the gastro-
intestinal tract visible on their X-rays, were
considered to be in the indeterminate phase
of the disease. Patients suffering from
dysphagia, regurgitation, constipation and/
or abnormal relaxation of the lower sphinc-
ter (ARLES), as defined by manometric
criteria, formed group III. Group IV was
formed of patients who had ECG abnorm-
alities but no dilated cardiomyopathy, and
group V of patients with dilated cardiomyo-
pathy, who presented with dyspnoea,
syncope, dysphagia and palpitations or
other cardiac abnormalities seen in Chagas
disease.
Sample Collection
To standardize the samples, venous blood
was obtained from each subject after an
overnight fast of >12 h, and allowed to clot
125
at room temperature for 2 h. The serum was
then separated by centrifugation (12006g
for 10 min), frozen at -20uC, and subse-
quently stored at -70uC until further use.
Testing for Anti-Tr. cruzi Antibodies
ANTIGEN
To obtain autochthonous antigen, Tr. cruzi
was isolated, by xenodiagnosis, from an
infected patient who lived in the state of
Puebla. The parasites were characterised, by
multilocus enzyme electrophoresis (MLEE)
and RAPD, as Tr. cruzi I, corresponding
to the HUM/ME/1997/MEX/RyCH1
reference strain (Pérez-Fuentes et al.,
2003). An antigen extract of the parasites
was prepared by culturing the parasites, in
liver-infusion–tryptose medium supplemen-
ted with 10% foetal calf serum, harvesting
them while they were still in the logarithmic
phase of growth, sonicating them in the
presence of protease inhibitors, and then
centrifuging the sonicate for 30 min at
10,0006g and 4uC. The concentration of
protein in the supernatant solution, which
was used as the crude antigen extract, was
quantified by the method of Lowry and then
adjusted to 1 mg/ml before the extract was
stored at 270uC.
ELISA
For the ELISA (Pérez-Fuentes et al., 1998),
the wells of polystyrene plates (Dynatech,
Chantilly, VA) were sensitized with the
crude antigen extract diluted in carbonate
buffer, at pH 9.5, to give 100 mg protein/ml,
and then blocked with 1% foetal bovine
serum in a solution of 0.01% (v/v) Tween
20 in phosphate-buffered saline (PBS) at
pH 7.2. Test sera, diluted 1:100, were then
added to the wells before the plates were
incubated at 37uC for 1 h. After several
washes in PBS (pH 7.2), a conjugate of
horseradish peroxidase with anti-human-
IgG was added. After the colorimetric
reaction was developed with orthophenyle-
nediamine and hydrogen peroxide and then
stopped, the absorbance of the contents of
126 PÉREZ-FUENTES ET AL.
each well at 490 nm was read in an ELISA
reader (Multiskan MS; Labsystems OY,
Helsinki).
IHAT
For the IHAT (Pérez-Fuentes et al., 1998),
sheep erythrocytes (2.5% whole blood in
Alsever’s solution) were tanned with a
1:60,000 dilution of tannic acid, sensitized
with the Tr. cruzi antigen extract at 37uC for
20 min, washed with PBS (pH 7.2) and
resuspended in the saline to give 0.2 mg
erythrocytes/ml. This suspension was mixed
with a test serum, to give serum dilutions of
1:8, 1:32 and 1:64, incubated for 2 h at room
temperature, and then checked for haemag-
glutination. A sample that caused haemag-
glutination at a titre of 1:8 was considered
positive for anti-Tr. cruzi antibodies. Samples
with IHAT titres of 1:8, 1:32 and 1:64 were
scored z, zz and zzz, respectively. All
samples were tested in triplicate in each of
two independent experiments.
Measurement of Serum Concentrations
of NO and TNF
The concentration of NO in each serum
sample was evaluated by reducing any
serum nitrate to nitrite, using spongy cad-
mium (5- to 20-mesh; Sigma), and then
estimating the concentration of nitrite by the
Griess method (Schmidt et al., 1989).
Absorbance at 540 nm was measured in
the plate reader used for the Tr. cruzi
ELISA. Sodium nitrite was used as a
standard. At the time the sera were col-
lected, none of the subjects had ingested any
exogenous dietary nitrate for at least 48 h.
Serum levels of TNF were determined
using serial dilutions of each test serum in a
commercial, two-site, sandwich ELISA that
uses purified and biotinylated anti-TNF
antibodies (Quantikine HSTA50 kit; R&D
Systems, Minneapolis, MN). Briefly, after
incubation with alkaline phosphatase
coupled to streptavidin, and development
with p-nitrophenyl phosphate, absorbance
was read, at a test wavelength of 405 nm
and a reference wavelength of 495 nm, on
the microplate reader used for the Tr. cruzi
ELISA. The TNF ELISA has a detection
limit of 0.01 ng/ml.
Statistical Analysis
Non-parametric Mann–Whitney U-tests
were used to compare TNF and NO
concentrations in the five groups of subjects.
In the two-tailed tests of significance, a
P-value of ,0.05 was considered indicative
of a statistically significant difference.
RESULTS
Subjects
The 50 controls (assigned to group I) were
apparently healthy blood donors aged 16–65
years whereas the 71 ‘patients’, who were
either asymptomatic blood donors in the
indeterminate phase of Chagas disease
(N534: assigned to group II) or cases with
chronic, symptomatic Chagas who had pre-
sented at the gastro-enterology or cardiology
clinics at the CMN (N537; assigned to
groups III–V), were aged 12–84 years (see
Table). Of the 37 symptomatic cases, 12
(group III) had ARLES and oesophageal
aperistalsis, and/or abnormality of the gastro-
intestinal tract, visible by radiological imaging,
characteristic of achalasia, 17 (group IV) had
ECG that showed conduction-system distur-
bances (mainly primary alterations of repolar-
ization and complete left-bundle-branch
blocks) but no evidence of dilated cardiomyo-
pathy, and the other eight (group V) had
cardiomegaly (see Table). All the seropositive
patients lived in areas of Mexico with endemic
Tr. cruzi and were assumed to have been
infected in Mexico.
Serum Concentrations of TNF
The mean (S.D.) serum concentration of
TNF in the seropositive patients was found
to be significantly higher than that in the
seronegative controls [7.65 (1.32) v. 4.24
(1.53) ng/ml; P(0.001]. Furthermore,
 TNF, NITRIC OXIDE AND SEVERITY OF CHAGAS DISEASE 127

among the patients, those with dilated

cardiomyopathy [8.82 (1.47) ng/ml] and
the other patients with ECG alterations
[8.37 (1.26) ng/ml] — but not those with
gastro-intestinal alterations — had signifi-
cantly higher serum concentrations of TNF
than those with the indeterminate, asympto-
matic form of the disease [6.38 (1.35)
ng/ml; P,0.01 for each; Fig. 1].

Serum Concentrations of NO
The concentrations of NO in the sera of the
seropositive patients were also found to be
significantly higher than those in the sera of
the seronegative controls, with means (S.D.)
of 114 (40) and 74 (21) mM, respectively
(P(0.0001). As seen with TNF, NO
concentrations in the sera of patients with
dilated cardiomyopathy [142 (45)] and
those in the sera of the other patients with
ECG alterations [134 (53) mM] — but not
those with gastro-intestinal alterations —
were significantly higher than those
recorded in the patients in the indeterminate
phase of Chagas disease [99 (28) mM;
P,0.01 for each; Fig. 2].
FIG. 1. The mean serum concentrations of tumour
necrosis factor (TNF) observed in the seronegative
control subjects (group I) and the seropositive patients
who were either in the asymptomatic/indeterminate
phase of their Trypanosoma cruzi infection (group II) or
in the symptomatic chronic phase (groups III–V). The
symptomatic patients had gastro-intestinal alterations,
such as achalasia and other oesophageal abnormalities
(group III), electrocardiographic abnormalities but no
signs of dilated cardiomyopathy (group IV) or dilated
cardiomyopathy (group V). The concentrations in each
of the groups of patients were significantly higher than
those in the healthy controls (P(0.001 for each) and
those in groups IV and V were significantly higher than
those in the asymptomatic patients of group II (P(0.01
for each). The vertical lines indicate S.D.

disease is still increasing (Luquetti, 1999).

DISCUSSION All the seropositive patients investigated in
the present study lived, and had probably
In countries without adequate control, the been infected, in Mexico. As recommended
reported incidence of new cases of Chagas by the World Health Organization, to help
TABLE. The demographic characteristics and ELISA and IHAT results of the seronegative control subjects (group
I) and the seropositive patients who were either in the asymptomatic/indeterminate phase of their Trypanosoma cruzi
infection (group II) or in the symptomatic chronic phase (groups III–V)

Group

Characteristic I II III* IV{ V{

No. of individuals 50 34 12 17 8
Mean (S.D.) age (years) 37.2 (8.6) 37.7 (18.2) 47.7 (22.6) 39.8 (17.1) 40.9 (8.4)
Range of ages (years) 16–65 12–90 20–84 19–63 26–53
Gender (% male) 66 50 58.3 58.8 62.5
Mean (S.D.) ELISA absorbances1 - 0.096 (0.023) 0.132 (0.045) 0.109 (0.047) 0.133 (0.031)
IHAT result (score) - z– zz z– zz zz zzz

*With gastro-intestinal alterations, such as achalasia and other oesophageal abnormalities.

{
With electrocardiographic abnormalities but no signs of dilated cardiomyopathy.
{
With dilated cardiomyopathy.
1
For a 1:100 serum dilution.
128 PÉREZ-FUENTES ET AL.
FIG. 2. The mean serum concentrations of nitric
oxide (NO) observed in the seronegative control
subjects (group I) and the seropositive patients who
were either in the asymptomatic/indeterminate phase of
their Trypanosoma cruzi infection (group II) or in the
symptomatic chronic phase (groups III–V). The symp-
tomatic patients had gastro-intestinal alterations, such
as achalasia and other oesophageal abnormalities
(group III), electrocardiographic abnormalities but no
signs of dilated cardiomyopathy (group IV) or dilated
cardiomyopathy (group V). The concentrations in each
of the groups of patients were significantly higher than
those in the healthy controls (P(0.001 for each) and
those in groups IV and V were significantly higher than
those in the asymptomatic patients of group II (P(0.01
for each). The vertical lines indicate S.D.
exclude any false results from the use of
a single testing method based on non-
standardized antigens (Camargo et al., 1986),
the seropositivity of each of these patients
was confirmed using two tests (IHAT and
ELISA) run in parallel. Clinical diagnoses
were based on the abnormalities previously
found to be associated with chronic Chagas
disease.
The apparent correlations between the
serum concentrations of TNF and NO and
disease severity in those with chronic
Chagas disease (Figures 1 and 2) are
interesting. Experimental infestation of mice
with Tr. cruzi generally induces cytokine
production, which modulates host resis-
tance against the parasite. The pro-
inflammatory cytokine TNF, for example,
mediates specific resistance to Tr. cruzi
infection (Silva et al., 1995). Curiously,
however, there is now a considerable body
of evidence indicating that alterations in the
quantity and/or quality of cytokine produc-
tion are strongly associated with the devel-
opment of chronic Chagas disease (Laucella
et al., 1996). Excessive TNF production
contributes to the immunopathology and
severity of some human infectious diseases,
such as AIDS (Barbaro et al., 2001),
tuberculosis (Zhou et al., 2000), malaria
(Grau et al., 1989) and human African
trypanosomiasis (Okomo-Assoumou et al.,
1995), and has also been implicated in the
cachexia seen during acute Tr. cruzi infec-
tion in mice (Truyens et al., 1995). TNF has
also been found to modulate the expression
of the adhesion molecules involved in the
recruitment of lymphocytes into inflamma-
tory sites, thus contributing to the progres-
sion of the local inflammatory reaction in
chagasic cardiomyopathy (Bachmaier et al.,
1997). In the present study, the high TNF
concentrations observed in the patients with
severe cardiomyopathy (group V) may
indicate that abnormalities in TNF balance
participate in the evolution of the pathogen-
esis of chronic Chagas disease.
Pro-inflammatory cytokines, including
TNF, are involved in the generation of
iNOS, which produces a continuous and
potentially large supply of NO in tissues that
normally experience only low and tightly
controlled levels of this ubiquitous molecule
(Gazzinelli et al., 1996). In Tr. cruzi infec-
tion, NO appears to play a pivotal role in the
host’s immune response, as a first line of
defence against the parasite (Vespa et al.,
1994). NO is released during the acute
phase of Tr. cruzi infection in mice, and
treatment of such mice with an inhibitor of
NO synthase exacerbates the infection
(Petray et al., 1994). In mice with acute
Tr. cruzi infections, the production of NO
appears crucial for the trypanocidal activity
of the host’s macrophages and for host
survival (Holscher et al., 1998). During the
chronic stages of infection, immunological
mechanisms involved in resistance to Tr.
cruzi may be associated with inflammation
 TNF, NITRIC OXIDE AND SEVERITY OF CHAGAS DISEASE
and the severity of pathology induced by the
parasite (Garcia et al., 1999).
In diseased humans, excessive NO may
cause pathology (Farrell et al., 1992; Miller
et al., 1993). NO is associated with the severity
of cerebral malaria (Grau et al., 1989), and
high serum concentrations have been
observed in people who are seropositive for
Tr. cruzi (Pérez-Fuentes et al., 1998; present
study). During acute, experimental infection
of rats with Tr. cruzi, NO is involved in the
development of the lesions seen in the
peripheral autonomic neurons (Garcia et al.,
1999). Pro-inflammatory cytokines generated
by activated immune cells can induce an
increase in NO in isolated rat myocytes
(Tsujino et al., 1994). At least in a mouse
model, NO released from cardiac iNOS may
participate in the pathogenesis of chagasic
heart disease (Huang et al., 1999). When they
investigated rats experimenatlly infected with
Tr. cruzi, Chandrasekar et al. (2000) found
that the hosts’ myocardium expressed high
levels of iNOS and NO metabolites at a time
when no parasite or immune-cell infiltration
could be detected. More recently, it was
demonstrated that NO derived from inducible
nitric oxide synthase plays an important role
in the development and progression of ven-
tricular dilatation and systolic dysfunction in
acute murine chagasic myocarditis (Chandra
et al., 2002). In the present study, interest-
ingly, the patients with dilated cardiomyo-
pathy had the highest serum concentrations of
NO observed (although these were not
significantly higher than those recorded in
the patients with gastro-intestinal alterations).
It seems highly unlikely that the between-
group differences in serum NO seen in the
present study reflect differences in nitrate/
nitrite ingestion. Because serum levels of NO
can be influenced by dietary intake, all the
study participants were recruited from
the same geographical location: Puebla state.
The water supply and human diet in this area
(the latter based mainly on rice, corn and
beans) are low in nitrates/nitrites. In addition,
the half-life of nitrate after an oral nitrate load
is only 6–7 h (Jungersten et al., 1993) and the
129
blood samples for the present study were each
collected after a fast of at least 12 h.
As a result of their in-vitro studies,
Machado et al. (2000) believed that high
levels of NO, pro-inflammatory cytokines
and chemokines produced by cardiomyo-
cytes limited the multiplication of Tr. cruzi
in mammalian hosts, and that inflamma-
tory-cell influx could contribute to the
pathogenesis of chagasic cardiomyopathy.
The present results confirm the potential
importance of systemic TNF and NO in
dilated cardiomyopathy (Habib et al.,
1996). It seems possible that high concen-
trations of TNF, associated with the infec-
tion, induce high levels of NO that cause
damage, mainly by oxidative processes, in
parasite-infected cells. The role that TNF
and NO may play in the development of
chronic Chagas disease is still unclear.
Although NO-related activity has been
shown to be protective against Tr. cruzi,
excessive local production of NO may affect
the normal function of various organs and,
in the long-term, NO-attributable oxidative
stress may lead to the typical manifestations
of chronic Chagas disease. Confirmation of
this hypothesis awaits further studies to
evaluate the roles that TNF and NO play
in the development of Chagas disease.
ACKNOWLEDGEMENTS. The authors thank the
medical staff of the cardiology, gastro-
enterology and radiology services at the
Centro Medico Nacional Manuel Avila
Camacho, for carrying out the clinical studies.
This work was financially supported, in part,
by the Fondo del Sistema de Investigación Ignacio
Zaragoza of the Consejo Nacional de Ciencia y
Tecnologı́a (FOSIZA–CONACyT), via grant
19980802019. E.T-R. and H.S-R. are the
recipients of fellowships from CONACyT.
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