Experimental Parasitology 172 (2017) 44e50

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Research brief

Antiprotozoal drug nitazoxanide enhances parasitemia, tissue lesions

and mortality caused by Trypanosoma cruzi in murine model
Juan Salvador Valle-Reyes a, b, Valery Melnikov a, *, Oxana Dobrovinskaya b,
Alejandrina Rodriguez-Herna
ndez a, Cristina Wookee-Zea a, Víctor Pimientel-Rodrigez a,
a
Gabriela Rueda-Valdovinos , Iva
n Delgado-Enciso a, Uriel A. Lo

pez-Lemus a, c,
Francisco Espinoza-Go
mez a

a
School of Medicine, University of Colima, Colima, Colima, Mexico
b
Center for Biomedical Research, University of Colima, Colima, Colima, Mexico
c
Center for Gene Therapy, Hematologic Malignancies & Stem Cell, Transplantation Institute, City of Hope, Duarte, CA, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Oral Nitazoxanide caused high mor-

tality in acute T.cruzi infection in
mice.

High dose nitazoxanide didn't cause
mortality nor tissues damage in
healthy mice.

Both, parasitemia and tissue damage
were higher in nitazoxanide treated
animals.

Parasitemia and tissues damage are
directly related to nitazoxanide dose.

Nifurtimox and nitozaxonide combi-
nation was less effective than nifur-
timox only.

a r t i c l e i n f o a b s t r a c t

Article history: Chagas' disease is caused by unicellular parasite Trypanosoma cruzi (T. cruzi). It is endemic throughout
Received 21 May 2016 Latin America, but nowadays has become a global challenge due to tourism and migration. Non-treated
Received in revised form infection may result in health-threatening complications and lead to death. Current medications for this
15 December 2016
infection are nifurtimox (NFT) and benznidazol. Both drugs may cause side effects and are ineffective in
Accepted 18 December 2016
Available online 21 December 2016
the chronic phase. Therefore, new antichagasic compounds are urgently required. Nitazoxanide (NTZ) is a
broad spectrum antiparasitic drug, proposed recently as a potential candidate to be added to the list of
essential medicines for integrated neglected tropical disease control and elimination. Although the effect
Keywords:
Trypanosoma cruzy
of NTZ against T. cruzi epimastigotes in vitro was reported, the corresponding experiments in animal
Nitazoxanide models of T. cruzi infection have never been undertaken. The present work was designed to fill this gap
Mortality and evaluate the effect of NTZ on experimental murine trypanosomiasis, in comparison with classical
antichagasic agent NFT. Highly sensitive to T. cruzi BALB/c mice were infected using Albarrada T. cruzi
strain, recently isolated in Mexico. Experimental groups were either left untreated, or otherwise treated
with NFT, NTZ (100 and 1000 mg/kg), or with both drugs simultaneously. The severity of the infection
was estimated based on criteria such as parasitemia, lesions in target tissues (heart, muscles and lungs)
and mortality. Despite the expected protective effect, NTZ drastically aggravates the course of T. cruzi
infection. Namely, parasitemia, tissue lesions and mortality caused by T. cruzi infection were significantly

* Corresponding author.
E-mail address: melnik@ucol.mx (V. Melnikov).

http://dx.doi.org/10.1016/j.exppara.2016.12.013
0014-4894/© 2016 Elsevier Inc. All rights reserved.
 J.S. Valle-Reyes et al. / Experimental Parasitology 172 (2017) 44e50
higher in NTZ-treated mice groups, even in comparison with untreated infected animals. NTZ by itself no
produced mortality o tissue damage, and NFT showed an expected protective effect. Our results indicate
that NTZ cannot be considered for Chagas’ disease treatment. Moreover, NTZ should be used with caution
in patients positive for T. cruzi infection.
1. Introduction
Chagas' disease, or American trypanosomiasis, is a tropical
parasitic disease caused by the protozoan hemoflagellate T.cruzi
(Chagas, 1909) and transmitted mostly by insects belonging to
Triatominae subfamily (Chagas, 1909). Because the triatominae
vectors are mainly found and widespread in South and Central
America, Chagas’ disease is considered endemic to these regions,
but significant number of cases attributed to immigrants is also
reported in the United States, Europe and Australia (WHO., 2016).
In recent years, by applying various programs aimed at vector
control and at preventing transmission by blood transfusions,
impressive progress has been attained in reducing Chagas’ disease
(WHO., 2016). However, T.cruzi infection still remains a serious
threat to public health in many regions of the Americas (WHO.,
2016). The World Health Organization has estimated that 6 to 7
million new cases occur annually, and about 100 million people are
at risk of infection (WHO., 2016). Given this situation, there is a
need to develop protocols for effective pharmacological treatment
of these already infected people. Effective diagnostics and phar-
macological treatment in the early stages of infection are required
to prevent the progression of the disease to severe and irreversible
stages, and to reduce transmission (WHO., 2016). Only two drugs of
limited efficacy at the chronic T.cruzi infection are actually available,
namely NFT and benznidazol (WHO., 2016). Both drugs are effective
in the acute phase and act through the formation of free radicals
and/or electrophilic metabolites (Mitelman et al., 2013). Although
T.cruzi possesses various mechanisms to detoxify free radicals and
their metabolites (Piacenza et al., 2013), they are not effective
enough to avoid the oxidative stress produced by aforementioned
antichagasic drugs that results in parasite death. However,
damaging effect of these radicals on healthy organs and tissues may
be also significant (Mitelman et al., 2013). Indeed, even at the
therapeutic doses both drugs cause numerous side effects (in up to
40% of all treated patients) (WHO., 2016), such as anorexia,
insomnia, nausea, headache, dizziness, asthenia, peripheral
neuritis, exfoliative dermatitis, thrombocytopenia, allergic purpura,
nervousness, hallucinations and convulsions; and as a conse-
quence, forcing treatment withdrawal (Molina et al., 2015; Forsyth
et al., 2016). Furthermore, many strains of T.cruzi were recently
reported to demonstrate drug resistance (Caldas et al., 2014)
Summing up, current antichagasic medication is costly and asso-
ciated with the risk of severe side effects; in many endemic coun-
tries, the access to these drugs is limited. Undoubtedly, new
effective and inexpensive antichagasic compounds are urgently
required.
A broad-spectrum antiparasitic drug NTZ is used to treat a wide
range of intestinal infections caused by helminthes, protozoa and
bacteria (Rossignol, 2014). In particular, it is often prescribed to
patients who have been diagnosed with cryptosporidiosis and/or
giardiasis. In the last years there are a growing number of reports
about the antiviral activity of this compound (Adagu et al., 2002).
Antiparasitic effect was shown to be due to selectively inhibition of
pyruvateferredoxinoxidoreductase, an enzyme essential for energy
metabolism of all intracellular parasites (Rossignol, 2014), blockade
45
© 2016 Elsevier Inc. All rights reserved.
of a glutamate-gated chloride ion channel known for its role in
ivermectin susceptibility against nematodes and nitro-reduction
among with free radical production (Adagu et al., 2002;
Somvanshi et al., 2014). Despite the widespread use of NTZ as an
antiparasitic drug, and considering its wide therapeutic window
and accessibility to the population, we have found only a single
report concerning its effect on T.cruzi. In this regard, Chan-Bacab
and coworkers (2009) have demonstrated that NTZ inhibits the
in vitro growth of kinetoplastid parasites such as Leishmania mex-
icana and T.cruzi; even more, against T.cruzi it was more effective
than benznidazole (Chan-Bacab et al., 2009). However, the effect
was proven only against epimastigotes, the morphologic form that
is present in triatomine vector. The question about its effectiveness
against morphological forms of T.cruzi found in the mammalian
host, namely extracellular trypomastigotes in the bloodstream
(parasitemia) and intracellular amastigotes in the tissues (parasite
burden), was not addressed yet. On the other hand, it is mandatory
to test drug activity against these two forms. NTZ have been safely
employed for the long term treatment (3e24 month) of the
disseminated cystic Echinococcosis in human patients (Pe
rez-
Molina et al., 2011), the fact what proves the safe administration
of NTZ for more than one week and its effectiveness in dissemi-
nated parasitosis. In this concern, the side effects displayed by NTZ
are not so common (<10%) and are less severe (Drugs.com Know
more. Be sure, 2000) compared to the both actual antichagasic
drugs, so NTZ is potentially eligible for replacing them. Considering
that the NTZ's mechanism of antiparasitic effect is somehow similar
to that of benznidazole, it is logic to hypothesize that NTZ in the
same manner hardly could be effective in chronic Chagas disease.
Therefore, the main aim of the present study was to explore the
effectiveness of NTZ against T.cruzi acute infection in the murine
model.
2. Materials and methods
Male BALB/c mice of six-to eight-week old were obtained from
HARLAN LABORATORIES, S.A. DE C.V., and housed in 12/12 h dark/
light cycle with controlled temperature conditions (22e24  C), with
water and aliments ad libitum. Before the experiment, they were
allowed to acclimate for seven days. T.cruzi strain TPAP/MX/2002/
Albarrada used in this study was isolated in the state of Colima,
Me
xico and belongs to TcI group (Melnikov et al., 2013) according to
the New Consensus for Trypanosoma cruzi intraspecific nomencla-
ture (Zingales et al., 2012). Mice were infected by intraperitoneal
injection with 1  105/mice of T.cruzi bloodstream trypomastigotes
previously obtained by cardiac puncture from previously infected
BALB/c mice.
NFT (Bayer®) and NTZ (Daxon®) were used in this study. The
drugs were dispersed in deionized water, and administrated orally.
NFT was administrated in the dosage of 200 mg/kg during first four
days, and then 50 mg/kg during next 16 days, as was reported
elsewhere (Z
arate et al., 1980).
Two experimental series were undertaken: in the first series, the
animals were monitored for 21 day, until the peak of parasitemia
was reached; in the second series, animals were monitored during
46 J.S. Valle-Reyes et al. / Experimental Parasitology 172 (2017) 44e50
50 days post-infection, until the parasitemia level was almost un-
detectable. All animals of first series and surviving mice of second
series were euthanized for histopathological studies. In both series,
the animals were allocated to 7 experimental groups, 5 (first series)
or 10 (second series) animals each, by simple aleatorization,
applying EPIDAT 3.1 software developed by the Department of
Public Health and Health Council of Spain (2006). The groups were
the following: 1) uninfected mice; 2) uninfected mice treated with
NTZ (1000 mg/kg); 3) mice infected with T.cruzi, without any
pharmacological treatment; 4) mice infected with T.cruzi, treated
with NFT; 5) mice infected with T.cruzi, treated with NTZ (100 mg/
kg); 6) mice infected with T.cruzi, treated with NTZ (1000 mg/kg);
7) mice infected with T.cruzi, treated with both NTZ (100 mg/kg)
and NFT. In groups 4,5,6 and 7 both drugs were administered for 20
days since the first day of T.cruzi parasitemia (3rd day after T.cruzi
inoculation). NTZ was administered orally daily, as previously
described (Caldas et al., 2014). When both drugs were used (group
No. 7), they were administered with 30 min interval.
The level of parasitemia was monitored by counting trypomas-
tigotes in the blood samples collected from the tail vein. Hemocy-
tometer was used to determinate the numbers of parasites per mm
of blood (Za
rate et al., 1980). At the end of the experiments the PCR
method published by Moser et al. (1989) was employed in order to
detect the parasitemia in all cases with parasites undetectable by
microscopic blood observation.
Hearts, muscles and lungs were collected from the animals
euthanized with an overdose of sodium pentobarbital at 21st or
50th day of post-infection, fixed in 10% buffered formalin solution
for 3 days, and processed in routine for paraffin embedding. Lungs
sampling is based on our previous report (Melnikov et al., 2005)
where damage in lungs parenchyma was shown in the acute
infection with strain TPAP/MX/2002/Albarrada in mice. Three mme
thick tissue sections were cut, stained with hematoxyline eosin
and analyzed by optical microscopy using digital imaging (Micro-
scope Axioscop Carl Zeiss, Digital Camara MR5 Carl Zeiss, Software
Axiovision 4.1). Pathologic damage and immune cells infiltrations
in muscles and hearts in different experimental groups were
compared applying Dallas scale designed for the semiquantitative
analysis of myocarditis and myositis (Aretz, 1987). In every sample
three parameters were quantified: inflammatory infiltration
severity (from 0 to 3 where the 3 is the most severe infiltration),
inflammatory infiltrate distribution (four grades from absent to
diffuse) and necrosis (absent/present) (Brasso et al., 2013).
2.1. Statistical analysis
Data were expressed as mean ± SEM and analyzed using
descriptive statistics for each variable, through analysis of means
and variances assuming a normal distribution. For comparisons of
groups and possible interactions between them, the two-way
ANOVA with posthoc analysis according to 2  2 factorial design
was used. p-values less than or equal to 0.02 were considered
statistically significant, based on the Bonferroni adjustment for
posthoc tests. SPSS19.0 software (IBM, Armonk, NY, USA) was used.
Program MedCalc 15.8.was used to analyze mortality and area
under the parasitemia curve. Logistics univariate correlation was
used to associate the level of parasite and mortality, and Sperman's
rank correlation coefficient - to evaluate risk factor of each
treatment.
2.2. Ethics statement
This study was carried out in accordance with the recommen-
dations in the Mexican Official Standard NOM-062-ZOO-1999
(Technical specifications for the production, care and use of
laboratory animals). All experimental procedures were reviewed
and approved by the Committee for Bioethics and Biosafety of the
University of Colima (Permit number: 2012e17).
3. Results and discussion
Efforts of various researchers around the world are now focused
on the search of safe and effective antichagasic compounds (Urbina,
2010). NTZ, a low cost antiprotozoal, antibacterial and antiviral
drug has been shown to be effective against T.cruzi epi-
mastigotes in vitro (Chan-Bacab et al., 2009). In the present work,
the effect of NTZ on the course of T. cruzi infection was studied in
the murine model.
Contrary to expectations, the results presented here suggest that
NTZ drastically aggravates T.cruzi infection in mammalian host,
enhancing mortality, parasitemia, tissue parasitism and
damage.
Infected experimental groups were either left untreated, or
otherwise treated with NFT, NTZ (100 and 1000 mg/kg), or with
both drugs simultaneously. Both the evolution of parasitemia and
survival in different experimental groups were monitored during a
period of 50 days post-infection (Figs. 1 and 2). The level of para-
sitemia reached in infected group without treatment was similar to
that reported earlier for BALB/c infected with TPAP/MX/2002/
Albarrada strain (Melnikov et al., 2005). At 16 days post-infection,
NFT treatment completely suppressed parasitemia in both infec-
ted groups (Fig. 1A). Accordingly, TPAP/MX/2002/Albarrada strain is
highly susceptible to NFT, similar to various T.cruzi strains reported
earlier (Andrade et al., 1985). Interestingly, in the group with
combined treatment (NTZ þ NFT), the parasitemia profile was
similar to that obtained in animals treated with NFT alone except
for the slight reactivation of parasitemia 2 days after the conclusion
of drug treatment (Fig. 1A). Surprisingly, both experimental groups
treated with different concentrations of NTZ showed a worse
evolution of the infection when compared to NFT group, and even
to untreated mice. In the first place, a drastic enhancement of
parasitemia was observed (Fig. 1A).
The area under the curve of parasitemia is a criterion commonly
used to evaluate both the parasitemia level and its persistence
(Becker et al., 2011). This value was significantly lower in the groups
treated with NFT as monotherapy, or in combination with NTZ, as
compared with untreated animals. In contrast, it was drastically
elevated in the group treated with the high dose of NTZ, whereas in
the group treated with lower dose of NTZ, this value did not differ
from those obtained in untreated group (Fig. 1B).
The criterion of accumulated mortality is used routinely in
murine models to determine T.cruzi strains’ virulence. TPAP/MX/
2002/Albarrada strain is of mild virulence in BALB/c murine model,
so that 90% (9/10) of infected mice had survived the 50-day period
of acute infection (Fig. 2). However, infected animals treated with
NTZ not only exhibited significantly higher parasitemia, almost all
of them succumbed between days 15 and 50. Two different doses of
NTZ were used in our study, and the higher dose (1000 mg/kg) was
lower but close to those (1400 mg/kg) reported as lethal dose 50%
(Murphy and Friedmann, 1985; Malesuik et al., 2012). But NTZ itself
(experimental group 2) even at the highest dose did not cause
mortality or tissue damage in uninfected treated animals. This
observation rules out the hypothesis about the possible toxic effect
of NTZ as a cause of mortality not related to infection in the groups
of mice injected with T.cruzi.
Summing up, NTZ treatment significantly increased the para-
sitemia level, persistence of bloodstream forms and overall mor-
tality of infected animals. According Spearman rank correlation
test, the NFT group showed the lowest risk, followed by a positive
(untreated) control, then group treated with NTZ 100 mg/kg and
 J.S. Valle-Reyes et al. / Experimental Parasitology 172 (2017) 44e50 47

Fig. 1. Treatment with NTZ enhances parasitemia in T. cruzi-infected mice. Male BALB/c mice were infected by intraperitoneal injection with 1  105/mice of bloodstream T.cruzi
trypomastigotes (TPAP/MX/2002/Albarrada strain). All medicamental treatments were initiated at the first day of parasitemia (3rd day postinfection) and finished 20 days after. A)
Parasites were counted by light microscopy in samples of peripheral blood during 50 days post infection, and parasitemia was calculated by Brenner method. Data are present as
(mean ± SEM)x105 of parasite/ml. Comparisons of parasitemia values in groups treated with NTZ or NFT regarding to untreated group were performed at 21st day post-infection
using two-way ANOVA 2  2, Tuckey post-test. Protective effect of NFT was observed, whereas NTZ alone caused enhanced parasitemia. *p ¼ 0.02;***p ¼ 0.001. B) Area under the
parasitemia curve was calculated by using MedCalc program. Comparisons were performed between untreated group and drug-treated groups (*), and between group treated with
NFT (standard treatment) and others groups (þ). **p ¼ 0.01; þp ¼ 0.019, þþþp ¼ 0.001.

finally, as the highest risk, the group treated with 1000 mg/kg,
being significant (r2 ¼ 0.22, p ¼ 0.000001). However, logistics
univariate correlation analysis did not reveal the association be-
tween enhanced parasitemia level and mortality in NTZ treated
groups (OR ¼ 1.2, 95% CI: 0.87e2.1). The correlation seems to be
more complex, and include additional factors such as tissue lesions
in target organs considered to be the factor of primary importance
in disease progression and prognosis.
3.1. T.cruzi-infected mice treated with NTZ present severe tissue
lesions when compared with untreated animals
For pathological studies, two series of experiments were un-
dertaken. No tissue lesions (both, infiltration nor necrosis were
found) were observed in both uninfected control groups, either
untreated or treated with NTZ. In regard to the T.cruzi infected mice,
all animals were euthanized at 21 days post-infection when the
peak of parasitemia was reached (Fig. 3), and at 51 days post-
infection when parasitemia in animals surviving the acute phase
48 J.S. Valle-Reyes et al. / Experimental Parasitology 172 (2017) 44e50
Fig. 2. Treatment with NTZ enhances mortality in T. cruzi-infected mice. Male BALB/c mice were infected by intraperitoneal injection with 1  105/mice of bloodstream T.cruzi
trypomastigotes (TPAP/MX/2002/Albarrada strain). All medicamental treatments were initiated at the first day of parasitemia (3rd day postinfection) and finished 20 days after.
Mortality was followed during 50 days post-infection and subjected to statistical analysis by Kruskal-Wallis and Langley post-test represents difference between T.cruzi group and
groups treated with NTZ,###p ¼ 0.001.
was almost undetectable by microscopic observation. Lesions
characteristic of experimental trypanosomiasis (Melnikov et al.,
2013) were found in the group of infected mice that were left un-
treated. In particular, amastigotic nests were present in myocar-
dium and skeletal muscles. Myocarditis, moderate to severe
according to Dallas criteria, with myocytes destruction, mono-
nuclear and fibroblastic infiltration was observed in the heart. Light,
moderate and severe myositis with mononuclear and fibroblastic
infiltrations was present in equal proportion in the skeletal mus-
cles. Mild inflammatory infiltrate and scarce parasites were found
in lungs. The blood of all animals was positive for T.cruzi on 21st and
on 50th day of infection, according to PCR analysis.
Tissue parasitism was not found in the T. cruzi infected mice
treated with NFT, tissue lesions were significantly less pronounced:
namely, light, if any, focal mononuclear infiltration in the heart and
striated muscles were detected. Although the blood of all mice was
T.cruzi positive on 21st day, only 30% were positive on 50th day.
On the other hand, parasites were present in myocardium and in
the striated muscles, but not in lungs of mice treated with NTZ and
NFT. Mild and focal myocarditis according to Dallas criteria was
detected in 100% of cases. Eighty percent of animals presented light
inflammatory changes in the striated muscles and 20% of animals
had moderate and confluent (gravity 1 and extension 2 according to
Dallas criteria) mononuclear infiltrations in this tissue. In the ani-
mals subjected to the combined treatment and euthanized at the
day 51 post-infection, scarce parasitism and bigger proportion of
moderate inflammatory changes were found in the same tissues
(moderate confluent infiltrations alongside with necrosis). At this
point, our observations suggest a reactivation of the infection due
to NZT, even in NFT-treated animals.
In contrast to the NFT-treated groups, the mice infected with
T.cruzi and treated with different concentrations of NTZ showed
even more severe grade of tissue damage compared to the un-
treated infected mice. Accordingly, in the groups treated with NTZ
only, the number of animals was limited, due to high mortality. But
it was impossible to undertake post mortem tissue analysis because
of rapid autolysis.
Different hypotheses may be proposed to explain the enhanced
level of parasitemia in mice treated with NTZ. According to the
earlier reports the virulence and parasitemia levels directly depend
on the internalization of the metacyclic trypomastigotes in a
membrane-bounded vacuole in the host cells (Romano et al., 2012).
This process was shown to be dependent on energetic status of the
host cell governed by mammalian target of rapamycin (mTOR).
Invasion by tissue culture trypomastigotes may be increased upon
host cell pre-starvation or mTOR inhibition by rapamycine
(Miyazawa et al., 2012). On the other hand, NTZ was shown to
inhibit mTORC1 signaling (Lam et al., 2012), and then may cause
increased invasion with consequent increased liberation of para-
sites into the bloodstream.
Cellular immunity is another crucial factor capable to control
T.cruzi infection (Machado et al., 2012). mTOR plays a crucial role in
the adaptive immunity, maintaining the balance between T cell
activation and quiescence. Antigen recognition by naïve T cells
leads to mTOR activation and promotes the differentiation of these
cells into effector T cells (revised in (Chi, 2012; Saleiro and Platanias,
2015)). Rapamicine, an mTOR inhibitor, is widely considered as an
immunosuppressor, and is prescribed to prevent graft rejection or
to treat autoimmune diseases (Dobashi et al., 2011). While NTZ is
capable to inhibit mTOR, it may demonstrate significant immuno-
suppressive effect and decrease specific immune response to T.cruzi
infection.
Although it was performed on a murine model, our study has
revealed a phenomenon of important clinical relevance. NTZ,
originally developed and commercialized as an antiprotozoal agent
(Rossignol and Maisonneuve, 1984; Gilles and Hoffman, 2002), later
was recognized as a broad-spectrum antibacterial and antiviral
drug; so it has been inclusively repurposed for the treatment of
influenza (Rossignol, 2014). Considered to be very safe, it has been
used for treating of a broad spectrum of intestinal parasitic in-
fections in the Latin America countries endemic for Chagas' disease.
Recently NTZ was proposed as a potential candidate to be added to
 J.S. Valle-Reyes et al. / Experimental Parasitology 172 (2017) 44e50
Fig. 3. Histopathological findings in tissues of T. cruzi-infected mice on 21stday of post-infection. Representative hematoxylin & eosin stained sections demonstrating heart,
muscle and lung histological lesions. Top panel: untreated animals; middle panel: NFT-treated animals; bottom panel: NTZ-treated (100 mg/kg) animals. Arrows indicate in-
flammatory infiltrates. Amastigotic nests are delimited by squares. A: alveoli; B: bronchi; V: vessels.
the list of essential medicines for integrated neglected tropical
disease control and elimination, as well as to be used in mass drug
administration programs (Hotez, 2014). It is important to stress that
Chagas disease was not listed as possible target for NTZ. However,
our findings indicate that in T.cruzie infected persons, NTZ
employed against the comorbidities treatable with this compound
may cause unexpected health-threatening effects and then should
not be considered as a safe drug. Moreover, precautions should be
taken bearing in mind that T.cruzi infection, is under-diagnosed in
many regions, especially in the acute and asymptomatic (indeter-
minate) chronic phases (Hotez et al., 2012). We suggest that a
reliable diagnostic for Chagas’ disease is required before taking a
NTZ treatment, which it may completely prevent undesired
complications.
Furtherer studies are required in order to reveal the mechanism
of this phenomenon in more detail. It would be of special interest to
address the question about NTZ effect in chronic phase of disease as
well as undertake new studies with other T.cruzi strains.
Acknowledgments
We would like to acknowledge Victor Montan ~ o Plascencia for
his technical assistance in processing of tissues samples. We are
thankful to Mr. Scott Hayes for his indispensible help in editing the
manuscript for English language and style.
49
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