Acta Tropica 103 (2007) 195200

Development of a real-time PCR assay for Trypanosoma cruzi

detection in blood samples
Maria Piron a, , Roser Fisa b , Natalia Casamitjana a , Paulo Lopez-Chejade b ,
Llus Puig a , Mireia Verges b , Joaquim Gascon c , Jordi Gomez i Prat d ,
Montserrat Portus b , Slvia Sauleda a
a Transfusion Safety Lab., Banc de Sang i Teixits, Barcelona, Spain
Department of Parasitology, Faculty of Pharmacy, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
Department of Tropical Medicine, Hospital Clnic i Provincial de Barcelona, C/Rossello 132, 2-2, 08026 Barcelona, Spain
d Unitat de Medicina Tropical i Salut Internacional Drassanes, Av. Drassanes, 19, 08001 Barcelona, Spain

b
c

Received 8 February 2007; received in revised form 4 May 2007; accepted 29 May 2007
Available online 23 June 2007

Abstract
The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients.
Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood
(7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1
blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic
areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time
PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human
RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR
contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as
the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive
hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients
were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides
an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in
chagasic patients.
2007 Elsevier B.V. All rights reserved.
Keywords: Trypanosoma cruzi; Real-time PCR; Chagas disease

1. Introduction
Chagas disease is a protozoan infection caused by
Trypanosoma cruzi. The disease is widespread in Central

Corresponding author at: Transfusion Safety Laboratory, Banc

de Sang i Teixits, Passeig Vall dHebron 119-129, 08035 Barcelona,
Spain. Tel.: +34 93 2749025; fax: +34 93 2749027.
E-mail address: mpiron@vhebron.net (M. Piron).
0001-706X/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2007.05.019

and South America and the number of infected people is

estimated at 1214 million. Transmission of T. cruzi to
humans occurs after the bite of the reduviid bug, when
the excreta containing the parasites contaminate the bite
wound and mucosa, usually after scratching. The parasite can also be transmitted from infected mothers to
their children, through blood transfusion or organ transplantation, and in secondary ways, by oral transmission
or laboratory accidents (WHO, 2002). Due to migration

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M. Piron et al. / Acta Tropica 103 (2007) 195200

movements from rural to urban areas and from endemic

countries to North America and Europe, chagas disease
is now found in non-endemic areas where, even in the
absence of the vector, the infection can still be transmitted congenitally, by blood transfusion and by organ
transplantation.
Chagas disease passes through two successive stages,
the acute and the chronic phase. After the acute clinical manifestations disappear, the infection rests with
a long period of clinical latency, called the indeterminate form period, which may last throughout life or
evolve to a chronic phase with cardiac or gastrointestinal involvement. The chronic phase is characterised by
high specific IgG antibody production and low, intermittent parasitaemia, which results in the low sensitivity of
the classic parasitological techniques. Diagnosis in this
stage mainly relies on serological techniques, despite
their lack of specificity when crude T. cruzi antigens
are used (Luquetti and Rassi, 2000). Also falsenegative
serological results have been reported, which may be
related to the antigen used, the parasite strain involved
in the infection, or poor immune response of the patient
(Luquetti and Rassi, 2000). In addition, serology is not
accurate enough in the evaluation of treatment efficacy, as it remains positive from 6 months to some
years after successful treatment, particularly in adults,
and has low positive predictive value in the diagnosis of congenital Chagas disease in the first months
of life due to transfer of antibodies from mother to
child.
Molecular-based assays, in particular amplification
by the polymerase chain reaction (PCR), provide a
more sensitive alternative to traditional parasitological
techniques. Some PCR protocols have been described,
leading to unequal results, probably due to differences
in the volume of blood processed, the DNA extraction procedure, or the DNA region of T. cruzi amplified
(Junqueira et al., 1996; Virreira et al., 2003).
Nested PCR (N-PCR) provides higher sensitivity than
single-run PCR assay and has already been reported for
supplementary diagnosis of Chagas disease (Marcon et
al., 2002). This technique is highly sensitive, but time
consuming and entails a high risk of false positive results
due to contaminating amplicons.
In contrast, real-time PCR technology uses fluorescent labels for continuous monitoring of amplification
throughout the reaction. The main advantages are the
rapid throughput of results (amplification and detection
in one step) and reduced risk of carry-over contamination (minimal manipulation of samples and use of UNG).
Real-time PCR can be optimised both as a qualitative and
quantitative assay.

The aim of this study was to develop and evaluate the

efficacy of a real-time PCR assay to detect and eventually
quantify T. cruzi in blood of chagasic patients.
2. Patients and methods
2.1. Positive controls
Epimastigotes from T. cruzi (Maracay strain) cultured
in LIT medium with 10% heat-inactivated foetal calf
serum were used to prepare the positive controls. The
number of parasites was assessed by microscopic examination and was adjusted to 7.8 epimastigotes/mL in
human EDTA whole blood from pooled healthy individuals (Control 1) and frozen at 20 C. For the sensitivity
analysis (qualitative assay), two-fold serial dilutions of
Control 1 in human EDTA whole blood were performed
down to 0.06 epimastigotes/mL. To assess the dynamic
range of the real-time PCR technique, 10-fold serial
dilutions of blood spiked with T. cruzi epimastigotes
were obtained (106 101 epimastigotes/mL). DNA was
extracted as described below and the real-time PCR reactions were performed as triplicates for each sample.
2.2. Patients and samples
Thirty-eight blood samples from patients with
chronic Chagas disease, who were native to endemic
areas (33 Bolivians, 3 Argentines, 1 Brazilian, 1 Honduran) and living in Barcelona, Spain were included.
One millilitre serum sample and 1 mL of EDTAblood
were frozen at 20 C until analysis. All of these patients
arrived at Spain between 2000 and 2004. The patients
included in our study were all adults, diagnosed as chronically infected by T. cruzi, and had not received any prior
specific treatment. All provided signed informed consent for inclusion in the study. Patients were diagnosed
by two serological techniques, the Bioelisa Chagas assay
(Biokit, Barcelona, Spain), which uses recombinant antigens, and a conventional in-house ELISA with whole T.
cruzi antigens. Blood from a newborn with congenital
Chagas disease (Riera et al., 2006) was also included
in the study. Blood samples from 100 healthy individuals from endemic areas, and 24 healthy individuals from
non-endemic areas were used as negative controls for
the specificity study. Peripheral blood buffy coat and
bone marrow from 20 patients with Leishmania infantum
visceral leishmaniasis (VL) were also studied.
2.3. DNA extraction
DNA was extracted from 100 L of 1 mL thawed
EDTAblood, 200 L buffy coat and 200 L bone

M. Piron et al. / Acta Tropica 103 (2007) 195200

197

Table 1
Primers and probe selected for the T. cruzi nested PCR and for the real-time PCR
PCR

Primer or probe

Sequencea (5 3 )

Nucleotide positionb

Nested

TCZ 1 (forward)
TCZ 2 (reverse)
TCZ 3 (forward)
TCZ 4 (reverse)

CGAGCTCTTGCCCACACGGG
CCTCCAAGCAGCGGATAGTTCAGG
TGCTGCASTCGGCTGATCGTTTTCGA
CARGSTTGTTTGGTGTCCAGTGTGTGA

120
165188
2146
142168

Real-time

Cruzi 1 (forward)
Cruzi 2 (reverse)
Cruzi 3 (probe)

ASTCGGCTGATCGTTTTCGA
AATTCCTCCAAGCAGCGGATA
CACACACTGGACACCAA

2746
172192
143159

a
b

R, A/G; S, C/G.
Nucleotide position in the DNA satellite sequence (GenBank accession no. AY520036).

marrow with the High Pure PCR Template Preparation kit (Roche, Basel, Switzerland), and eluted in
200 L of elution buffer according to the manufacturers instructions. Five microliters of extracted DNA
was amplified in triplicate in both the real-time PCR and
N-PCR.
2.4. Nested PCR assay
The N-PCR assay is based on a previously described
procedure (Marcon et al., 2002) using primers TCZ
1 and TCZ 2 for the first reaction and TCZ 3 and
TCZ 4 for the nested amplification, with some modifications (Table 1). PCR was carried out in 20 L
reaction mixture containing 1.5 mM MgCl2 and 1 U of
RedTaq polymerase (Sigma), with annealing temperatures of 63 C (40 cycles) and 57 C (25 cycles) in the
first and second amplification runs, respectively. The
PCR programs were run on an MJ Research thermocycler (PTC-200), and the 149-nucleotide amplicon was
separated by electrophoresis on 3% agarose gel and visualised by ultraviolet transillumination after staining with
ethidium bromide.

conditions in the PCR mixture were 1 Universal Master Mix with UNG (Applied Biosystems), 0.1 RNase
P detection reagent, 750 nM each T. cruzi primer and
250 nM for the T. cruzi probe in a 20 L reaction. The
samples were amplified in a thermocycler ABI Prism
7700 (Applied Biosystems) with the following PCR conditions: first step (2 min at 50 C), second step (10 min at
95 C) and 45 cycles (15 s at 95 C and 1 min at 58 C). A
sample was considered valid when the internal control
was efficiently amplified, and was considered positive
for T. cruzi when the threshold cycle (Ct) for the T. cruzi
target was <45. The Ct for a given sample is the first
cycle of the PCR reaction where fluorescence is detected
above the baseline. A non-template control was included
in each run as the real-time PCR negative control.
2.5.1. Statistical analysis
The sensitivity of the real-time PCR assay was calculated by PROBIT analysis (SPSS v13, Chicago, IL),
and the 95% and 50% positive hit rates are reported. The
Ct values are expressed as the mean and standard deviation. The coefficient of variation was calculated as the
percentage of the Ct standard deviation divided by its
mean.

2.5. Real-time PCR assay

3. Results
Primers and probe were selected from the same repetitive sequences of satellite DNA amplified in the N-PCR
protocol and designed according to the Primer Express
Software (Applied Biosystems). The primers Cruzi 1
and Cruzi 2 amplify a 166-bp segment. The probe Cruzi
3 was labelled with 5 FAM (6-carboxyfluorescein) and
3 MGB (minor groove binder). Table 1 describes the
position of primers and probe for both the N-PCR and
real-time PCR. The pre-developed reagent for the RNase
P human gene (TaqMan Human RNase P detection
reagent, Applied Biosystems) was included in the PCR
reaction as internal control of amplification. The final

3.1. Analytical sensitivity and specicity

The sensitivity of the real-time PCR was assessed
by analysing 24 aliquots of each two-fold serial dilution of parasite-spiked whole blood (Table 2). The 24
PCR results for each dilution were obtained from two
independent runs. After PROBIT analysis, the 95%
detection limit was found at 2.07 parasites/mL (95%
CI 1.682.80) and the 50% detection limit at 0.80 parasites/mL (95% CI 0.621.03). Therefore, a negative
result in the real-time PCR assay should be reported

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M. Piron et al. / Acta Tropica 103 (2007) 195200

Table 2
Results for the sensitivity assay for the real-time PCR using two-fold
dilutions of blood sample spiked with a known T. cruzi concentration

Table 3
Description of the positive and discrepant samples in the real-time PCR
and nested PCR in blood from patients with Chagas disease

Parasites/mL

Positive/tested

Sample

Origin

Real-time PCR (mean Ct)

N-PCR

7.81
3.91
1.95
0.98
0.49
0.24
0.12
0.06

24/24
24/24
21/24
17/24
10/24
6/24
4/24
2/24

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Argentina
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Honduras
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Spaina

34.63
28.73
32.92
35.48
36.37
39.21
35.43
39.36
36.33
33.50
30.60
39.35
39.22
39.22
35.55
Negative
Negative
14.74b

Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Positive
Positive

inferior to 2.8 parasites/mL. The sensitivity of the realtime PCR was similar to that obtained with the N-PCR
assay, which in our hands yields a clear band down to 2
parasites/mL.
As for the quantitative results, the real-time PCR reactions performed in triplicate with 10-fold serial dilutions
of DNA from blood spiked with T. cruzi epimastigotes (106 101 epimastigotes/mL) showed a linear curve
from 105 down to 10 parasites/mL of blood, with a fourlog dynamic range (Fig. 1).
As for the reproducibility of the assay, single-use
aliquots of Control 1 were extracted and amplified in 28
independent runs. Mean Ct values were 33.73 1.68 for
the T. cruzi target and 28.91 1.36 for the human RNase
P gene, for a resulting coefficient of variation of 4.97%
and 4.87%, respectively. We observed no contamination
of the negative controls or non-template controls of the
assay due to carry-over during either the DNA extraction
or the real-time PCR set-up.
3.2. Clinical sensitivity and specicity
Negative samples have been tested to determine the
specificity of the real-time PCR and the new primers
described here. None of the 124 T. cruzi seronegative

a
b

Congenital case, mother from Bolivia.

From buffy-coat sample.

samples or 20 samples from VL patients tested positive

by real-time PCR. In our study, the specificity of the
real-time PCR was, therefore, 100%. When the chagasic
patients were analysed, we found that 23 out of 39 samples were negative by real-time PCR. All these samples
had an acceptable signal for RNase P gene amplification
(mean Ct 28.94 2.65), thus indicating that no false negative results were generated by PCR inhibitors, nor were
there errors in sample dispensing into the PCR reaction.
Conversely, 16 samples out of the 39 chagasic patients
(41%) were positive by the real-time PCR for T. cruzi.
The mean Ct value for the T. cruzi target in positive samples from chronic patients was 35.54 3.29, being all
of them near or below the lower limit of the dynamic
range (Fig. 1). When compared to the N-PCR, agreement between the techniques was 90%. Four samples
had discrepant results, two positives by N-PCR and two
positives by real-time PCR (Table 3).
Finally, the sample from the newborn with congenital
infection was positive with a mean Ct equal to 14.74,
which is over the dynamic range.
4. Discussion

Fig. 1. Dynamic range of the real-time PCR. DNA of 10-fold serial

dilutions of blood spiked with T. cruzi epimastigotes were amplified as
triplicates (106 101 epimastigotes/mL). The linear regression curve
and regression coefficient are indicated.

Herein, we describe the development of a new realtime PCR method based on TaqMan technology for the
diagnosis of T. cruzi infection in blood samples. Previously described PCR methods for the diagnosis of
Chagas disease were based on one-step PCR, with or

M. Piron et al. / Acta Tropica 103 (2007) 195200

without hybridisation, or N-PCR followed by gel electrophoresis. Consequently, they were time-consuming
and produced false positive results due to contamination
of the samples by carry-over, or false negative results due
to inhibition in the amplification process, when PCRs
were performed in the absence of parallel amplification
of a human gene as a control.
More recently, some works have described real-time
PCR methods using SybrGreen technology which is
based on incorporation of a fluorescent dye into the
double-strand DNA (Cummings and Tarleton, 2003;
Virreira et al., 2006). We chose to use a TaqMan probe
that better guaranties the specificity of the measured signal.
Our real-time PCR method is based on amplification
of a genomic DNA sequence which had been previously
described as specific for all T. cruzi lineages (Moser et
al., 1989; Virreira et al., 2003). It has been designed
to include a decontamination step and an internal control for the quality of amplification, and results can be
interpreted both qualitatively and, with the appropriate
standard curve, quantitatively. As is shown in our study,
the real-time PCR has a sensitivity similar to that of the
N-PCR, with a concordance in the clinical samples tested
of 90%. Discrepant results between real-time PCR and
N-PCR can be explained by low parasitaemia, probably
below the limit of detection of both PCR techniques.
Indeed, we noted that both samples with a positive realtime PCR but negative N-PCR result presented relatively
late Ct for the real-time technique (39.35 and 39.22,
Table 3), which actually correspond to low parasitaemia
according to our standard curve (Fig. 1). Therefore, sensitivity could be further improved if a larger volume of
blood were processed for DNA extraction. The samples
included in this study are representative of the T. cruziinfected population in a non-endemic area, that is, young
adults in an indeterminate phase of the disease with low
or absent parasitaemia. We must mention that all the
selected patients were diagnosed on serological findings
and had not received any treatment before enrolment
in the study. All patients with Chagas chronic disease
arrived at Spain between 2000 and 2004 and there was
no significant difference in the time spent outside the
endemic area between patients who presented a positive result by PCR (real-time or N-PCR) and those who
presented a negative result. Use of the real-time PCR
in this context will be as a qualitative assay to accompany the serological diagnosis. Although the quantitative
assay is of limited value in the indeterminate phase of
the disease, the true potential of the real-time PCR will
eventually be better recognised in other situations for
which PCR-based techniques have been promoted, such

199

as congenital infections (Mora et al., 2005; Schijman et

al., 2003; Virreira et al., 2003), monitoring parasitaemia
during and after treatment (Apt et al., 2005; Britto et al.,
2001; Russomando et al., 1998; Sanchez et al., 2005;
Schijman et al., 2003), early detection of relapses after
heart transplantation (Maldonado et al., 2004), and other
immunosuppressive circumstances. As expected, a low
Ct value (Ct = 14.74), even over the dynamic range of the
technique, was found in the sample from the newborn
with acute congenital infection included in this study.
To be quantifiable, this sample should be diluted before
the extraction step in order to enter the dynamic range
of the technique. However, for more accurate results
in any PCR assay, an international reagent with properly quantified T. cruzi DNA load would be necessary
for each experiment. International standards for various
infectious agents (human immunodeficiency virus, hepatitis C virus) (Saldanha et al., 1999) are available for
molecular biology assays, in order to standardise and
compare methods and laboratory performance. Agencies providing such materials should be encouraged to
make properly characterised T. cruzi reagents available
for this purpose.
In summary, this new real-time PCR system is simpler, faster and more reliable than conventional PCR
techniques. Moreover, the possibility of quantification
and the reduced risk of contamination are added values
to this method.
Acknowledgements
This study has been partially supported by grant
024/13/2004 from the Ag`encia dAvaluacio de Tecnologies I Recerca M`ediques (AATRM, Catalunya, Spain).
The T. cruzi Maracay strain was kindly provided by
Prof. A. Osuna (Granada, Spain). We are grateful to
Celine Cavallo for the English revision of the manuscript
and to Marta Espelt for technical assistance.
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