Comparison of Trypanosoma cruzi detection by
PCR in blood and dejections of Triatoma infestans
fed on patients with chronic Chagas disease
Ximena Coronado a , Ines Zulantay a , Eduardo Reyes c , Werner Apt a ,
Juan Venegas a , Jorge Rodriguez b , Aldo Solari a , Gittith Sanchez a,∗
a
Programa de Biologı́a Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina,
Universidad de Chile, Independencia 1027, Correo 70086, Santiago 7, Chile
b Escuela de Salud Pública, Facultad de Medicina, Universidad de Chile, Santiago, Chile
c Estudiante de Medicina, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Abstract
In this study, we compare the sensitivity of detecting Trypanosoma cruzi in dejections of Triatoma infestans nymphs that had fed
on the blood of chronic chagasic patients, with detection of T. cruzi in peripheral blood, using a polymerase chain reaction assay
(PCR-D and PCR-B, respectively). Fifty-seven chronic patients were evaluated who were positive (group I) or negative (group II)
by xenodiagnosis (XD). Patients showed 84.8 and 75% positive PCR results in both kinds of samples in groups I and II, respectively.
Six cases (10.5%) showed positive PCR-D and negative PCR-B, five of them belonged to group I. In contrast, five cases of group
II showed negative PCR-D and positive PCR-B. Overall, the PCR-D assay gave positive results in 52 out of 57 samples (91.2%),
while 51 out of 57 (89.5%) were positive by PCR-B. In comparison, only 57.9% were positive by XD (p = 0.0001). In conclusion,
PCR performed in dejection or blood was more sensitive for the parasite detection than xenodiagnosis. All patients (100%) were
detected positive when both, PCR-D and PCR-B, were applied.
Keywords: Chronic Chagas disease; Polymerase chain reaction; Xenodiagnosis
Chagas disease, caused by the protozoan parasite
Trypanosoma cruzi (T. cruzi), is a major public health
problem in Latin America. T. cruzi detection during the
chronic phase is difficult due to the small number of
circulating trypomastigote forms, which are recovered
by xenodiagnosis (XD) only from a limited number of
patients (Castro et al., 2002). The polymerase chain
reaction (PCR) in human blood or serum has been
∗
Corresponding author. Tel.: +56 2 9786757; fax: +56 2 7355580.
E-mail address: [email protected] (G. Sanchez).
considered an alternative technique for parasitological
diagnosis (Britto et al., 2001). T. cruzi kinetoplast
DNA detection by PCR has been applied in dejections
of experimentally and naturally infected triatomines
(Chiurillo et al., 2003), and in dejections of insect
vectors fed with blood from infected patients (Silber
et al., 1997). Kinetoplast DNA of T. cruzi is abundant
and can be easily extracted (Junqueira et al., 1996).
The use of the vector fecal drop as template for
PCR assays was reported, although, others suggest
that fresh tissue obtained by dissection of the bug is
preferable (D’Alessandro-Bacigalupo and Saravia,
X. Coronado et al.
1992). PCR using these two types of samples for the
detection of T. cruzi are equivalent. Also the detection
capacity of T. cruzi by microscopy and PCR has been
evaluated. PCR was significantly more sensitive than
microscopy for the detection of the parasite (Dorn et
al., 2001; Botto-Mahan et al., 2005). Other authors
applied improved DNA extractions and PCR strategies
for screening and identification of T. cruzi lineages
directly from faeces of triatomines. They found that
in the faecal sample was detected a mixed of two
clones while in the culture sample only showed one
of them. This fact demonstrates the importance of
lineage identification of T. cruzi in field-collected
triatomine (Marcet et al., 2006). The aim of the present
work was to compare PCR sensitivity for detection
of T. cruzi in dejections of Triatoma infestans (T.
infestans) nymphs that had fed on the blood of chronic
chagasic patients, with PCR performed in peripheral
blood of the same patients. Oligonucleotides 121 (5 AAATAATGTACGGG (T/G) GAGATGCATGA3 ) and
122 (5 -GGTTCGATTGGGGTTGGTGTAATATA3 ),
that amplify a 330 bp T. cruzi DNA fragment from
minicircles were used (Wincker et al., 1994).
Fifty-seven chronic chagasic individuals, 25 females
and 32 males (mean 22 years old) from endemic regions
of Chile (IV and V regions) (Apt et al., 2003) were
selected on the basis of positive serologic results (IFI and
ELISA), and grouped as XD positive (group I, n = 33) or
XD negative (group II, n = 24). The XD test was carried out using two wooden boxes with seven T. infestans
nymphs stage III each (free of infection). Microscopic
examination of insect dejection was performed at 30, 60
and 90 days after feeding, and a pool of all the insects
dejection was collected to be used in the PCR assay,
no matter whether the XD was positive or negative at
these time (Schenone, 1999). As negative control, noninfected triatomines dejections were used for PCR. Two
millilitres of whole blood was mixed with a similar volume of the buffer Guanidine HCl 6 M and EDTA 0.2 M,
and DNA phenol extracted. PCR were made in triplicate in blood or dejection and performed as described
(Solari et al., 2001). A positivity sample was considered
when the three assays were positive. However, in case of
discordance other four assays were performed. Positive
samples were considered when at least five out of seven
assays were positive. Results were analyzed using the ␹2
goodness of fit statistical test, and p-values <0.05 were
considered to be significant.
Conventional parasitological methods rarely reach
100% sensitivity in the chronic phase of the Chagas disease using serology as gold standard, since, this parameter depends on the number of parasites in peripheral
blood (Castro et al., 2002). Recently, molecular assays as
PCR, which amplify certain repetitive sequences of trypanosome kDNA, have been proposed as a good alternative tool for detection of T. cruzi in human blood (Britto
et al., 1995; Chiari, 1999; Zulantay et al., 2004). The
PCR assays have shown a variable degree of efficiency,
thus, a negative result does not necessarily mean that
the chagasic patient is free of infection (Chiari, 1999).
In the present study, T. cruzi kDNA detection was considered positive when a band of 330 bp was detected.
Fifty-seven chronic patients were evaluated who were
positive (group I) or negative (group II) by xenodiagnosis. Patients showed 84.8 and 75% positive PCR results
in both kinds of samples in groups I and II, respectively.
Six cases showed positive PCR-D and negative PCR-B,
five of these belonged to group I. In contrast, five cases
of group II showed negative PCR-D and positive PCRB (Table 1). Therefore, 10.5% of the cases were only
detected by PCR-D and 8.8% only by PCR-B. The PCRD assay gave positive results in 52 out of 57 (91.2%),
while in 51 out of 57 (89.5%) were positive by PCR-B. In
contrast, only 57.9% were positive by XD (p = 0.0001).
Other authors reported 59.4% sensitivity with PCR in
blood samples of serologically positive chronic chagasic individuals compared with 35.6% obtained by XD
(Junqueira et al., 1996). However, PCR has been reported
to provide 90% of T. cruzi detection efficiency with
seropositive results for Chagas disease in other endemic
regions (Britto et al., 1995). A study that detected T.
cruzi DNA by PCR in dejection of insect vectors fed
Table 1
PCR-B and PCR-D in chronic chagasic patients with positive or negative xenodiagnosis
Group
Cases
XD
n
I
II
33
24
Total
57
+
−
[PCR-B (+) PCR-D (+)]
[PCR-B (−) PCR-D (+)]
[PCR-B (+) PCR-D (−)]
n
%
n
%
n
%
28
18
84.8
75
5
1
15.2
4.2
0
5
0
20.8
46
80.7
6
10.5
5
8.8
X. Coronado et al.
with blood from infected patients using nuclear oligonucleotides BP1/BP2, found that this technique was only
10% more sensitive than XD (Silber et al., 1997).
Our results using 121 and 122 primers in these type
of biological samples, showed a better overall sensitivity than XD. Our results demonstrate that PCR applied
in both types of samples, peripheral blood or in dejection of triatomines, is significantly more sensitive for
detection of T. cruzi than microscopically examination
used in XD and confirm the importance of applying
PCR in these biological samples. We have demonstrated
that PCR applied in faecal samples is more precocious
and sensitive than xenodiagnosis (manuscript in preparation).
The presence of T. cruzi in 100% of patients was only
detected by PCR when using both samples. In 15.2%
(XD+) and 4.2% (XD−) of the cases were PCR-D (+)
and PCR-B (−), possibly for the presence of T. cruzi
clones better adapted in triatomines than in human blood.
Also, this result is likely due to the parasitemias being
below the detection limit of the assay. We discarded the
possibility of PCR inhibition in the DNA lysates obtained
from these blood samples because a protocol was used as
PCR-internal control for amplification of the human ␤globin gene sequences, as described (Saiki et al., 1985).
On the other hand, 8.8% of the cases were PCR-B (+)
and PCR-D (−) suggesting very low parasitemia, therefore, diminishing the T. cruzi detection. According to
our results, we estimated that the detection limit was
one parasite in 2 ml of blood samples, the volume of T.
infestans feeding was 0.1 ml/triatomine (two boxes with
seven nymphs each = 1.4 ml of blood) and the volume of
blood used for PCR was 2 ml, therefore, the volume of
samples were in the same order of magnitude.
T. cruzi populations appear to have a multiclonal
structure (Barnabé et al., 2005), therefore, it is important to analyze parasites amplified in the XD test (Apt
et al., 2003) because a probably selection of some T.
cruzi genotypes and the amplification in T. infestans of
minor parasite clones (Breniere et al., 1998). Recent
studies of our group support this idea of a preferential selection of T. cruzi genotypes in vertebrate and
invertebrate hosts (Coronado et al., 2006). In previous
studies, PCR was used to analyze T. cruzi directly in
biological samples: feces of infected Triatomine bugs,
blood samples of experimentally infected mice and artificially infected human blood samples. The diagnosis of
T. cruzi infection and simultaneous direct identification
of the different natural clones, circulating in vectors and
mammalian blood without isolation of the stocks was
performed (Breniere et al., 1992). In other studies, PCR
was employed to detect T. cruzi DNA in both insects
fed on experimentally infected monkeys and insects.
They found that monkeys with parasitemia levels below
the limit of detection show delayed results than monkeys with higher parasitemia. These results suggest that
PCR can be used to speed the xenodiagnosis results with
great sensitivity (Russomando et al., 1996). Our results
demonstrated that T. cruzi was detected in all patients
only when both, PCR-D and PCR-B were applied. This
fact show the importance of using XD dejection samples
for PCR, specially in patients with negative PCR-B in
order to evaluate chemotherapeutical efficacy and/or to
improve diagnosis.
Acknowledgements
This study was supported by FONDECYT 1040731,
and fellowship PG/83/2003, Post grade Department and
Postitle, University of Chile.
References
Apt, W., Arribada, A., Zulantay, I., Sanchez, G., Vargas, S.L.,
Rodriguez, J., 2003. Itraconazole or allopurinol in the treatment of
chronic American trypanosomiasis: the regresion and prevention
of electrocardiographic abnormalities during 9 years of follow-up.
Ann. Trop. Med. Parasitol. 97 (1), 23–29.
Barnabé, C., Tibayrenc, M., Marcondes, C.B., 2005. Genetic characterization of Trypanosoma cruzi natural clones from the state of
Paraiba. Braz. Mem. Inst. Oswaldo Cruz. 100 (3), 273–275.
Botto-Mahan, C., Ortiz, S., Rozas, M., Cattan, P., Solari, A., 2005.
DNA evidence of Trypanosoma cruzi in the Chilean wild vector
Mepraia spinolai (Hemiptera: Reduvidae). Mem. Inst. Oswaldo
Cruz. 100 (3), 237–239.
Breniere, S.F., Bosseno, M.F., Revollo, S., Rivera, M.T., Carlier, Y.,
Tibayrenc, M., 1992. Direct identification of Trypanosoma cruzi
natural clones in vectors and mammalian hosts by polymerase reaction amplification. Am. J. Trop. Med. Hyg. 46 (3), 335–341.
Breniere, S.F., Bosseno, M.F., Telleria, J., Bastrenta, B., Yaksic, N.,
Noireau, F., Alcazar, J.L., Barnabe, C., Wincker, P., Tibayrenc, M.,
1998. Different behavior of two Trypanosoma cruzi major clones:
transmission and circulation in young Bolivian patients. Exp. Parasitol. 89, 285–295.
Britto, C., Cardoso, M.A., Monteiro, Van, C.M., Hasslocher-Moreno,
A., Xavier, S.S., Oeleman, W., Santoro, A., Pirmez, C., Morel,
C., Wincker, P., 1995. Polymerase chain reaction detection of
Trypanosoma cruzi in human blood samples for diagnosis and treatment evaluation. Parasitology 110, 241–247.
Britto, C., Silvera, C., Cardoso, M.A., Marques, P., Luquetti, A.,
Macedo, V., Fernandez, O., 2001. Parasite persistance in treated
chagasic patients revealed by xenodiagnosis and polymerase chain
reaction. Mem. Inst. Oswaldo Cruz. 96, 823–826.
Castro, A.M., Luquetti, A.O., Rassi, A., Rassi, G.G., Chiari, E., Galvao, L.M.C., 2002. Blood culture and polymerase chain reaction
for the diagnosis of the chronic phase of human infection with
Trypanosoma cruzi. Parasitol. Res. 88, 894–900.
Chiari, E., 1999. Chagas disease diagnosis using polymerase chain
reaction, hemoculture and serologic methods. Mem. Inst. Oswaldo
Cruz. 94 (Suppl. 1), 299–300.
X. Coronado et al.
Chiurillo, M.A., Crisante, G., Rojas, A., Peralta, A., Dias, M., Guevara,
P., Anez, N., Ramirez, J.L., 2003. Detection of Trypanosoma cruzi
and Trypanosoma rangeli infection by duplex PCR assay based
on the telomeric sequences. Clin. Diagn. Lab. Immunol. 10 (5),
775–779.
Coronado, X., Zulantay, I., Albrech, H., Rozas, M., Apt, W., Ortiz,
S., Rodrı́guez, J., Sánchez, G., Solari, A., 2006. Variation in Trypanosoma cruzi clonal composition detected in blood patients and
xenodiagnosis triatomines: implications in molecular epidemiology. Chil. Am. J. Trop. Med. Hyg. 74 (6), 1008–1012.
D’Alessandro-Bacigalupo, A., Saravia, N.G., 1992. Trypanosoma
rangeli. In: Kreier, J., Baker, J. (Eds.), Parasitic Protozoa. Academic Press, San Diego, pp. 1–55.
Dorn, P.L., Flores, J., Brahney, B., Gutierrez, A., Rosales, R., Rodas,
A., Monroy, C., 2001. Comparison of polymerase chain reaction
on fresh tissue samples and fecal drops on filter paper for detection
of Trypanosoma cruzi in Rhodnius prolixus. Mem. Inst. Oswaldo
Cruz. 96 (4), 503–505.
Junqueira, A.C., Chiari, E., Wincker, P., 1996. Comparison of the polymerase chain reaction with two classical parasitological methods
for the diagnosis of Chagas disease in a endemic region of northerneastern Brazil. Trans. R. Soc. Trop. Med. Hyg. 90 (2), 129–132.
Marcet, P.L., Duffy, T., Cardinal, M.V., Burgos, J.M., Lauricella, M.A.,
Levin, M.J., Kitron, U., Gurtler, R.E., Schijman, A.G., 2006. PCRbased screening and lineage identification of Trypanosma cruzi
directly from faecal samples of triatomine bugs from northwestern
Argentina. Parasitology 132 (Pt 1), 57–65.
Russomando, G., Rojas de Arias, A., Almiron, M., Figueredo, A.,
Ferreira, M.E., Morita, K., 1996. Trypanosoma cruzi: polymerase
chain reaction-based detection in dried feces of Triatoma infestans.
Exp. Parasitol. 83 (1), 62–66.
Saiki, R.K., Scharf, S., Faloona, F., 1985. Enzymatic amplification of
␤-globin sequences and restriction site analyses for diagnosis of
sickle cell anemia. Science 230, 1350–1354.
Schenone, H., 1999. Xenodiagnosis. Mem. Inst. Oswaldo Cruz. 94
(Suppl. I), 289–294.
Silber, A.M., Bua, J., Porcel, B.M., Segura, E.L., Ruiz, A.M., 1997.
Trypanosoma cruzi: specific detection of parasites by PCR in
infected humans and vectors using a set primers (BP1/BP2) targeted to a nuclear DNA sequence. Exp. Parasitol. 85 (3), 225–232.
Solari, A., Ortiz, S., Soto, A., Arancibia, C., Campillay, R., Contreras, M., Salinas, P., Rojas, A., Schenone, H., 2001. Treatment
of Trypanosoma cruzi infected children with nifurtimox: a 3 year
follow-up by PCR. J. Antimicrob. Chemoth. 48 (4), 515–519.
Wincker, P., Britto, C., Borges Pereira, J., Cardoso, M.A., Oelemann,
W., Morel, C.M., 1994. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from
chronic chagasic patients in a rural endemic area. Am. J. Trop.
Med. Hyg. 51 (6), 771–777.
Zulantay, I., Honores, P., Sánchez, G., Apt, W., Ortı́z, S., Osuna, A.,
Rojas, A., Lopez, B., Solari, A., 2004. Use of polymerase chain
reaction and hybridization assay to detect Trypanosoma cruzi in
chronic chagasic patients treated with itraconazole or allopurinol.
Diagn. Microbiol. Infect. Dis. 48 (4), 253–257.