Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach.

Abstract

The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens.
Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results.
These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.

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Sensitive and Specific Detection of Trypanosoma cruzi
DNA in Clinical Specimens Using a Multi-Target Real-
Time PCR Approach
Yvonne Qvarnstrom1, Alejandro G. Schijman2, Vincent Veron3, Christine Aznar3, Francis Steurer1,
Alexandre J. da Silva1*
1Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2LabMeCh,
INGEBI-CONICET, Buenos Aires, Argentina, 3Laboratoire hospitalier et Universitaire-CH Andre ´e Rosemon, Faculte ´ de Me ´decine H. Bastaraud-EA 3593, Cayenne, Guyane
Franc ¸aise
Abstract
Background: The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests
will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR
can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three
TaqMan PCR assays to detect T. cruzi DNA in clinical specimens.
Methods/Principal Findings: Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue
specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both
whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected
exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from
immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose
acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital
case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood
analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for
discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false
positive results.
Conclusions/Significance: These data strongly indicate that at least two PCR assays with different performances should be
combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen’s
buffy coat to increase the sensitivity of PCR analysis.
Citation: Qvarnstrom Y, Schijman AG, Veron V, Aznar C, Steurer F, et al. (2012) Sensitive and Specific Detection of Trypanosoma cruzi DNA in Clinical Specimens
Using a Multi-Target Real-Time PCR Approach. PLoS Negl Trop Dis 6(7): e1689. doi:10.1371/journal.pntd.0001689
Editor: Elodie Ghedin, University of Pittsburgh, United States of America
Received January 17, 2012; Accepted April 30, 2012; Published July 3, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was supported by US federal funds, allocated at the Centers for Disease Control and Prevention (CDC) in the Division of Parasitic Diseases
and Malaria. 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: abs8@cdc.gov
Introduction
Chagas disease is a vector-borne infectious disease caused by the
parasite Trypanosoma cruzi. It is endemic in several countries of
Central and South America. In endemic areas the disease is spread
by certain species of triatomine bugs that excrete the parasites in
their feces while feeding on human hosts. Humans get infected
when feces from infected triatomines contaminates wounds,
allowing the parasite to enter the bloodstream. Other routes of
infection include congenital transmission, blood transfusion, organ
transplantation, accidental inoculation of the parasite during
laboratory research and by consuming food and juice contami-
nated with the parasite. As efforts to control vector-borne and
blood transmission are successful, congenital and oral transmission
paths are becoming increasingly important [1].
After a short acute phase when the parasite can be found
circulating in the blood, the disease enters the chronic phase when
the amastigote stage develops and multiplies in organ tissues,
primarily in the heart. The chronic phase is characterized by two
forms; patients first develop the indeterminate form of chronic
infection which can last for decades and the patients are typically
asymptomatic during this time. An estimated 30–40% of patients
may develop clinical disease, with manifestations such as
cardiomyopathy or digestive megasyndromes [1]. Chronically
infected patients that become immunosuppressed may experience
a reactivation of the disease, a condition characterized by
increasing parasitemia and atypical presentations such as epider-
mic lesions and compromise of the central nervous system [2].
The options for laboratory diagnosis of Chagas disease depend
on the disease phase. Serology is the method of choice to diagnose
chronic infections. Acute infections can be diagnosed by detecting
motile organisms in fresh blood preparations, by culture or by
detection of parasite DNA by PCR [3]. The latter methods are
also recommended to detect increasing parasitemia in cases of
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reactivation following immunosuppression [4]. In cases of acute
infections or reactivation of chronic disease it is important to use
sensitive diagnostic methods since early detection and treatment
results in a more favorable outcome. PCR-based methods are
generally considered to be more sensitive than microscopy and
have lately been increasingly used to diagnose Chagas disease [4].
However, the use of PCR is also challenging as there is no ‘‘gold
standard’’ method for the diagnosis of Chagas disease [5,6] and
the diagnostic performance can vary widely depending on the type
of PCR assay. The most widely used PCR assays used for
diagnostic purposes target either the kinetoplast genome (kDNA),
also called the minicircle, or a nuclear mini-satellite region
designated TCZ [4,6,7,8,9,10,11,12,13,14,15,16]. Both of these
targets are present in multiple copies in the parasite genome,
which increases the sensitivity of detection [15,17]. However,
assays that target these regions have been reported to cross-amplify
non-T. cruzi DNA [10,16,18,19,20]. Assays that amplify other
genes may show better specificity but they are generally less
sensitive [4,9,16,21].
One important use of PCR as a diagnostic tool is to provide a
sensitive method to detect reactivation in chronically infected
patients with immunosuppression. Patients with chronic Chagas
heart disease often require a heart transplant [22]. Current
recommendations state that these patients should be monitored at
regular intervals after the transplant for signs of increasing
parasitemia [3]. Another category of patients for whom PCR
testing is beneficial is patients who receive organs from chronically
infected donors. Since only a fraction of organ recipients will
develop an acute T. cruzi infection, preventive drug treatment is
not recommended. In such cases the use of PCR can allow for
early detection of those cases where transmission has occurred.
Recently, an international collaborative study focusing on
standardization and validation of PCR for diagnostic detection
of T. cruzi DNA was conducted [13]. The study relied on the use of
DNA specimens from genetically distinct cultured T. cruzi strains
plus blood specimens from chronically infected patients. The
specimens were coded at a coordinating laboratory and shipped
to 26 participating laboratories that performed PCR testing
according to their own standard operating procedures. Results
were then sent back to the coordinating laboratory and
performance characteristics were calculated for each PCR assay.
The study found a high degree of variability in accuracy and
performance among the included PCR tests and identified and
further evaluated two DNA extraction methods and four PCR
assays that performed better than the others. Two of the best-
performing assays were real-time PCR assays.
To continue these efforts we here present results from a
diagnostic testing algorithm involving three of the real-time PCR
assays included in the international validation study mentioned
above. Real-time PCR has several advantages over conventional
PCR, e.g. shorter turnaround times and less risk of amplicon
carry-over contamination [23], both of which can be advanta-
geous in diagnostic laboratories. One of the real-time PCR assays
included in this study was ranked among the four best-performing
assays in the international validation study; a real-time PCR assay
targeting the mini-satellite TCZ region. The second real-time
PCR assay was selected because it was the best-performing real-
time PCR assay targeting the kDNA included in the international
validation study. The third real-time PCR assay was included in
this study because it targets the small subunit ribosomal RNA
(18 S rRNA) gene, which is generally suitable for diagnostic assays
because it is highly conserved.
In contrast to the international validation study we mainly used
specimens from patients with suspected acute or reactivating
Chagas disease since PCR testing is more relevant for early
diagnosis or monitoring in this patient group than in chronic
patients, whose diagnosis relies on serological methods. The
majority of the specimens tested were EDTA blood samples; we
performed real-time PCR on DNA extracted from buffy coat
preparations in addition to whole blood to determine the effect of
buffy coat concentration on the sensitivity of the PCR analysis.
Methods
Clinical specimens
All the specimens used in this study were submitted to CDC for
confirmatory diagnosis of Chagas disease during years 2008–2010
from state public health laboratories, hospitals and private clinics
in the United States. The tests were performed on 349 laboratory
specimens from 119 patients, who lived in the United States at the
time of specimen collection. A breakdown of the specimen types
and the conditions that prompted the diagnostic requests are
presented in Table 1. Samples analyzed in this study were
anonymized by removing identifiers after diagnostic results were
reported, in accordance with the CDC IRB, protocol number
3580, entitled ‘‘Use of Human Specimens for Laboratory Methods
Research’’. All of the patients included in this study were evaluated
for serology status using the Chagatest recombinante v. 3.0
(Wiener Laboratorios, Rosario, Argentina) and a CDC in-house
IIF test.
DNA extraction
DNA extraction was performed from all specimens within
24 hours of arrival at the laboratory. DNA was extracted from
whole blood specimens using the QIAamp blood mini DNA kit
(QIAGEN, Valencia, Calif.). The volume of whole blood used was
0.2 ml and if the remaining volume exceeded 1 ml, the buffy coat
fraction was separated as follows: up to 2 ml of whole blood was
centrifuged at 2,5006g for 10 minutes. The plasma was removed
and the buffy coat layer plus some of the erythrocyte pellet were
transferred to a clean tube. DNA was then extracted from that
material in parallel with the whole blood aliquot using the same
Author Summary
Chagas disease is endemic in several Latin American
countries and affects approximately 8 to 11 million people.
The protozoan parasite, Trypanosoma cruzi, is the agent of
Chagas disease, a zoonotic disease that can be transmitted
to humans by blood-sucking triatomine bugs. Other routes
of infection include congenital transmission, blood trans-
fusion, organ transplantation, accidental inoculation of the
parasite during laboratory research and by consuming
food and juice contaminated with the parasite. This study
focused on the evaluation of three quantitative PCR
(QPCR) assays for the diagnosis of Chagas disease. The
evaluation was based on the analysis of 349 specimens
submitted for confirmatory diagnosis of Chagas disease to
the Centers for Disease Control and Prevention from 2008
to 2010. By using such assays we were able to diagnose
acute and Chagas disease reactivation in 20 patients,
including one case of organ-transmitted infection and one
congenital case. The paper also highlights the benefit of
extracting DNA from the blood specimen’s buffy coat to
increase the sensitivity of diagnostic PCR analysis. The
results obtained in this study strongly indicate that at least
two QPCR assays with different performance characteris-
tics should be combined to increase diagnostic accuracy.
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method mentioned above. Three EDTA blood specimens had
enough volume left after initial DNA extraction to allow for one or
more additional buffy coat preparations. Two-ml aliquots of these
specimens were stored at 4uC for one, two or four weeks and then
processed as described above. DNA from tissue specimens was
extracted with the DNeasy blood and tissue DNA kit (QIAGEN).
For cerebrospinal fluids (CSF), approximately half of the total
volume received (0.5–1 ml) was centrifuged for 5 minutes at
60006g. Most of the supernatant was carefully removed until
0.2 ml remained and DNA was extracted from this remaining
volume (plus any pellet) with the DNeasy blood and tissue DNA kit
(QIAGEN). All the DNA extraction procedures were performed
following manufacturer’s instructions for the different types of
samples. Previous experiences with these methods in our
laboratory had ensured that they efficiently removed potential
PCR inhibitors from the specimen types included in this study
(data not shown). One negative extraction control was included in
each batch of DNA extractions to monitor for potential cross-
contamination among samples and contamination of kit reagents.
PCR protocols
All three real-time PCR assays were included in the interna-
tional validation study [13]. Table 2 summarizes validation data
for the PCR assays as presented in that study, plus specificity data
for two other Trypanosoma spp. obtained in our laboratory. The
real-time PCR assays were performed and analyzed in an
Mx3000P QPCR system (Agilent Technologies, Calif.). Each
DNA sample was added to the PCR mix in two different
concentrations (corresponding to 5 ml and 1 ml of undiluted DNA).
All PCR runs included two or more negative amplification
controls (adding water instead of template DNA) plus two positive
amplification controls (DNA extracted from a culture of the Y
strain in two different dilutions). The risk of false positive results
due to contamination was minimized by the following procedures:
using separate rooms for DNA extraction, pre-and post-amplifi-
cation processes; having a uni-directional workflow; and using
enzymatic removal of contaminating amplicons before real-time
PCR amplification.
TCZ TaqMan real-time PCR (designated as method LbF1 in
the international validation study [13]): This TaqMan assay was
performed as described in Piron 2007 [11], except that the
Platinum qPCR supermix was used instead of the Universal
mastermix from Applied Biosystems.
kDNA TaqMan real-time PCR (designated as method LbG/3
in the international validation study [13]): The reaction mix
consisted of 16Platinum qPCR supermix, 0.4 mM of each PCR
primer 32F, 59-TTT GGG AGG GGC GTT CA-39, and 148R,
59-ATA TTA CAC CAA CCC CAA TCG AA-39, plus 0.1 mM of
the LNA TaqMan probe 71P, 59-CA TCTC AC CCG TACA
TT-39, where the LNA nucleotides [24] are underlined. Total
reaction volume was 20 ml. Thermocycling structure was as
follows: 2 minute incubation at 50uC to activate UDG degrada-
tion, 2 minute incubation at 95uC to activate the hot-start DNA
polymerase, and 40 cycles of 95uC for 15 seconds and 58uC for
60 seconds.
18 S rRNA TaqMan real-time PCR (designated as method
LbS/4 in the international validation study [13]): The reaction
mix consisted of 16 Platinum qPCR supermix, 0.2 mM of each
PCR primer TcF1042, 39-GCA CTC GTC GCC TTT GTG-39,
and TcR1144, 59-AGT TGA GGG AAG GCA TGA CA-39 plus
0.05 mM of the TaqMan probe TCP1104, 59-AA GAC CGA
AGT CTG CCA ACA ACA C-39. Total reaction volume was
20 ml. Thermocycling structure was as follows: 2 minute incuba-
tion at 50uC to activate UDG degradation, 2 minute incubation at
95uC to activate the hot-start DNA polymerase, and 40 cycles of
95uC for 15 seconds and 60uC for 60 seconds.
Results
This study focused on diagnostic specimens tested from 2008 to
2010. Fifty of the 349 samples produced positive results in at least
one of the real-time PCR assays. The three real-time PCR assays
produced identical results for 329 samples (94%), of which 30 were
PCR positive, while the remaining 20 samples had discrepant
results in the three assays.
Of the 50 samples with at least one positive PCR result, 37
samples were collected from 13 chronically infected patients with
reactivation disease, six samples from three transplant recipients of
organs from chronically infected donors, four samples from a
patient with acute Chagas disease acquired during travel in an
endemic region, one sample from a congenitally transmitted
Table 1. Specimens included in this study.
reason for testingnumber of patients
Number of specimens
EDTA blood cord bloodheart tissue skin tissueCSFtotal
reactivation due to heart transplant 18630 151079
reactivation due to AIDS5600028
reactivation due to other condition2500005
symptoms of cardiomyopathy13 100300 13
symptoms of acute Chagas disease 18260011 28
bites from triatomines but symptom-free13 130000 13
congenital transmission20 16600022
recipients of organs from Chagas positive donors141390000 139
laboratory accidents9 350000 35
serologically positive but symptom-free*7700007
total 1193206 1823 349
*=these included three mothers in investigations of possible congenital transmission and four organ donors with chronic Chagas disease. PCR-testing was performed
on these chronically infected persons to aid in the evaluation of disease transmission risk.
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infection and two samples from two patients under evaluation for
severe cardiomyopathy. Tables 3, 4, and 5 list the detailed real-
time PCR results from specimens collected from a selection of
these patients, as outlined below.
Detecting reactivation disease in immunosuppressed
patients using real-time PCR
Specimens from 25 patients with chronic Chagas disease (as
determined by positive serology) were received for evaluation of
reactivation disease during the study period. Eighteen of the
patients had received a heart transplant, five were HIV infected
and two had undergone a bone marrow transplant. Sixteen
patients had one or more PCR-positive samples, including
sporadic PCR-positive results in seven patients who had received
a heart transplant prior to 2008. Table 3 lists a selection of the
samples analyzed from the remaining nine patients with at least
one PCR-positive test result during this study. Seven patients were
tested for reactivation following transplants; four of these were
monitored on a regular basis by PCR. Two of the five HIV-
positive patients were diagnosed with re-activated Chagas disease
(patients 8 and 9); one of them had cerebral Chagas, confirmed by
the presence of T. cruzi DNA in CSF.
Using real-time PCR to detect T. cruzi transmission via
organ transplants
Real-time PCR was used to test blood specimens from 14
previously non-infected transplant patients who received organs
from a donor with suspected or confirmed chronic Chagas disease.
It is recommended to closely monitor these patients with PCR or
other sensitive technique in order to detect potential transmission
as soon as possible. Three organ recipients had one or more PCR
positive results (see Table 4). However, only patient 12, a heart
recipient, was actually infected with T. cruzi. The PCR positive
results for the other two patients (patients 10 and 11) were
reported as equivocal and were most likely false positive results
because of the following circumstances. Patient 10 received a
kidney from a donor with borderline positive serology results with
the Ortho T. cruzi ELISA test (Ortho-Clinical Diagnostics,
Raritan, New Jersey). Since this could have been interpreted as
indicative of infection in the donor, regular PCR testing was
started on patient 10. However, subsequent serology testing of the
donor associated with this case could not confirm the preliminary
results; i.e., the T. cruzi RIPA was indeterminate and both the
Wiener and the IIF test were negative on repeated serum samples.
It was therefore concluded that the donor was not infected with T.
cruzi and additional PCR follow up of patient 10 was unnecessary.
However, before the final donor serology status had been
determined, weak positive signals in the kDNA and TCZ TaqMan
assays were verified in blood samples from patient 10. Unexpect-
edly, each subsequent specimen obtained from this patient showed
a signal that was weaker than the signal obtained for the previous
sample; i.e. the opposite of what was expected from an acute T.
cruzi infection in an immunocompromised patient. At six weeks
post-transplant patient 10 was no longer positive in any of the real-
time PCR assays. Patient 11 received a kidney from a donor that
was confirmed to be serologically positive for T. cruzi. The blood
sample from patient 11 collected on the 3rdweek post-transplant
tested weakly positive in the kDNA and TCZ TaqMan assays,
with only the whole blood aliquot being positive and not the buffy
coat fraction. The blood collected a week later was PCR negative
in both whole blood and buffy coat. Neither patient 10 nor 11 had
any clinical signs of T. cruzi infection. Their blood smears were
constantly negative for parasites and they did not receive anti-
trypanosomal drugs.
Using real-time PCR to diagnose acute infections
We received 48 blood samples from 13 healthy patients who
had been bitten or in close contact with triatomine bugs plus 9
laboratory workers that had been accidentally exposed to T. cruzi
via needle stick accidents or animal bites during research activities.
None of these were PCR positive. We tested 22 specimens from 20
children (aged newborn to 8 years) with sero-positive mothers for
possible congenital transmission and detected T. cruzi DNA in the
blood of a 19-days-old infant (patient 14 in Table 5). Twenty-eight
specimens were received from 18 adult patients with symptoms of
acute T. cruzi infection (fever and malaise after traveling to
endemic region and/or having close contact with triatomine bug;
three had a swollen eye that could be chagoma). Only one of these
patients tested positive for T. cruzi by PCR and was treated for
acute Chagas disease (patient 13 in Table 5). Follow-up specimens
from this patient again tested positive in PCR after completed
drug treatment but unfortunately the patient was lost to follow-up.
Effect of buffy coat examination on the sensitivity of PCR-
based detection
Thirty-five of the PCR-positive blood specimens (from 16
patients) had enough volume to allow for DNA extraction from
both whole blood aliquots and buffy coat fraction. Of these, 26
specimens (from 10 patients) had PCR-detectable levels of T. cruzi
DNA in both whole blood and buffy coat, with a relatively higher
concentration in the buffy coat based on the quantitative output
(the Cq value) from the real-time PCR assays. The remaining 9
specimens (from 6 patients) were positive only in the buffy coat
fraction. Thus, 26% of the PCR-positive specimens would have
been reported as being negative for T. cruzi if no buffy coat analysis
had been performed. For three patients the analysis of buffy coat
was crucial: Chagas disease reactivation in two patients was
detected two weeks earlier by testing the buffy coat sample as
Table 2. Validation data for the real-time PCR assays.
kDNA TaqManTCZ TaqMan18 S rRNA TaqMan reference
diagnostic sensitivity (using serology as comparison method) 78%63% 6% [13]
diagnostic specificity (using serology as comparison method)40% 100% 100%[13]
analytical sensitivity (detection limit) for DTU I 0.1 fg/ml 0.1 fg/ml 10 fg/ml [13]
analytical sensitivity (detection limit) for DTU IV1 fg/ml1 fg/ml 10 fg/ml[13]
DNA from T. rangeli cultures (n=2)positivenegative negativethis study
DNA from T. theileri-infected tissue (n=3)negativenegativenegative this study
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Table 3. Detailed PCR findings from patients with reactivation disease.
Patient
IDpatient descriptiontime of specimenspecimen type
results individual assays
Reported
results
kDNA TaqManTCZ TaqMan
18 S rRNA
TaqMan
1chronic patient with heart transplant time of heart transplant explanted heart
tissue
positivepositivepositivepositive
time of heart transplant heart biopsy negativenegativenegativenegative
1 month post-transplantheart biopsypositivepositivepositivepositive{
2chronic patient with heart transplant 2.5 months post-transplant EDTA bloodpositivepositivenegativepositive
3 months post-transplant EDTA blood positivepositivenegativepositive{
3chronic patient with heart transplanttime of heart transplant EDTA bloodnegativenegativenegative negative
3 weeks post-transplant EDTA blood negativenegativenegativenegative
5 weeks post-transplantEDTA blood BC positive BC positive negativepositive
6 weeks post-transplantEDTA bloodBC positivenegativenegative equivocal
7 weeks post-transplantEDTA bloodpositivepositivepositivepositive
9 weeks post-transplantEDTA blood positivepositive positivepositive
3 months post-transplantEDTA bloodnegativenegativenegativenegative{
7 months post-transplantEDTA bloodnegativenegativenegativenegative
4chronic patient with heart transplanttime of heart transplantEDTA bloodpositivepositivepositivepositive
3 months post-transplantEDTA bloodnegative negativenegativenegative
5chronic patient with heart transplant1 week post-transplantEDTA blood positivepositivenegativepositive
2.5 weeks post-transplant EDTA bloodpositivepositivenegativepositive
3.5 weeks post-transplantEDTA blood positivepositivepositivepositive
1 month post-transplantEDTA bloodpositivepositive positivepositive
1.5 months post-transplantEDTA bloodnegativenegativenegativenegative{
5 months post-transplant EDTA bloodnegativenegativenegativenegative
6chronic patient with heart transplanttime of heart transplantexplanted
heart tissue
positivepositivenegativepositive
1 week post-transplant EDTA bloodnegativenegativenegativenegative
2 weeks post-transplantheart biopsy negative negative negative negative
3 weeks post-transplantheart biopsy negativeBC positive negativeequivocal
4 weeks post-transplantEDTA bloodBC positiveBC positive negativepositive
5 weeks post-transplantEDTA bloodpositivepositivepositivepositive
3 months post-transplantEDTA blood negativenegative negativenegative{
6 months post-transplantEDTA bloodnegativenegativenegativenegative
7chronic patient with organ transplant1 week post-transplantEDTA bloodnegativenegativenegativenegative
2 weeks post-transplantEDTA bloodpositive negative negativeequivocal
3 weeks post-transplant EDTA bloodpositivepositivepositivepositive
4 weeks post-transplantEDTA bloodpositivepositivepositivepositive
2.5 months post-transplant EDTA bloodnegativenegativenegativenegative{
3.5 months post-transplantEDTA bloodnegativenegativenegativenegative{
7 months post-transplantEDTA bloodnegativenegativenegativenegative
8reactivation due to AIDSfirst sample EDTA bloodpositivepositivepositivepositive
sample 1 month later EDTA bloodpositivepositivepositivepositive{
sample 1 month later CSFpositivepositivepositive
positive{
9reactivation due to AIDS N/AEDTA bloodpositivepositive positivepositive
All of the patients in Table 3 tested positive in serology for Chagas disease and no decrease in antibody titer was detected during the monitoring period.
BC=buffy coat; only the buffy coat fraction was PCR positive for these samples.
{indicate specimens that were collected during drug treatment (benznidazole or nifurtimox). Drug treatment information is missing for patients #4 and #9.
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compared to whole blood (patients 3 and 6 in Table 3) and the
patient who acquired Chagas disease through transplantation
(patient 12 in Table 3) was identified as positive one week earlier
by testing buffy coat as compared to whole blood.
Three of the PCR-positive blood samples had enough volume to
allow for analysis of more than one buffy coat preparation.
Aliquots of these three samples were stored at 4uC for up to four
weeks and then processed as described. Figure 1 depicts the
quantitative real-time PCR results obtained from these samples
over time. The results suggested that storage of EDTA blood for a
limited time had minor effect on the quality of T. cruzi DNA
obtained from buffy coat preparations, at least for the kDNA and
TCZ genetic regions. Although these are only preliminary data
that need confirmation with a larger set of samples, it removes
some of the uncertainty whether to accept EDTA-blood samples
that for various reasons are delayed in transport to the diagnostic
laboratory.
Discussion
The laboratory diagnosis of Chagas disease relies mainly on
serology, microscopic identification of trypomastigotes in blood or
buffy coat, hemoculture and PCR [3]. Several PCR assays with
variable diagnostic sensitivity and specificity have been developed
and used as diagnostic tests [4,7,8,9,10,11,15,16,21]. A compli-
cating factor for PCR assays is the high genetic variability of T.
cruzi strains; there are currently six genotype groups or discrete
typing units (DTU) described that differ significantly in genetic
content and gene copy numbers [25,26]. Since some DTUs are
more common than others in various endemic regions, the same
PCR assay can perform differently depending on the geographic
origin of the specimen [16,25,27,28,29]. One way to circumvent
these accuracy problems is to combine two or more PCR assays
that target different genes.
The reference diagnostic laboratory at CDC employs a multi-
target PCR testing algorithm consisting of three real-time PCR
assays that are performed in parallel on all specimens. The three
assays target different genomic regions in T. cruzi and have
therefore variable sensitivity and specificity. The rationale for
including all three assays in the testing algorithm is to ensure the
highest accuracy possible by combining assays that complement
each other. The kDNA TaqMan assay seems to be the most
sensitive assay but it can amplify non-T. cruzi DNA, e.g. T. rangeli,
and thus lead to false positives. The TCZ TaqMan assay has better
specificity but as shown in this study can produce false positive
PCR results as well. The kDNA and TCZ TaqMan assays are
both much more sensitive than the 18 S rRNA TaqMan but the
main advantage of including the 18 S rRNA assay in the testing
algorithm is that it seems to be 100% specific. According to the
CDC protocol, if a specimen tests positive in all three real-time
PCR assays it will be reported as positive for T. cruzi, but any
specimen that is only positive in the kDNA and/or TCZ TaqMan
Table 4. Detailed PCR-findings for recipients of organs from donors with suspected/confirmed chronic Chagas disease.
Patient ID Patient descriptiontime of specimen Specimen typeresults individual assaysReported result
kDNA TaqMan TCZ TaqMan 18 S rRNA TaqMan
10 kidney recipient2 weeks post-transplantEDTA bloodnegativenegativenegativenegative
3 weeks post-transplantEDTA bloodpositive positivenegativeequivocal
4 weeks post-transplant EDTA bloodpositivepositive negativeequivocal
5 weeks post-transplantEDTA bloodpositivenegativenegativeequivocal
6 weeks post-transplantEDTA bloodnegativenegativenegative negative
7 weeks post-transplantEDTA blood negativenegativenegativenegative
9 weeks post-transplant EDTA bloodnegativenegativenegativenegative
11 weeks post-transplant EDTA bloodnegativenegativenegativenegative
11kidney recipient1 week post-transplant EDTA bloodnegativenegativenegative negative
3 weeks post-transplantEDTA bloodpositive positivenegativeequivocal
4 weeks post-transplantEDTA blood negativenegativenegative negative
5 weeks post-transplantEDTA bloodnegativenegativenegativenegative
3 months post-transplantEDTA bloodnegativenegativenegativenegative
6 months post-transplant EDTA bloodnegativenegative negativenegative
12 Heart recipient2 weeks post-transplant EDTA bloodnegative negativenegativenegative
3 weeks post-transplant EDTA bloodnegative negativenegativenegative
4 weeks post-transplantEDTA bloodBC positive BC positivenegative positive
5 weeks post-transplantEDTA blood positivepositivenegativepositive
6 weeks post-transplantEDTA bloodnegativenegativenegative negative{
7 weeks post-transplantEDTA blood negative negativenegativenegative{
3 months post-transplantEDTA bloodnegativenegativenegativenegative
5 months post-transplant EDTA bloodnegativenegativenegativenegative
8 months post-transplantEDTA bloodnegativenegativenegativenegative
All of the patients in Table 4 were serologically negative at time of transplant and none sero-converted during the time they were monitored at CDC.
BC=buffy coat; only the buffy coat fraction was PCR positive for these samples.
{indicate specimens that were collected during drug treatment (benznidazole or nifurtimox). Drug treatment information is missing for patients #4 and #9.
doi:10.1371/journal.pntd.0001689.t004
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assays and negative in the 18 S rRNA TaqMan assay require
additional confirmation by other tests or clinical data in order to
be reported as positive for T. cruzi. If confirmatory data is absent or
does not support a diagnosis of T. cruzi infection, the PCR results
are reported as equivocal and a new specimen is requested to
repeat the molecular analysis.
Diagnostic sensitivity can be enhanced by maximizing the
amount of target DNA in the aliquot used for DNA extraction.
For multi-copy PCR targets this can be obtained by mixing blood
specimens with guanidine HCl-EDTA solution that lyses the
parasites and releases their genetic content, thus making it possible
to detect as little as one parasite in a large volume of blood [30,31].
It has also been reported that sensitivity could be enhanced if blood
clot was used as starting material [32]. An alternative method is to
concentrate the parasites in the buffy coat fraction [33] prior to
DNA extraction; this has been reported to increase the sensitivity
compared to analysis of frozen EDTA-blood and guanidine HCl-
EDTA treated blood [32,34]. During this study, we compared the
PCR results obtained from buffy coat concentration with results
from fresh EDTA- blood and found that analysis of buffy coat
allowed earlier detection of increasing levels of circulating parasite
genome in three cases: two reactivation cases and one organ-
transmitted acute infection. Thus, appropriate drug treatment for
these patients could be initiated 1–2 weeks sooner.
Analyzing both the buffy coat fraction and a whole blood
aliquot in parallel can also give helpful information to ensure test
validity and to troubleshoot suspicious false positive PCR results.
DNA extraction from the buffy coat fraction of a blood sample
containing T. cruzi trypomastigotes should produce more T. cruzi
DNA than the corresponding volume of whole blood. If that is not
the case, there could be a problem with the quality of the blood
specimen, the DNA extraction process or the PCR accuracy. One
of the false positive PCR results obtained in this study was
immediately flagged as suspicious because only the whole blood
fraction was positive while buffy coat was negative. Nevertheless,
more data must be accumulated during a longer period of time for
a more robust assessment about the advantages of analyzing both
whole blood and buffy coat.
In conclusion, we propose that in reference laboratories with the
adequate infrastructure, the use of two or more real-time PCR
Table 5. Detailed PCR findings for patients with acute Chagas disease.
Patient ID patient descriptiontime of specimen specimen typeresults individual assaysReported results
kDNA TaqMan TCZ TaqMan18 S rRNA TaqMan
13 acute infection after triatomine
contact in endemic region1
sample 1EDTA bloodpositivepositivepositivepositive
1 week laterblood clot positivepositivepositivepositive{
3 weeks later EDTA bloodpositivepositivepositivepositive{
2 months later EDTA bloodnegativenegativenegativenegative{
3 months laterEDTA bloodnegativenegativenegativenegative
4 months laterEDTA bloodpositivepositivenegativepositive
4.5 months laterEDTA bloodpositive negative negativeequivocal
14 congenital transmission2
19 days oldEDTA bloodpositivepositive positive positive
2 months oldEDTA bloodnegativenegative negativenegative{
1Patient was serologically positive for Chagas disease by the time she was tested at CDC.
2Patient was serologically positive when 19 days old due to maternal antibodies. Another sample collected at 10 months of age tested negative in serology.
{indicate specimens that were collected during drug treatment (benznidazole or nifurtimox).
doi:10.1371/journal.pntd.0001689.t005
Figure 1. Effect of storage of EDTA blood specimens on real-time PCR results. The lower the Cq (quantitative cycle) value, the better
recovery was obtained from the DNA extraction process.
doi:10.1371/journal.pntd.0001689.g001
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tests with different performance characteristics combined with the
analysis of buffy coat and whole blood can strengthen the use of
PCR for accurate diagnosis of Chagas disease.
Supporting Information
Checklist S1
(DOC)
STARD checklist.
Acknowledgments
The authors would like to thank Lori Hall from the CDC Drug Service for
help with drug treatment data for the study patients. Susan Montgomery,
Caryn Bern and Patricia Wilkins from the Division of Parasitic Diseases
and Malaria, CDC, provided invaluable comments during the preparation
of this manuscript.
Author Contributions
Conceived and designed the experiments: YQ AGS VV CA AJDS.
Performed the experiments: YQ AGS VV CA FS AJDS. Analyzed the
data: YQ AGS FS AJDS. Contributed reagents/materials/analysis tools:
YQ AJDS. Wrote the paper: YQ AGS VV CA FS AJDS.
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