Am. J. Trop. Med. Hyg., 75(6), 2006, pp. 1082–1084
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: AMNIOTIC FLUID IS NOT USEFUL FOR DIAGNOSIS OF
CONGENITAL TRYPANOSOMA CRUZI INFECTION
MYRNA VIRREIRA, SABRINA MARTINEZ, CRISTINA ALONSO-VEGA, FAUSTINO TORRICO, MARCO SOLANO,
MARY CRUZ TORRICO, RUDY PARRADO, CARINE TRUYENS, YVES CARLIER, AND MICHAL SVOBODA*
Laboratoire de Chimie Biologique, Faculté de Médecine, Université Libre de Bruxelles (U.L.B.), Brussels, Belgium; Facultad de
Medicina, Universidad Mayor de San Simon (U.M.S.S.), Cochabamba, Bolivia; Laboratoire de Parasitologie, Faculté de Médecine,
Université Libre de Bruxelles (U.L.B.), Brussels, Belgium
Abstract. Although Trypanosoma cruzi can be transmitted transplacentally and induce congenital infection, no data
are available about the presence of this parasite in human amniotic fluid. We examined 8, 19, and 4 amniotic fluid
samples (collected at delivery or by aspiration of gastric content of neonates) from control uninfected mothers (M−B−),
infected mothers delivering uninfected newborns (M+B−), and mothers of confirmed congenital cases (M+B+), respectively. Polymerase chain reaction (PCR), using nuclear and kinetoplastic DNA primers (Tcz1-Tcz2 and 121-122), were
negative for all control M−B− samples, but positive for 5 of 19 M+B− and 2 of 4 M+B+ samples. To determine the
number of parasites in the positive samples, real-time PCR using S35/S36 kinetoplastic DNA was performed. Only one
M+B+ sample presented a high parasitic DNA amount, whereas the other six PCR-positive samples displayed traces of
T. cruzi DNA. In conclusion, the release of parasites in amniotic fluid is probably a rare event that cannot be helpful
for the routine diagnosis of congenital Chagas disease.
specific serological tests, and congenital infection with T.
cruzi was sought for by direct microscopic examination of
blood buffy coat collected in microhematocrit heparinized
tubes, or hemoculture, as previously described.8 The absence
of T. cruzi infection in parasitologically negative newborns
was confirmed by PCR performed on umbilical cord blood.9
Clinical data of newborns were collected as previously described.8 This study was approved by the scientific/ethic committees of the “Universidad Mayor de San Simon” and the
“Université Libre de Bruxelles,” and written consent of the
informed mothers was obtained before sample collection.
Parasites were not detected by direct microscopic examination of centrifugation pellet of eight AF samples of 5 mL
from M+B− mothers, and the other samples were not examined using this parasitological technique. All AF/GAF
samples (0.5–5 mL) were mixed with the same volume of the
buffer guanidine-HCl, 6 mol/L, and EDTA 0.1 mol/L and
boiled for 15-minute DNA extraction was performed on 200
␮L of guanidine-mixed samples, using the “QuiAmp DNA
blood” kit (Quiagen) according to the manufacturer’s instructions. PCR amplifications were made using primers targeting
either nuclear (primers TCZ1/TCZ2) or kinetoplastic parasite
DNA (primers 121/122), as previously described.9 In all PCR
amplifications, DNA extracted from a negative sample (AF
from uninfected mother) was included. All PCR were performed at least twice in duplicates. A PCR amplification of a
fragment of the human ␤-globin gene was systematically performed to assess the integrity of extracted DNA.9
To get information on the parasite amount detected in
PCR-positive samples, the relative intensity of their kDNA
amplicons (obtained with primers 121/122) was compared
with those of amplicons obtained, in the same PCR assay,
from DNA prepared from known amounts of T. cruzi parasites. Parasite amounts equivalent to 0.0002, 0.02, and 2 parasites/assay corresponded to 0.4, 40, and 4,000 parasites/mL of
extracted fluid, taking in account the dilution with guanidine
and that performed during extraction of DNA (Figure 1). To
obtain more accurate quantitative information on PCRpositive samples, real-time PCR (qRT-PCR) using kinetoplastic DNA primers (modified S35/S36) was performed as
previously described,10 increasing the hybridization tempera-
Trypanosoma cruzi, the causative agent of Chagas disease,
is transmitted mainly by insect vectors, but also by alternative
routes such as blood transfusion and congenital transmission.1
The maternal–fetal transmission rate of T. cruzi infection in
the Southern cone countries varies widely from 1% in Brazil
to 4–12% in Argentina, Bolivia, Chile, and Paraguay.2 As
recently shown in studies of placentas from Bolivian mothers
infected with T. cruzi, the trans-placental transfer of maternal
blood parasites mainly occurs through the placental membranes rather than by crossing villous trophoblast.3 During
the parasite multiplication in the chorial plate, amniotic cells
can be infected,3 making possible the release of T. cruzi parasites in amniotic fluid (AF). If this occurs, AF could be considered as a possible biologic sample for the diagnosis of congenital infection, as is the case for the diagnosis of congenital
toxoplasmosis and viral diseases.4–7 However, as far as we
know, study of T. cruzi in human amniotic fluid has not been
reported, and its predictive or diagnostic value remains unknown. This study aims to investigate the presence of parasitic DNA in amniotic fluids of T. cruzi–infected mothers,
using polymerase chain reaction (PCR) methods.
Mothers were admitted to the Bolivian maternity German
Urquidi (Universitary Hospital Viedma, Universidad Mayor
de San Simon) in Cochabamba. Samples of AF were collected
either at the time of membrane rupture before delivery, with
precautions to avoid maternal blood contamination, or by
aspiration of the gastric fluid content (GAF) in newborns,
immediately after birth. A total of 31 AF/GAFs have been
studied: 4 (all GAF) from congenital cases of T. cruzi infection (mothers and newborns are infected, M+B+: cases
1–0311 and 1–0480 were asymptomatic; cases 1–0098 and
1–0899 displayed splenomegaly and hepatomegaly, respectively), 19 (16 AF and 3 GAF) from infected mothers having
delivered uninfected babies (M+B−), and, 8 (all AF) from
control cases (mothers and newborns are uninfected, M−B−).
Infection in mothers was assessed using standard parasite-
* Address correspondence to Michal Svoboda, Laboratoire de
Chimie Biologique, Faculté de Médecine, U.L.B., 808 route de Lennik CP 611, B-1070 Bruxelles, Belgium. E-mail: [email protected]
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TRYPANOSOMA CRUZI IN AMNIOTIC FLUID
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FIGURE 1. Amplification of T. cruzi kDNA from standard dilutions and from amniotic fluid. DNA extracted from T. cruzi parasites
(“Tulahuen” strain of sublineage TcIIe), amounts equivalent to
0.0002, 0.02, and 2 parasites/assay, was amplified in parallel with extract from LA/GAF samples.
ture to 63°C, to reduce amplification of unspecific bands
originating from human DNA. The SyberGreen system
(Roche Diagnostic, Vilvoorde, Belgium) in LightCycler ver.2
apparatus (Roche Diagnostic, Vilvoorde, Belgium) was used
according to the manufacturer’ instructions. The same DNA
amount of negative AF sample was added to all reference
standard samples. As shown in the Figure 2A, a reproducible
correlation could be obtained between the number of cycles
and the number of parasites ranged from 0.0002 to 2 parasites/
assay (corresponding to 0.4–4,000 parasites/mL).
All the eight control AF samples were negative in PCR for
both TCZ1/TCZ2 and 121/122 primers. Five of the 19 (26.3%)
M+B− and 2/4 (50%) of M+B+ AF/GAF samples were PCR
positive for both kinds of primers. Comparisons of amplicon
intensities of all the positive PCR samples with amplicons
from known amounts of parasites showed only one M+B+
sample (GAF from the asymptomatic case 1-0311) displaying
a strong intensity corresponding to several thousands of parasites per milliliter. All the other positive samples (one of the
M+B+ group 1-0899 and five of the M+B− group 1-0343, 868,
962, 1025, 1057) showed lower amplicon intensities corresponding roughly to 1 parasite/mL. Complementary studies
by qRT-PCR confirmed these observations. The M+B+ GAF
sample 1-0311 showed a high concentration of parasites
(18,400 parasites/mL), whereas all the other samples that
were weakly positive in PCR displayed very low T. cruzi
DNA concentrations, around the limit of reliable qRT-PCR
detection, corresponding to < 0.4 parasites/mL (Figure 2B).
However, the electrophoretic analysis of these qRT-PCR
products showed the presence of the expected amplicon,
showing the actual presence of T. cruzi DNA traces in these
samples.
Altogether our results show T. cruzi parasites being hardly
detectable in AF (collected from amniotic sac or aspirated
from newborn stomach), because 1) only one of the four
samples from mothers of congenital cases (M+B+) displayed
a significant and PCR-detectable parasite DNA amount and
2) the other M+B+ PCR-positive sample contained only
traces of parasitic DNA. Moreover, 26% of AF/GAF from
M+B− mothers delivering uninfected babies also displayed
such DNA traces. This might suggest that placentas of M+B−
FIGURE 2. (A) Standard curve of quantitative real-time-PCR of
T. cruzi. Amplification of DNA isolated from known amount of parasites was performed in the presence of extract of amniotic fluid from
M−B− negative sample (AF 43) using modified S35/S36 primers.
Equivalent of 1 ␮L of extract was added to each reaction. Mean ± SE
of five separate experiments is represented. (B) Quantitative realtime-PCR of T. cruzi DNA in amniotic fluid. Fluorescence of amplification curves of extract from AF/GAF samples, performed as
above, is represented. The AF-43 sample (M−B−) was used as negative control to asses background limit of amplification in the presence
of human DNA.
mothers might also be infected without parasite transmission
to the fetus, in agreement with previous reported data.11
However, detection of such traces of parasitic DNA raises the
question of the actual presence of living parasites in samples.
Indeed, sample contamination by maternal blood cannot be
formally excluded. Although serious precautions have been
taken for AF collection from amniotic sac, GAL might have
been contaminated by swallowing maternal blood during
vaginal delivery of neonates (all PCR-positive samples came
from vaginally delivered babies). The GAF containing significant parasite amount was from an asymptomatic congenital
case, suggesting that parasite detection in AF probably does
not relate to Chagas disease severity. Moreover, parasites can
be more easily detected in umbilical cord blood.2,8 Therefore,
if T. cruzi can be released in AF after placental invasion, this
is probably a rare event that cannot be helpful for the routine
diagnosis of congenital Chagas disease.
Received June 21, 2006. Accepted for publication July 24, 2006.
Acknowledgments: The authors thank Marisol Cordova and the staff
of the maternity German Urquidi (Cochabamba, Bolivia) for the
management of patients; Miguel Guzman, Myrian Huanca, Rudy
Parrado, and Marco Antonio Solano (CUMETROP/LABIMED,
U.M.S.S., Cochabamba, Bolivia) for the serological and parasitological diagnosis of patients.
Financial support: M. Virreira is fellow of the “Xénophilia” grant
(ULB, Belgium). C. Alonso-Vega is fellow of the “Association pour
la Promotion de l’Education et la Formation à l’Etranger” (APEFE,
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VIRREIRA AND OTHERS
“Communauté Française de Belgique”). This study was supported by
the Conseil Interuniversitaire de la Communauté Française de Belgique (CIUF), the Fonds National de la Recherche Scientifique
Médicale (Belgium, conventions 3.4.595.99/3.4.615.05), and the Community and Child Health (CCH) Chagas control program (USAID).
Authors’ addresses: Myrna Virreira, Sabrina Martinez, and Michal
Svoboda, Laboratoire de Chimie Biologique, Faculté de Médecine,
Université Libre de Bruxelles (U.L.B.), Route de Lennik 808, CP
611, B-1070 Brussels, Belgium. Cristina Alonso-Vega, Faustino Torrico, Marco Solano, Mary Cruz Torrico, and Rudy Parrado, Facultad
de Medicina, Universidad Mayor de San Simon (U.M.S.S.), Avenida
Aniceto Arce 371, casilla 922, Cochabamba, Bolivia. Carine Truyens
and Yves Carlier, Laboratoire de Parasitologie, Faculté de Médecine,
Université Libre de Bruxelles (U.L.B.), Route de Lennik 808, CP
611, B-1070 Brussels, Belgium.
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