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Journal of Clinical Microbiology, August 2000, p. 2929-2932, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Real-Time PCR for Diagnosis and Follow-Up of
Toxoplasma Reactivation after Allogeneic Stem Cell Transplantation
Using Fluorescence Resonance Energy Transfer Hybridization
Probes
J.-M.
Costa,1
C.
Pautas,2
P.
Ernault,1
F.
Foulet,3
C.
Cordonnier,2 and
S.
Bretagne3,*
Laboratoire de Biologie Moléculaire,
Hôpital Américain, BP 109, 92202, Neuilly,1 and Service
d'Hématologie Clinique, Hôpital H. Mondor-APHP,2 and Laboratoire de
Parasitologie, Hôpital H. Mondor-APHP et Université
Paris XII,3 94010, Créteil, France
Received 5 January 2000/Returned for modification 5 April
2000/Accepted 19 May 2000
 |
ABSTRACT |
Toxoplasma reactivation is a life-threatening complication of
allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of
parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect
and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from
0.75 × 106 to 0.75 parasites per PCR mixture. Serial
samples from four patients with toxoplasma reactivation were evaluated.
Three patients had several consecutive PCR-positive samples which
corresponded to 
0.75 parasites. These three patients became PCR
negative during trimethoprim-sulfamethoxazole therapy but never
developed clinically apparent toxoplasmosis. In contrast, one patient
had an increasing PCR signal, from 1 to 396 parasites in 12 days, and
developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment.
Real-time quantitative PCR is useful for diagnosis and follow-up of
toxoplasma reactivation.
| |
INTRODUCTION |
Toxoplasmosis is a worldwide
infectious disease caused by the protozoan Toxoplasma
gondii. In humans, the parasite exists in two stages: the
tachyzoite stage and the bradyzoite stage (7). Tachyzoites
are responsible for the acute infection, and their differentiation into
bradyzoites correlates with the onset of protective immunity.
Bradyzoites are located within cysts, which are believed to persist for
life. These quiescent stages are able to reconvert into active
tachyzoites when the immune system fails. This reactivation is thought
to be the main source of cerebral or disseminated toxoplasmosis in
immunocompromised individuals.
In allogeneic stem cell transplant recipients, the infection is usually
life-threatening and is often diagnosed postmortem (6, 13,
15), probably because the diagnosis is difficult, being based on
the detection of evidence of parasites in tissue. Serological tests are
of little help in these patients and can be used to say only that the
patient, if positive, is at risk of reactivation as he or she harbors
some toxoplasma cysts. Therefore, empiric treatment is often initiated
on the basis of clinical suspicion and cerebral computed tomography
(CT) scan abnormalities. This treatment, based on pyrimethamine and
sulfadiazine or clindamycin, can have multiple side effects. Efficacy
cannot be clinically assessed for several days. The risk is then of
missing other diagnoses.
A positive PCR result with blood is expected to improve the prognosis
because treatment can be initiated when the parasite burden is still
low and allows avoidance of invasive procedures such as brain biopsy
(2, 9, 10-12, 14). Quantitative PCR gives precious clues
that support the choice of specific treatments for patients harboring
several pathogens. Therefore, we have developed a quantitative PCR for
the diagnosis of T. gondii infection based on the
LightCycler technology (16). This technology relies on hybridization of amplicon-specific probes with adjacent fluorophores capable of fluorescence resonance energy transfer when they bind to the
target sequence. This technology provides a real-time measure of the
amplification product. We subsequently analyzed serum samples from
allogeneic stem cell transplant recipients.
| |
MATERIALS AND METHODS |
Development of LC-PCR.
The LightCycler PCR (LC-PCR) test was
targeted at the T. gondii B1 gene (4). The B1
gene amplicon comprises a 126-bp fragment. The primers used for
amplification were the 23-mer 5'-GGAGGACTGGCAACCTGGTGTCG-3' (sense) and the 25-mer 5'-TTGTTTCACCCGGACCGTTTAGCAG-3'
(antisense). Two hybridization probes (Tib MolBiol, Berlin,
Germany) were designed that recognize adjacent internal sequences
within the target B1 gene sequence. One
(5'-ACGGGCGAGTAGCACCTGAGGAGAT-3') was labeled at the 5' end
with LC-Red 640 and was phosphorylated at the 3' end to prevent probe
elongation by the Taq DNA polymerase. The other one
(5'-CGGAAATAGAAAGCCATGAGGCACTCC-3') was labeled at the 3'
end with fluorescein.
T. gondii DNA (RH strain) was extracted by a conventional
phenol-chloroform procedure from purified parasites obtained from ascitic fluids of inoculated mice and was quantified by UV
spectrophotometry. Because some mouse DNA can contaminate the T. gondii DNA, we measured the mouse DNA in the total DNA extracted
using a real-time quantitative PCR assay that we have developed for
other purposes. This TaqMan PCR assay is targeted at a single copy of
the mouse GALT gene (GenBank access number M96265). Therefore, we
extracted the mouse DNA from the total DNA for further measurements. We
estimated that one parasite corresponded to 0.1 pg of DNA, assuming
that the haploid T. gondii genome size is 80 Mb
(5) and that the B1 gene, the target for the LC-PCR test, is
repeated 35 times (4). After extraction, one 10-fold serial
dilution of T. gondii DNA was made, with parasite
concentrations ranging from 0.75 × 106 to 0.75 parasites per 10 µl. This series of 10-fold dilutions was included in
each amplification run.
LC-PCRs were set up in a final volume of 20 µl with the DNA Master
Hybridization Probes Kit (Roche Molecular Biochemicals,
Meylan,
France), each primer at a concentration of 0.5 µM, each
probe at a
concentration of 0.25 µM, and 10 µl of extracted DNA
sample. A
hot-start procedure was systematically used by the addition
of an
anti-
Taq DNA polymerase antibody (Clontech, Paris, France)
to the amplification reaction mixture. Carryover was prevented
by using
heat-labile uracil-DNA-glycosylase (UNG; Roche Molecular
Biochemicals,
Meylan, France). The reaction mixture was initially
incubated for 1 min
at room temperature to allow the UNG to act.
This incubation was
followed by a 2-min step at 95°C to denature
the DNA, to inactivate
the UNG, and to free the
Taq DNA polymerase
from the
anti-
Taq DNA polymerase antibody. Amplification was
performed
for 50 cycles of denaturation (95°C for 5 s; ramp
rate, 20°C/s),
annealing (60°C for 10 s; ramp rate, 20°C/s),
and extension (72°C
for 15 s; ramp rate, 20°C/s).
The amplification was carried out in a LightCycler Instrument (Roche
Molecular Biochemicals), and a single fluorescence reading
for each
sample was taken at the annealing step. Quantitative
results were
expressed by determination of the threshold of detection,
or the
crossing point (Cp), which marked the cycle when the fluorescence
of a
given sample significantly exceeded the baseline signal.
They were
expressed as a fractional cycle number. Then, the Cp's
were plotted
against the known concentration of the parasite to
obtain the standard
curve. The parasite count for a given clinical
sample was calculated by
interpolation from this standard
curve.
Patients.
Several serum samples were obtained from four
allogeneic stem cell recipients known to be toxoplasma PCR positive by
our routine test (3, 8). Serum samples were collected in
Vacutainer SST tubes (Becton Dickinson) at least once a week, frozen
within 4 h of receipt in the laboratory, and stored at 
20°C.
After thawing, DNA from 200 µl of serum was extracted with the High
Pure PCR Template Preparation kit (Roche Molecular Biochemicals)
according to the manufacturer's recommendations. PCR amplification was
performed with 10 µl of the eluate. The PCR results were secondarily
compared to the clinical data in the medical records.
| |
RESULTS |
Development of LC-PCR.
Figure 1
shows the amplification of the purified T. gondii DNA in
10-fold serial dilutions with fluorescence plotted against cycle
number. The higher the initial copy number, the earlier the fluorescent
signal appears. As the initial template concentration decreases, the
curves are shifted to the right. The sensitivity of the system is such
that a single parasite can be distinguished from the background noise.
The intra-assay coefficient of variation was below 1% for the
high-concentration DNA (Cp, 24.24 ± 0.07) and 1.6% for the
low-concentration DNA (Cp, 33.74 ± 0.55) by using commercial
T. gondii DNA (Advanced Biotechnologies Inc., Columbia, Md.)
tested 10 times at two different concentrations in the same run.
Reproducibility was estimated by testing the 10-fold dilution 10 times
in independent runs. The interassay coefficients of variation were 6.4, 12.3, 13.8, and 36% for samples with 103, 102,
10, and 1 parasite(s), respectively.
View larger version (26K):
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|
FIG. 1.
Real-time quantitative LC-PCR test using fluorescence
energy transfer. (A) Duplicate amplification plots obtained for
T. gondii DNA dilutions from 0.75 × 106
(right) to 0.75 parasite in 10 µl (left). Each slope corresponds to a
particular input target quantity. (B) Plot of the duplicate Cp against
the input target quantity (common log scale) showing the linearity of
the results. The computer-calculated correlation coefficient is 1.
|
|
Quantitative analysis of clinical samples.
Real-time
quantitative analysis by the LC-PCR was carried out with DNA extracted
from serum samples from four patients (Table 1). All samples positive by our routine
PCR test (3, 8) were also positive by the quantitative PCR
test. Moreover, the serum samples were also proved to be free of PCR
inhibitors since the internal control for our competitive PCR test was
correctly amplified each time (3, 8).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Main features, pretransplant toxoplasma serological
status of the recipient and donor, antitoxoplasma treatment, and
outcomes for four allogeneic stem cell transplant recipients with
toxoplasma PCR-positive blood samples studied by quantitative
LC-PCR testa
|
|
Patients 613, 685, and 696 did not develop full-blown toxoplasmosis
(Table
1). The first and last samples were PCR negative.
These patients
were febrile at the time of detection of the first
PCR-positive sample
but chest X ray, fundoscopic examination,
and cerebral CT scan were
normal. The quantified PCR signal corresponded
to less than 0.75 parasite. For these three patients the positive
PCR signal disappeared
in 22, 9, and 11 days, respectively, after
trimethoprim-sulfamethoxazole was given orally at doses of 160
mg of
trimethoprim and 800 mg of sulfamethoxazole three times
weekly.
In contrast, patient 708 (Table
1) had encephalitic symptoms compatible
with toxoplasmosis, and this raised several therapeutic
issues. Because
of known allergy to sulfonamide, aerosolized pentamidine
instead of
trimethoprim-sulfamethoxazole was given for
Pneumocystis carinii prophylaxis. On day 61, the patient was febrile and
agitated.
The fever was not documented, and he was empirically given
antibiotics.
A cerebral CT scan was normal, and the behavior troubles
were
attributed to steroid therapy (2 mg/kg of body weight/day). A
couple of days later, abdominal pain developed and ultrasonic
examination showed signs of cholecystitis. A cholecystectomy was
performed on day 70. After surgery, the patient did not recover
consciousness. A new cerebral CT scan showed numerous cerebral
lesions
compatible with herpes, toxoplasma encephalitis, or metabolite
disorders. Pyrimethamine (100 mg/day) and clindamycin (1.2 g/day)
were
given on day 75. The patient died on day 91 without recovery
of
consciousness. An autopsy was not
performed.
The retrospective analysis of nine serum specimens by LC-PCR showed
that the first PCR-positive serum was retrieved on day
61 after
transplantation, when psychiatric troubles and fever
occurred. LC-PCR
showed that the number of parasites increased
from 1 per 200 µl of
serum on day 61, to 4 per 200 µl of serum
on day 69, and to 397 per
200 µl of serum on day 75, when antitoxoplasma
therapy was started.
Then, the parasite number declined sharply
in 3 days to six parasites
per 200 µl of serum and then remained
positive below the last point
of the standard curve from day 82
until death. On day 75 only the
cerebrospinal fluid (CSF) was
available, and the results of cytologic
and biochemical analyses
of the CSF were normal. The number of
parasites was 4,240 per
200 µl of CSF, 10 times higher than the
number in serum. In addition,
a PCR with CSF for detection of herpes
simplex virus was
negative.
| |
DISCUSSION |
The real-time quantitative LC-PCR system that we have developed
for determination of the concentration of T. gondii DNA in serum gives reproducible quantitative results over a wide dynamic range
and has a fast turnaround time of less than 2 h, including the DNA
extraction steps. The risk of a false-positive result due to
contamination with previously amplified products, the main concern in
PCR, is dramatically reduced since there is no need to open the tube at
the end of the amplification. Furthermore, the enzymatic method of
prevention of contamination, based on the use of dUTP instead of dTTP
in the reagent mixture and the systematic use of the enzyme UNG,
increases the safety of the test.
The most important observation in this study is that the parasite count
could be determined and correlated with the clinical symptoms and
treatment. In the three PCR-positive patients who did not develop
full-blown toxoplasmosis, the parasite count was always low and PCR
became negative when trimethoprim-sulfamethoxazole was given. This
finding might be frequent in allogeneic bone marrow transplant
recipients. Indeed, in a recent prospective study we observed that
12.5% (3 of 24) of Toxoplasma-seropositive allogeneic stem
cell transplant recipients had transient PCR-positive test results but
did not develop full-blown toxoplasmosis (1). In contrast,
for the patient in the present study who developed cerebral symptoms,
the parasite count increased until antitoxoplasma therapy was started.
Then, the parasite count decreased sharply. Because no autopsy was
performed, the cause of death was not ascertained, but we can conclude
that the antitoxoplasma therapy was efficient in reducing the parasite load.
The real-time quantitative LC-PCR assay developed in this study can be
used not only to detect the presence of T. gondii DNA but
also to provide precise evaluations of the parasite load in immunocompromised patients. This PCR test should be useful for the
monitoring of treatment efficacy and should help provide an understanding of the pathogenesis of toxoplasma reactivation.
| |
ACKNOWLEDGMENTS |
All the data presented here have been generated with a
LightCycler apparatus kindly provided by Roche Molecular Biochemicals, Meylan, France.
This research was supported in part by l'Association de Recherche
contre le Cancer (grant 4029 1997).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Parasitologie-Mycologie, Hôpital Henri Mondor, 51 avenue du
Général DeLattre de Tassigny, 94010, Créteil, Cedex,
France. Phone: 33-1-49-81-36-41. Fax: 33-1-49-81-36-01. E-mail:
bretagne{at}univ-paris12.fr.
| |
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Journal of Clinical Microbiology, August 2000, p. 2929-2932, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
