Diagnosis of Toxoplasmosis after Allogeneic Stem Cell Transplantation: Results of DNA Detection and Serological Techniques
Toxoplasmosis after Transplantation • CID
Diagnosis of Toxoplasmosis after Allogeneic Stem Cell Transplantation: Results of DNA Detection and Serological Techniques
H e´l e`ne Fricker-Hidalgo () 2
Claude-Eric Bulabois 1
Marie-Pierre Brenier-Pinchart 2
Rebecca Hamidfar 0
Fr e´d e´ric Garban 1
Jean-Paul Brion 3
Jean-Franc¸ ois Timsit 0
Jean-Yves Cahn 1
Herv e´ Pelloux 2
0 Re ́animation Me ́dicale
1 He ́matologie , Poˆle Cance ́rologie
2 Parasitologie-Mycologie, Poˆle Biologie
3 Maladie Infectieuse, Poˆle Me ́decine Aigu ̈e et Communautaire, Centre Hospitalier Universitaire , Grenoble , France
Background. The biological diagnosis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) is based on the detection of Toxoplasma gondii DNA in blood specimens or other samples. Serological testing is used mainly to defin the immunity status of the patient before HSCT. The aim of our study was to examine the performance of polymerase chain reaction (PCR) and serological techniques in the diagnosis of toxoplasmosis after HSCT. Methods. Seventy patients underwent allogeneic HSCT from September 2004 through September 2006. DNA was detected by PCR, and immunoglobulin G and immunoglobulin M were detected by enzyme-linked immunosorbent assay. Results. The results of immunoglobulin G detection before allogeneic HSCT were positive in 40 (57.1%) of the patients and negative in 30 (42.9%). After HSCT, 57 patients (81.4%) had test results that were negative for immunoglobulin M and had negative results of DNA detection, without toxoplasmosis infection. Four patients (5.7%) had at least 4 samples with positive PCR results and/or test results positive for immunoglobulin M against T. gondii; toxoplasmosis was then confi med by clinical symptoms. Nine patients (12.9%) with positive PCR results and 1 or 2 samples with test results negative for immunoglobulin M were considered to have asymptomatic T. gondii infection. Reactivation of latent infection was the cause of toxoplasmosis in 3 of the 4 patients, and toxoplasmosis occurred as a primary infection in 1 patient. The detection of specifi anti-T. gondii immunoglobulin M was the only biological evidence of toxoplasmosis in 2 patients, and samples were positive for immunoglobulin M before PCR was performed in 1 patient. Conclusions. Thus, after HSCT, all patients were at risk for toxoplasmosis; all patients who receive HSCTs should be followed up with biological testing that combines PCR and serological techniques. Toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) remains a cause of severe infection and is associated with a high mortality rate . Various studies have estimated a toxoplasmosis risk of 0.3%-5.0% after allogeneic HSCT, depending on the prevalence of Toxoplasma gondii in the population , but the true incidence of toxoplasmosis among immunocompromised patients is difficul to assess, be-
cause in many instances, the diagnosis is overlooked
. Recent single-center and multicenter retrospective
studies  have suggested that invasive disease may be
more common than was previously known, with
incidences among T. gondii–seropositive recipients of
allogeneic transplants of up to 4% and an estimated
mortality rate of 60%–90%. The major site of infection is
the brain, with a focal mass presentation or, less
commonly, diffuse encephalitis [3, 8]. Myocarditis,
pneumonitis , hepatitis, chorioretinitis, and disseminated
disease may also complicate the course of toxoplasmosis
in immunocompromised hosts [1, 4]. The clinical
presentation is nonspecific and the pleiomorphic
manifestation of toxoplasmosis makes it difficul to obtain
a definit antemortem diagnosis . Studies
underscore the need for rapid diagnostic tests in an effort to
improve the outcome . Serological testing is used
Toxoplasmosis after Transplantation • CID 2009:48 (15 January) • e9
PCR and IgM
PCR+ and IgM+
PCR and IgM+
PCR+ and IgM
1 patient had 4 positive samples;
1 patient had 5 positive samples
1 patient had 5 positive samples;
9 patients had 1 positive sample
NOTE. IgM detection was performed using ELISA (Vidas; bioMe´ rieux), immunosorbent
agglutination assay (bioMe´ rieux), and indirect immunofluorescence. Toxoplasma gondii DNA
detection was performed using PCR. +, Positive; , negative
a The patient with 5 positive samples.
mainly to identify at-risk patients prior to allogeneic HSCT
. PCR has been shown to be sensitive in
immunocompromised patients with disseminated toxoplasmosis [11–14] and
may represent a better modality for early identificatio of
infection . Routine PCR testing of peripheral blood
specimens may be an appropriate tool to guide preemptive therapy
for patients at very high risk of developing invasive disease [7,
The aim of this study was to retrospectively analyze the
combined results of PCR and serological techniques in a survey of
70 allogeneic HSCT recipients and to determine the prevalence
of invasive toxoplasmosis.
PATIENTS AND METHODS
Patients. Seventy consecutive adult patients received HSCTs
from September 2004 through September 2006 in the
Hematologic Department of Grenoble University Hospital (Grenoble,
France). Serological testing was performed on baseline
pretransplantation samples obtained from each patient. After
transplantation, the patients were systematically monitored by
PCR performed on blood samples obtained every 2 weeks
during immunosuppressive treatment and were monitored less
systematically by serological testing. If toxoplasmosis was
suspected, PCR was performed more frequently (every 2 days) on
blood samples and/or on another sample from the involved
organ (usually CSF or bronchoalveolar lavage fluid) After
engraftment, primary prophylaxis with
trimethoprim-sulfamethoxazole was recommended for all patients when possible (∼1
month after HSCT). Yet, if intolerance or persistent neutropenia
occurred, alternative prophylaxis decisions were made. If a
patient was found to have PCR results positive for T. gondii
without clinical symptoms, no treatment was initiated. If a patient
developed Toxoplasma disease, therapy including
pyrimethamine-sulfadiazine or pyrimethamine-clindamycin was initiated
on the basis of biological, clinical, and radiological evidence.
Methods. The parasite DNA was detected using a
qualie10 • CID 2009:48 (15 January) • Fricker-Hidalgo et al.
tative PCR system, described elsewhere , that was designed
to use the B1 target gene and that uses an internal control to
monitor inhibition, as well as an amplicon carry-over
contamination control with a sensitivity level of 1 parasite. Anti–T.
gondii IgM was detected by ELISA (Vidas; bioMe´rieux), indirect
immunofluo escence, and immunosorbent agglutination assay
(bioMe´rieux) . Anti–T. gondii IgG was detected by ELISA
(Vidas; bioMe´rieux) and indirect immunof uorescence .
Definitions T. gondii infection and disease were define
according to the European Group for Blood and Marrow
Transplantation–Infectious Diseases Working Party Guidelines .
In brief, T. gondii infection was define as being present in a
patient who had positive results of PCR performed on blood
samples and no evidence of organ involvement, with or without
fever. Definit toxoplasmosis was define as histological
evidence of active Toxoplasma disease found in a clinically and
radiologically involved organ, whereas probable toxoplasmosis
was define as positive results of PCR performed on a blood
sample and/or another sample from the involved organ (usually
CSF or bronchoalveolar lavage fluid with clinical signs and
symptoms and radiological evidence of active disease.
Biological test results. Before HSCT, serological testing was
performed to determine the immune status of the recipients.
The results of specifi IgG detection testing were positive for
40 (57.1%) of 70 HSCT recipients. The results of specifi IgM
detection testing were negative for 69 (98.6%) of 70 HSCT
recipients; the 1 HSCT recipient with positive results had
residual IgM detected. After HSCT, 4 recipients (5.7%) who had
Toxoplasma disease had at least 4 samples with positive
biological test results (i.e., presence of T. gondii DNA or IgM or
both) (table 1). The 57 patients (81.4%) with negative IgM test
results and negative DNA detection results had no T. gondii
infection. The 9 other patients (12.8%), who had 1 or 2 samples
with positive PCR results and negative IgM results, were
considered to have T. gondii infection without disease; these
patients experienced a favorable outcome without any specif c
antitoxoplasmic treatment. The predictive value of having 1
sample with a positive PCR result was 25%. Thus, the incidence
of Toxoplasma disease was 5.7%, and the incidence of T. gondii
infection was 12.8%.
Characteristics and outcome of Toxoplasma disease.
Characteristics and outcomes of the 4 recipients with
toxoplasmosis are shown in table 2. These 4 recipients received
suppressive treatment, but 3 recipients (patients 1, 2, and 3)
received transplants from unrelated donors and developed
graft-versus-host disease of grade I or II. None of the patients
had received anti-T. gondii prophylaxis. Two recipients (patients
1 and 2) developed cerebral toxoplasmosis, and the other 2
recipients (patients 3 and 4) developed disseminated
toxoplasmosis with respiratory distress. The disease onset was earlier
for the 2 recipients with pulmonary symptoms (patient 3, onset
at day 27; patient 4, onset at day 33) than for the 2 recipients
with neurological symptoms (patient 1, onset at day 114; patient
2, onset at day 153). Before the allograft was performed, 3
recipients (patients 2, 3, and 4) had serological test results that
were positive for T. gondii IgG. In patients 2, 3, and 4,
toxoplasmosis seemed to occur through reactivation of latent cysts
into invasive tachyzoites. In patient 1, the recipient and the
donor were seronegative, the results of PCR performed on 10
blood samples and 1 CSF sample were negative, and
toxoplasmosis occurred as a primary infection with a typical
seroconversion (figu e 1A). The symptoms included abnormal
movements of all 4 limbs and of the eyes. Treatment with
pyrimethamine-clindamycine was effective. Patient 2 had fever
and somnolence. Two peripheral blood samples yielded positive
PCR results on days 165 and 168 after transplantation (figu e
1B) and confi med the presence of toxoplasmosis. The results
of a PCR performed on 1 CSF sample were negative.
Subsequently, 2 relapses of toxoplasmosis were observed after
stopping antiparasitic treatment. The results of biological testing
performed after the firs relapse were negative, and the diagnosis
was confi med by a cerebral CT. A second relapse occurred on
day 800, and biological test results revealed an increased IgM
level, but PCR results were negative. Patient 3, who had
pulmonary toxoplasmosis, received a toxoplasmosis diagnosis on
the basis of positive results of PCR performed on
bronchoalveolar lavage fluid but the patient died before treatment could
be administered (figu e 1C). A retrospective analysis of the
serum samples obtained 27, 34, 43, and 49 days before this
patient’s death revealed the presence of IgM. Patient 4 received
a diagnosis on the basis of positive results of PCR performed
on bronchoalveolar lavage flui on day 36 after transplantation
(figu e 1D). Giemsa fast staining and microscopic observation
of the bronchoalveolar lavage flui sample obtained on day 46
revealed tachyzoites of T. gondii. In this case, tests to detect
IgM had negative results. Treatment with
pyrimethamine-clindamycin was started, but the patient died on day 59.
Characteristics and outcome of T. gondii infection.
Table 3 shows the demographic and clinical characteristics and
outcomes for the 9 patients with 1 or 2 blood samples with
positive PCR results. Before the allograft was received, 6 of 9
recipients had serological tests with results that were positive
for T. gondii (i.e., positive for IgG). In 6 patients, the serostatus
of the donor was positive, and 3 patients (patients 10, 11, and
12) received transplants from unrelated donors. Seven patients
(patients 5, 6, 7, 9, 10, 12, and 13) received suppressive
treatment, and 4 patients (patients 5, 9, 10, and 12) developed
graftversus-host disease of grade I or II. Only 1 of 9 patients received
anti-T. gondii prophylaxis. No signs of toxoplasmosis were
observed. The 9 patients did not receive treatment with anti–T.
gondii drugs, and the outcomes were favorable for all patients.
Few studies [1, 3, 20] have analyzed the incidence of T. gondii
infection and disease in patients after allogeneic HSCT. Martino
et al.  described the results of a prospective study of the
incidence of reactivation of toxoplasmosis that was performed
at 5 European transplantation centers and involved 106 T.
gondii–seropositive adult recipients of HSCT. They found an
incidence of 16% for T. gondii infection and 6% for Toxoplasma
disease. In our study, the incidence of T. gondii infection was
12.8%, and the incidence of Toxoplasma disease was 5.7%.
However, the patients in our study had positive or negative
serological test results obtained before HSCT. The seropositive
patients were at risk for reactivation of latent cysts, whereas
the seronegative patients were at risk for primary T. gondii
infection. Thus, toxoplasmosis is more common after HSCT
than has been previously suggested [2–5]. The incidence of
toxoplasmosis in our study approximated that in the study by
Martino et al. .
Toxoplasmosis after HSCT is a severe infection that is
associated with a high mortality rate [1, 10, 21]. Analysis of our
data also indicates that infections that occur late after
transplantation (e.g., patients 1 and 2; table 2) may have a better
prognosis than infections that occur soon after transplantation
(e.g., patients 3 and 4). The low rate of late reactivation is
probably attributable to the fact that immune recovery
improves with time after HSCT . The patients who received
HSCTs from unrelated donors (3 of the 4 recipients in our
study) possibly had a higher risk of toxoplasmosis than did
patients who received HSCTs from related donors. PCR
techniques to detect the presence of T. gondii DNA in peripheral
blood and other samples may help to establish the
toxoplasmosis diagnosis [14, 22]. The PCR used in this work was
qualitative. However, this is not a major drawback, because no clear
threshold level for therapy has been described to date. Our
Toxoplasmosis after Transplantation • CID 2009:48 (15 January) • e11
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study shows that the detection of IgM may be important for
obtaining a diagnosis of toxoplasmosis in some patients. For
2 patients, the detection of specifi anti–T. gondii IgM was the
only biological evidence of toxoplasmosis (patient 1) or relapse
(patient 2). For 1 patient (patient 3), IgM was detected before
positive PCR results were obtained. For the biological
followup of patients undergoing allogeneic HSCT, no consensus exists
on the optimal protocol to be used in clinical laboratories. We
recommend monitoring by PCR of blood samples and
serological testing once every 2 weeks during immunosuppressive
therapy after transplantation.
Reactivation of latent tissue cysts in previously infected
individuals is the usual mechanism involved in toxoplasmosis
that occurs after HSCT . Therefore, it is important to
determine the patient’s serostatus before transplantation and to
monitor the patient with negative specifi serological tests to
detect potential new infection. Three patients with
toxoplasmosis infection were seronegative before transplantation but
had test results that were positive for IgG after HSCT. A negative
test result obtained before allograft does not always mean the
absence of T. gondii immunity, because deep
immunosuppression may reduce the level of anti–T. gondii antibodies.
Therefore, all of the patients with test results negative for IgG before
allograft, as well as those with test results positive for IgG,
should be followed up with PCR and serological testing. The
need for T. gondii prophylaxis before HSCT in transplant
recipients should be better defined and T. gondii prophylaxis
should preferably be combined with Pneumocystis jiroveci
prophylaxis . For prophylaxis, we propose the use of
cotrimoxazole or pyrimethamine combined with pentamidin.
In conclusion, toxoplasmosis is a serious health problem
among HSCT recipients that can lead to death. Reactivation of
latent infection is the most common cause of toxoplasmosis in
such patients. However, toxoplasmosis can also occur as a
primary infection. After allogeneic HSCT, all patients are at risk
of toxoplasmosis and should be followed up with biological
testing. We recommend the use of PCR performed on blood
samples and serological testing to detect anti–T. gondii IgM and
We thank Catherine. Barois, Claudine Mur, and Agnes Meunier, for their
technical assistance, and Sabine Durville, for reading the manuscript.
Potential confl cts of interest. All authors: no conf icts.
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