Received on 19 July 2008; revised 7 December 2008.
Address for correspondence: Dr. Fábio Luís Nascimento Nogui. Rua das
Camélias, 321 ap 83. Zip code: 04048-060. São Paulo/SP, Brazil. Phone:
+55 11 5581-1200. Fax: +55
11 5087-9886. E-mail:
The Brazilian Journal of Infectious Diseases 2009;13(1):18-23.
© 2009 by The Brazilian Journal of Infectious Diseases and Contexto
Publishing. All rights reserved.
Neurotoxoplasmosis Diagnosis for HIV-1 Patients by Real-Time PCR of Cerebrospinal Fluid
Fábio Luís Nascimento Nogui1, Sandro Mattas2, Gilberto Turcato Júnior1 and David Salomão Lewi1
1Federal University of São Paulo, Division of Infectious Disease; 2Federal University of São Paulo, Division of Neurology; São Paulo, SP,
Encephalitis caused by Toxoplasma gondii is the most common cause of central nervous system damage in patients
with acquired immunodeficiency syndrome (AIDS). Toxoplasma may infect any of the brain cells, thus leading to
non-specific neurotoxoplasmosis clinical manifestations including focused or non-focused signs and symptoms of
central nervous system malfunction. Clinical development ranges from insidious display during weeks to
experiencing acute general confusion or ultimately fatal onset. Cerebral toxoplasmosis occurs in advanced stages
of immunodeficiency, and the absence of anti-toxoplasmosis antibodies by the immunofluorescence method does
not allow us to rule out its diagnosis. As specific therapy begins, diagnosis confirmation is sought through clinical
and radiological response. There are few accurate diagnosis methods to confirm such cases. We present a method for
T. gondii DNA detection by real time PCR-Multiplex. Fifty-one patients were evaluated; 16 patients had AIDS and
a presumptive diagnosis for toxoplasmosis, 23 patients were HIV-positive with further morbidities except
neurotoxoplasmosis, and 12 subjects were HIV-negative control patients. Real time PCR-Multiplex was applied to
these patients’ cephalorachidian liquid with a specific T. gondii genome sequence from the 529bp fragment. This
test is usually carried out within four hours. Test sensitivity, specificity, positive predictive value, and negative
predictive value were calculated according to applicable tables. Toxoplasma gondii assay by real time Multiplex of
cephalorachidian fluid was positive for 11 out of 16 patients with AIDS and a presumptive diagnosis for cerebral
toxoplasmosis, while none of the 35 control patients displayed such a result. Therefore, this method allowed us to
achieve 68.8% sensitivity, 100% specificity, 100% positive predictive value, and 87.8% negative predictive value.
Real time PCR on CSF allowed high specificity and good sensitivity among patients who presumably had cerebral
toxoplasmosis. Since this is a low invasive method, it could be included in the diagnosis algorithm of patients with
AIDS and central nervous system damage.
Key-Words: AIDS, cerebral toxoplasmosis, real time polymerase chain reaction.
Neurological complications occur in 39% to 70% of AIDS
patients [1-3]. The most frequent opportunistic infections are
cerebral toxoplasmosis, cryptococcal meningitis, progressive
multifocal leukoencephalopathy (PML), tuberculous
meningitis, and cytomegalovirus encephalitis (CMV). In
addition to infections, central nervous system primary
lymphoma is also an important cause of focused brain damage
in such patients. An accurate diagnosis of these neurological
complications is crucial, since most such complications are
likely to be treated, and prompt effective intervention may
eventually yield to longer survival or better quality of living.
Most commonly, encephalic toxoplasmosis in AIDS patients
is caused by reactivation of a chronic infection. The incidence
of cerebral toxoplasmosis in HIV-infected patients is
proportional to the prevalence of T. gondii latent infection
among the population in general. Prior to antiretroviral therapy,
one out of three HIV-infected persons could develop
encephalic toxoplasmosis with progressive immunological
deterioration if no specific prophylaxis were applied [1,4,5].
Current USA information, as well as information gathered from
other regions, has shown a decrease in brain toxoplamosis
among AIDS patients as a result of powerful antiretroviral
therapy and the use of sulfa derivatives as a primary
prophylaxis against Pneumocystis jirovecii pneumonia, which
indirectly prevents T. gondii infection. Such a decrease
evolved from 2.1/100 patients/year in 1992 to 0.7/100 patients/
year in 1997 .
In Brazil, there has also been a significant drop in the
number of encephalic toxoplasmosis cases within the last
years. According to the São Paulo State Epidemiology
Surveillance Bulletin, the number of cerebral toxoplasmosis
cases fell from 1,408 (17%) in 1980-1989 to 3,224 (10.1%) in
Among immunocompetent patients, toxoplasmosis
diagnosis can usually be attained either through direct parasite
detection or differences in specific antibody titers in serology
tests. In the case of AIDS patients, however, it is recommended
to use an algorithm based on imaging examination criteria, i.e.
brain CT scan and/or MRI, along with therapeutic proof for
around 14 days. Treatment failure usually leads to serious
clinical involvement due to misdiagnosis.
We evaluated a quick T.gondii encephalitis diagnosis
method using the cephalorachidian liquid of HIV-infected
patients by means of real time PCR-Multiplex.
Material and Methods
This project was approved by the research and ethics
committees of the institutions involved (São Paulo state AIDS/
STD Reference & Training Center, and the São Paulo Federal
BJID 2009; 13 (February)
University Hospital). Fifty-one patients were evaluated, among
which 30 were from the São Paulo Federal University Hospital
and 21 were from the São Paulo state AIDS/STD Reference &
Training Center). In this group, 16 subjects were HIV-positive
with a presumptive diagnosis of cerebral toxoplasmosis, which
included signs and symptoms of central nervous system
malfunction and mass damage detected by brain CT scan or
MRI. Response to specific treatment was exhibited two weeks
later. Thirty-five subjects were included as control patients,
among which 23 were HIV-infected patients and 12 were non-
HIV patients who displayed other diseases or neurological
The assay was targeted at the 529-bp repeat element
(GenBank accession numbers AF 487550 and AF 146527),
repeated more than 300-fold in the genome of T.gondii (26).
Human B-actin gene was co-amplified and detected as the
internal control for DNA isolation and PCR amplification. The
reactions were performed on the ABI PRISM 7700 Sequence
Detection System, ABI PRISM 7500 Sequence Detection
System (Applied Biosystems) or Roto-Gene 300 (Corbett
Research), using two different TaqMan MGB probes, one for
T.gondii 529-bp fragment and one for human B-actin gene. In
the sensitivity test, we applied serial T. gondii DNA dilutions.
The detection threshold was estimated as 80fg of DNA, which
is equivalent to one parasite per reaction. Negative controls
were used in order to achieve higher quality control. The result
was shown as detected, and was calculated by interposition
of the parasite standard dilution curve. The average duration
of this test was four hours.
Sensitivity, specificity, positive predictive value, and
negative predictive value were calculated according to
applicable tables. Differences in proportions were compared
by the Fischer’s exact test. The Kruskal-Wallis test was applied
to continuous variables. We applied a +/- one standard
deviation to variables in which the average was used. Odds
ratios (ORs) with a 95% confidence interval were calculated
for all variables. We considered p < 0.005 as a significant
value. The statistical analysis was carried out with the
Statistical Package for the Social Sciences, version 11.5 (SPSS,
Chicago, IL, USA).
Among the 51 patients evaluated from August 2004 to
November 2005, 16 displayed AIDS symptoms and had a
presumptive diagnosis of cerebral toxoplasmosis. Thirty-five
patients were evaluated as control patients, among which 23
were HIV-infected and 12 were HIV-negative.
All patients underwent fluid punction for Toxoplasma
gondii DNA assay by real time polymerase chain reaction.
Among the 16 patients who displayed neurological and
radiological clinical signs and symptoms of cerebral
toxoplasmosis, nine were males (56%). In all patients, serum
immunoglobulin (IgG and IgM) was detected through
immunoassay for T. gondii. Brain damage diagnosis was made
by neuroimaging examination. All patients underwent a skull
CT scan; three patients additionally underwent an MRI.
The therapy applied was a sulfadiazine-pyrimethamine
combination, except for a patient for whom clindamycin was
prescribed instead of sulfa. As foreseen in the inclusion criteria,
there was a response in all evaluated cases. This response
consisted of clinical and radiological improvement by the 14th
day of therapy. The number of helper T (CD4) lymphocytes
ranged from seven to 212 cells/mm³ (mean 72.75 cells/mm³;
median 44 cells/mm³). In nine patients (56%), the number of
CD4 T-cells was lower than 100 cells/ mm³.
In five among the 16 patients with a presumptive diagnosis
for cerebral toxoplasmosis who showed a response to
treatment, it was not possible to detect cerebral toxoplasmosis
DNA by real time PCR. In these patients, the CD4 T-cell count
ranged from 9 to 169 cells/ mm³ (mean 98.2 cells/ mm³; median
150 cells/ mm³). When we applied the Kruskall-Wallis test, we
found no significant difference between the two groups
(p=0.125) in the CD4 cell counts (Table 1).
The CSF collection date varied from the 1st to the 9th day
following therapy initiation. In 12 cases (75%), collection was
carried out within the first 72 hours of the patient’s admission
to the hospital.
Some clinical and laboratory variables were compared in
the five false negative patients and the 11 patients who gave
better results in positive real time PCR. When we considered
the difference in proportion between T. gondii-positive and
negative PCR patients, we found the following: Fever (OR, 0.250;
95% CI 0.03 - 2.32; p=0.299), headache (OR, 1.250; 95% CI 0.146
- 10.699; p=1.00), seizures (OR, 0.15; 95% CI 0.01 - 1.562; p=0.245),
motor deficit (OR, 0.56; 95% CI 0.06 - 4.75; p=1.00), and altered
consciousness (OR, 2.62; 95% CI 0.30 - 22.99; p=0.596). Thus,
we found no significant differences when we compared the
clinical symptoms of the two groups (Table 2).
We worked with two control groups. The first group
comprised 23 HIV-positive patients who underwent fluid
examination because they were likely to have other
neuroinfections than neurotoxoplasmosis. This age group
ranged from 27 to 60 years (mean = 39.9 years); 13 (56%) were
males and 10 were females. Of these patients, five (22%) were
diagnosed with neurosyphilis, four (17%) with
neurocryptococcosis, four (17%) with tuberculous
meningoencephalitis, two (9.5%) were reported to have
cephalea symptoms, two (9.5%) displayed seizures, and the
others were found to have hepatic enchephalopathy, bacterial
meningitis, progressive multifocal leukoencephalopathy
(PML), and chorioretinitis caused by toxoplasmosis with a
cerebral vascular accident. One AIDS patient underwent CSF
collection to fight acute lymphocytic leukemia through
All 12 non-HIV patients had been admitted to the São
Paulo Hospital. The age group ranged from 18 to 75 years
(mean = 39.4 years); eight of the 12 were males and four were
females. The neurological diseases that led to CSF collection
Real-Time PCR and Cerebral Toxoplasmosis
BJID 2009; 13 (February)
were as follows: four due to herpetic meningoencephalitis,
and the others due to politraumatism, meningioma, cerebral
venous thrombosis, Guillain-Barré syndrome, optic neuritis,
pineal tumor, chorioretinitis caused by toxoplasmosis, and
All HIV-positive control subjects yielded negative real time
PCR results, showing that the specificity of this method was
100% in this sample. Patients with chorioretinitis-
provokedtomoxoplasmosis (both HIV-positive and HIV-
negative) showed negative results in the real time PCR T.
Assessment of sensitivity, specificity, positive predictive
value, and negative predictive value were calculated in a 2x2
table. The following results were obtained:
Negative Predictive Value: 87.8%
Positive Predictive Value: 100.0%
There were no false positives, which confirms the
specificity of as high as 100%. However, five cases of patients
with cerebral toxoplasmosis and negative real-time PCR were
found, resulting in a sensitivity of 68.8%.
Neurological complications often occur in HIV-infected
people, and T. gondii encephalitis is the most frequent cause
of focal brain disease in AIDS patients . More than 50% of
HIV-positive individuals will eventually develop neurological
symptoms of this disease. Such complications are generally
observed in advanced stages of the disease, with severe
immunodepression. However, 10-20% of HIV-positive patients
may develop opportunistic infections even if there is no
immunological failure . Among our cases, only one patient
with a cerebral toxoplasmosis diagnosis had T-helper CD4
lymphocyte counts greater than 200 cells/mm³.
In such neurologically-affected patients, focal brain
damage (FBD) features among the main issues while reaching
a diagnosis in HIV-1 infected patients. Cerebral toxoplasmosis
is the most common cause of FBD among these individuals,
though differential diagnoses have also reported lymphomas,
TB, and cryptococcosis. According to the latest
Epidemiological Bulletin issued by the São Paulo State STD/
AIDS Program in 2005, 20,059 (14.9%) cases of
neurotoxoplasmosis have been reported in Brazil since 1980.
Toxoplasma gondii infection may be diagnosed either indirectly
through serological methods, or directly through such
methods as PCR, hybridization, and histology. Whenever
clinical manifestations suggest that there might be brain
damage, neuroimaging studies, such as skull CT scan or MRI
should be performed. Positive presumptive diagnosis, which
is broadly regarded as a suitable clinical practice for HIV
patients and is adopted as a rule in both national and foreign
hospitals, is based on the patient’s clinical conditions, imaging
study (CT scan/MRI), and response to treatment after two
weeks . This standard diagnosis usually does not consider
brain biopsy, which is regarded as the ultimate definite
diagnosis, in spite of the risk to which patients are then
exposed. A retrospective multicentric study carried out by
Antinori with 160 brain biopsies in HIV patients with focal
brain damage showed a sensitivity of as high as 87%.
Nevertheless, this study led to significant morbidity and
mortality (7.5 and 3.1%, respectively). The causes of such
morbidity and mortality were basically due to biopsy-related
Real-Time PCR and Cerebral Toxoplasmosis
Table 1. Mean and median number of T CD4 lymphocytes among patients with presumptive diagnosis for cerebral toxoplasmosis
demonstrated by real -time PCR
Lymphocytes TCD4 (cells/mm³)
PCR (No.)Mean Medianp
Table 2. Clinical features among patients with cerebral toxoplasmosis with positive and negative PCR
Signs and symptoms Cerebral Toxoplasmosis
(%) No. of
Abnormal level of consciousness
Cranial nerve abnormalities
BJID 2009; 13 (February)
surgical brain hemorrhage, permanent facial-brachial paralysis,
transitory neurological failure, surgical wound infection, and
osteomyelitis. Biopsy-related mortality occurred, on average,
14 days following surgery and was also related to the
stereotaxic method .
Moreover, if toxoplasmosis empirical therapy does not lead
to clinical improvement, it will eventually delay diagnosis of
other possible neuropathologies in these patients, thus
affecting prognosis and therapeutic success directly. We
evaluated a quick, accurate method for diagnosing cerebral
toxoplasmosis so patients do not need to undergo
unnecessary treatment and invasive diagnostic procedures.
We found that real time PCR of CSF is a relatively simple and
quick method that can be performed in developing countries
to confirm suspected cases. It is a less invasive procedure
when compared with brain biopsy. As opposed to conventional
PCR, real time PCR provides a lower false positive risk ,
since there is no technical need to open the sample tubes
when amplification is completed .
Other studies have also shown that conventional PCR on
CSF results in a sensitivity of from 12% to 70% (usually 50%
to 60%) and a specificity of nearly 100% in patients with
cerebral toxoplasmosis [17-19]. Most studies have
demonstrated the diagnostic role of PCR in blood samples,
although sensitivity has varied within a broad range, i.e. 25 -
A retrospective study performed by Antinori analyzed T.
gondii DNA presence in five HIV-infected patients who were
believed to have encephalic toxoplasmosis (Group 1), eight
HIV infected patients with other symptoms (Group 2), and
seven non-HIV patients with other neurological diseases
(Group 3). PCR was positive for two of four of the patients
with an ultimate diagnosis of encephalic toxoplasmosis,
whereas it was negative in the other groups. This small study
confirmed the low sensitivity and high specificity of PCR when
applied to encephalic toxoplasmosis diagnosis .
Another study analyzed 88 CSF samples from HIV-positive
patients, among which 56 had focal brain damage. The patients
were prospectively tested for T. gondii B1 gene nested PCR.
Six out of the 18 patients with cerebral toxoplasmosis (but no
patients with other brain disorders) showed a positive PCR
result, resulting in 33% sensitivity and 100% specificity. This
study also proved that early collection leads to higher
sensitivity. As the study considered solely encephalic
toxoplasmosis patients whose CSF was collected prior to or
during the first week of antitoxoplamosis therapy, sensitivity
increased to 50%. Antitoxoplamosis prophylaxis had no effect
on PCR results .
Vidal et al. have recently evaluated CSF samples from 12
AIDS patients with a first episode of cerebral toxoplasmosis,
and 18 AIDS patients with other neurological opportunistic
diseases and no previous cerebral toxoplasmosis. This
evaluation was carried out at the Emilio Ribas Infectology
Institute. Samples from all cerebral toxoplasmosis patients
showed positive conventional PCR results (sensitivity, 100%),
and the sample from one of the 18 AIDS patients with other
neurological diseases also showed positive PCR results (94.4%
In our study, we evaluated HIV-positive individuals who
attended the São Paulo Hospital emergency room as well as
patients who were admitted to the São Paulo State AIDS/STD
Reference & Training Center with signs and symptoms that
suggested focal brain damage. These patients underwent
neuroradiological examination and were admitted to the
hospital due to suspected encephalic toxoplasmosis. Fluid
punction was performed soon after admission. The
presumptive diagnosis of these 16 patients was then confirmed
as prescribed by the Centers for Disease Control and
We applied real time PCR based on 200 to 300-fold
amplification of the T. gondii DNA 529bp fragment. This
fragment was selected not only because it is the least repeated
one, but also because it bears a more diverging sequence
standard, thus causing sensitivity to be greater than that
resulting from using the B1 gene [26,27].
Our group of patients with a presumptive diagnosis of
neurotoxoplasmosis displayed the typical signs and symptoms
of HIV-infected patients, particularly altered consciousness
(56.3%), motor deficit (50%), and headache (43.8%). However,
no such symptoms prevailed in the group with cerebral
toxoplasmosis when compared with patients with other
infections. We found no statistical difference related to cerebral
toxoplasmosis clinical conditions between PCR positive
patients and false negative patients.
As mentioned above, serum immunoglobulin (IgG and
IgM) assay for T. gondii immunoassay in patients with a
presumptive diagnosis was positive for all subjects, which
confirms that this group had previously been exposed to the
protozoa. Skull CT scan (both with and without contrast) and/
or MRI showed a lesion compatible with cerebral
toxoplasmosis (skull CT was performed in 100% of the patients,
while skull MRI was performed in 18.75% of patients). The
prevailing lesion found on neuroimaging examination was
related to contrasted annular granuloma.
The prescribed therapy was a sulfadiazine-pyrimethamine
combination. We considered a definite diagnosis of cerebral
toxoplasmosis as we observed patients’ clinical and imaging
improvement 14 days following specific therapy initiation.
We found 68.8% sensitivity, 100% specificity, 100%
positive predictive value, and 87.8% negative predictive value.
Gianotti et al. applied PCR (either conventional or real time)
for T. gondii in 52 patients with a presumptive diagnosis of
cerebral toxoplasmosis; they achieved 100% specificity, 16%
sensitivity, 100% positive predictive value, and 40% negative
predictive value .
Cingolani et al. found 33.3% sensitivity in a study carried
out on 88 HIV-1 infected patients, by applying nested PCR.
Another study, which also applied nested PCR showed 100%
sensitivity because CSF was collected during the first week
of therapy .
Real-Time PCR and Cerebral Toxoplasmosis
BJID 2009; 13 (February)
When we reviewed the low sensitivity attained among
our toxoplasmosis patients, we found that T-helper/CD4+
lymphocyte counts ranged from 7 to 212 cells/mm³ (mean 51.7
cells/mm³, median 27 cells/mm³) in the 11 patients with real
time PCR detection, whereas T-helper/CD4+ counts ranged
from 9 to 171 cells/mm³ (mean 98.2 cells/mm³, median 150 cells/
mm³) in patients with a presumptive diagnosis of cerebral
toxoplasmosis but no real time PCR detection. Even though
the mean and median T CD4 counts in the real time PCR group
was lower, such a gap was not found to be significant. Thus,
false negative PCR tests would not be justified by this
Another factor that could otherwise distinguish
undetected PCR patients was the amount of brain lesions
seen on neuroimaging examination of such patients, since
four of 10 showed a single lesion. We could therefore assume
that the low parasite load upon fluid punction (notwithstanding
the signs and symptoms compatible with cerebral
toxoplasmosis) eventually affected T. gondii DNA
amplification adversely in these patients, who coincidentally
showed improved immunity (CD4 counts greater than 100
cells/mm³). This may have had an effect on sensitivity in our
For ethical reasons, we were unable to carry out brain
biopsy in patients with a presumptive diagnosis of
neurotoxoplasmosis in order to reach a definite etiological
diagnosis. However, there was no positive result for real time
PCR among control patients, which ultimately confirms the
excellent test specificity (100%). Therefore, finding a positive
real time PCR test for T. gondii in CSF is of great diagnostic
relevance, particularly in patients whose CT scan or MRI does
not appear to be typical. In this case, one would have better
reasons as to whether or not carry out such an invasive
procedure as a CNS biopsy.
Given the small sample size in our study, we believe that it
should be enhanced by further investigation with a greater
number of individuals. Carrying out real time PCR in AIDS
patients with a suspected diagnosis of cerebral toxoplasmosis
may soon become a powerful diagnostic tool, thus preventing
patients from undergoing such a high morbidity-mortality
examination as brain biopsy. Performing T. gondii assay by
means of a less invasive method that delivers quicker results
such as real time PCR would certainly be a very useful tool in
the disease algorithm.
Fábio L. N. Nogui was supported by the Coordenação de
Aperfeiçoamento de pessoal de Nível Superior (CAPES) do
Ministério da Educação do Brazil. The authors wish to thanks
Dr. Mauro Figueredo and Dra. Kozue Myashiro at Fleury
Medicine and Health, São Paulo, SP for techical support.
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