The introduction of highly active antiretroviral therapy (HAART) has decreased the incidence of cerebral toxoplasmosis. Currently, the prevalence of AIDS-related focal brain disorders still accounts for a considerable proportion of mortality and morbidity, especially in developing countries (33
). In Brazil, cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS patients, and it is the third most frequent AIDS-defining condition (37
). In clinical practice, treatment for cerebral toxoplasmosis is usually initiated upon a presumptive diagnosis, which is based on clinical and radiological features (12
During the last decade, significant progress has been made in immunological and molecular techniques for the diagnosis of infectious diseases. Several studies, have demonstrated the usefulness of PCR on cerebrospinal fluid (CSF) samples for the diagnosis of cerebral toxoplasmosis (11
). However, a lumbar puncture could be contraindicated in a subgroup of patients with expansive cerebral lesions (10
). In this setting, peripheral blood samples present an additional advantage.
In this study, we evaluated the immunological and molecular diagnosis of cerebral toxoplasmosis using peripheral blood samples from Brazilian AIDS patients.
During the first years of the HAART era, the incidence of cerebral toxoplasmosis had declined in AIDS patients (2
). However, at present, its occurrence still represents a determinant of a poor diagnosis in the natural history of HIV-infected patients, even in the HAART era (3
). Cerebral toxoplasmosis has caused high morbidity and mortality, particularly in Brazilian AIDS patients (37
). For these reasons, it is necessary to evaluate accurate, less invasive, and rapid diagnostic tools.
The evaluation of molecular diagnosis in cerebral toxoplasmosis is normally done in CSF samples. In general, the sensitivity of the PCR test is extremely variable (11.5 to 100%), but the specificity is high (96 to 100%) (9
). Nevertheless, CSF collection is invasive and is inappropriate in a subset of patients with expansive cerebral lesions.
PCR in peripheral blood samples has also been used with several reported sensitivities (16 to 86%) (4
). In addition, methodologies have been developed using different sets of primers for different DNA targets and small numbers of samples (4
). The present study presents good sensitivity and specificity (80% and 98%, respectively) using a considerable number of samples (from 192 AIDS patients). The sensitivity and specificity percentages were similar to those in a previous study by our group using CSF samples (38
), even though DNA extraction from the blood samples was more complex than from CSF samples. These results may be explained as follows. First, the usefulness of the PCR assay using the B1 gene sequences as primers for detection of T. gondii
DNA has been demonstrated (5
). B22 and B23 primers were able to amplify and detect the DNA of a single organism directly from a crude cell lysate or 10 parasites in the presence of 100,000 human leukocytes (5
). The high sensitivity and specificity of this primer pair were previously reported in comparison with other T. gondii
sequence primers (8
). Second, all blood samples from patients with cerebral toxoplasmosis were collected before or on day 3 of specific therapy. Previous studies reported that anti-Toxoplasma
therapy decreases diagnosis sensitivity, especially if samples are collected after the first week of treatment (9
). Finally, most of the studied patients with cerebral toxoplasmosis presented severe immunodeficiency (less than 200 CD4+
T lymphocytes/μl). Some of them also presented disseminated forms and probably great parasite burdens. In this setting, performing PCR on blood samples seems to be more sensitive (24
). Three patients without cerebral toxoplasmosis presented positive PCR results. Two of them with cryptococcal meningoencephalitis were Toxoplasma
antibody positive. Another, with progressive multifocal leukoencephalopathy, was Toxoplasma
antibody negative. We cannot exclude the possibility that these patients carried T. gondii
and other microorganisms in their blood.
The laboratory conditions are also essential to raise PCR sensitivity and specificity in peripheral blood. To avoid false results and the action of any Taq polymerase inhibitor, three conditions are important: (i) the blood samples must be collected with EDTA and be processed rapidly, (ii) the plasma must be removed and the erythrocytes lysed rapidly with a specific buffer, and (iii) the lysed cells must be removed before lysis of cells with nucleus for DNA extraction. To determine whether any Taq polymerase inhibitor could change the actual results, PCRs were performed using a mixture of a negative DNA sample with a positive DNA (from the positive control) as a template. A positive result confirmed the absence of inhibitory substances.
Earlier studies showed that different levels of anti-T. gondii
IgG antibodies were unable to determine a reactivation or to follow the course of cerebral toxoplasmosis (26
). However, others have suggested that high titers in patients might be indicative of the presence of cerebral toxoplasmosis or a higher risk of developing the disease (13
). Despite these controversial data, our results confirm the clinical utility of measuring anti-T. gondii
IgG antibody levels in the diagnosis of cerebral toxoplasmosis. Patients with cerebral toxoplasmosis presented significantly higher anti-T. gondii
IgG titers measured by IF and ELISA than patients without cerebral toxoplasmosis. Although these antibodies suggest the neurological disease, all patients presented high-avidity IgG antibodies. These data support the idea that the reactivation of the latent infection observed in immunocompromised patients occurs in the secondary immune response (28
The majority of patients with cerebral toxoplasmosis seem to develop high titers of anti-T. gondii
antibodies, as confirmed here and by others (13
). However, six patients from group I presented positive PCR and negative ELISA and IF results (for both IgG and IgM antibodies). Thus, we emphasize that a negative serological result does not exclude a positive diagnosis (3
In conclusion, our results suggest that quantitative serology and PCR in blood can be useful in cerebral toxoplasmosis diagnosis. Although a definitive diagnosis of cerebral toxoplasmosis requires the demonstration of parasites by a histopathological procedure, the clinical and radiological data could be complemented by a less invasive approach, such as molecular and immunological diagnoses.