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Department of Parasitology, Instituto Adolfo Lutz
Departments of Infectious Disease
Neurology, Instituto de Infectologia Emílio Ribas, Sao Paulo, SP, Brazil
Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands
ABSTRACT
Cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS and still accounts for high morbidity and mortality in Brazil. Its occurrence is more frequent in patients with low CD4+ T-cell counts. It is directly related to the prevalence of anti-Toxoplasma gondii antibodies in the population. Therefore, it is important to evaluate sensitive, less invasive, and rapid diagnostic tests. We evaluated the value of PCR using peripheral blood samples on the diagnosis of cerebral toxoplasmosis and whether its association with immunological assays can contribute to a timely diagnosis. We prospectively analyzed blood samples from 192 AIDS patients divided into two groups. The first group was composed of samples from 64 patients with cerebral toxoplasmosis diagnosed by clinical and radiological features. The second group was composed of samples from 128 patients with other opportunistic diseases. Blood collection from patients with cerebral toxoplasmosis was done before or on the third day of anti-toxoplasma therapy. PCR for T. gondii, indirect immunofluorescence, enzyme-linked immunosorbent assay, and an avidity test for toxoplasmosis were performed on all samples. The PCR sensitivity and specificity for diagnosis of cerebral toxoplasmosis in blood were 80% and 98%, respectively. Patients with cerebral toxoplasmosis (89%) presented higher titers of anti-T. gondii IgG antibodies than patients with other diseases (57%) (P < 0.001). These findings suggest the clinical value of the use of both PCR and high titers of anti-T. gondii IgG antibodies for the diagnosis of cerebral toxoplasmosis. This strategy may prevent more invasive approaches.
INTRODUCTION
Toxoplasma gondii infection occurs worldwide, and it is one of the most common infections in humans. The infection is mainly acquired by ingestion of undercooked or raw meat containing viable tissue cysts or by ingestion of food and water that is contaminated with oocysts shed by cats. The course of the primary infection is usually subclinical. The vast majority of the infected human population remains asymptomatic, and some patients present mild symptoms. However, the infection can cause significant morbidity and mortality. The reactivation of latent infection occurs in immunocompromised patients, causing life-threatening disease, especially encephalitis (14, 19).
Cerebral toxoplasmosis is one of the most common opportunistic neurological infections in AIDS patients, and it is typically observed in the later stages of human immunodeficiency virus (HIV) infection (26). It is also directly related to the prevalence of anti-T. gondii antibodies in the general population (21).
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, 27).
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, 15, 23, 30, 36, 38). 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.
MATERIALS AND METHODS
Patients and blood samples. We analyzed blood samples from 192 AIDS patients admitted and treated at the Instituto de Infectologia Emilio Ribas, So Paulo, Brazil. The patients were divided into two groups. Group I consisted of 64 patients with cerebral toxoplasmosis, defined by the Centers for Disease Control and Prevention criteria. The criteria for AIDS-related cerebral toxoplasmosis included clinical and radiological features: (i) recent onset of a consistent focal neurological abnormality with intracranial disease or reduced level of consciousness; (ii) a lesion having a mass effect evidenced by brain imaging (on computed tomography or magnetic resonance imaging) or a lesion whose radiographic appearance was enhanced by injection of contrast medium. These diagnoses were associated with a successful response to the specific treatment (7). Blood samples were collected before or on the third day of specific therapy for toxoplamosis. Group II consisted of 128 patients with other diseases. Sixty-four presented with neurological diseases: 25 with cryptococcal meningoencephalitis, 7 with progressive multifocal leukoencephalopathy, 14 with central nervous system tuberculosis, 12 with HIV-associated cognitive motor disorder, and 6 with syphilitic meningitis. The other 64 patients presented with nonneurological diseases: 18 with pulmonary tuberculosis, 12 with bacterial pneumonia, 7 with oral candidiasis, 5 with diffuse lymphoma, and 22 with Pneumocystis carinii pneumonia. Group II was considered the control group. The definitive diagnosis of these patients was determined by clinical, radiological, and laboratory assessments. From each patient, 10 ml of peripheral blood with EDTA was collected for DNA extraction and 5 ml for immunological tests. No patient had been receiving HAART before the blood sample collection. The institutional review boards of the Ethics Committees of the Instituto de Infectologia Emilio Ribas and the Instituto Adolfo Lutz approved this study. All patients gave informed written consent.
Parasite preparation. T. gondii tachyzoites (RH strain) were grown and maintained in Swiss mouse ascites by intraperitoneal inoculation. Every 2 or 3 days after infection, the peritoneal fluids from infected mice were collected in phosphate-buffered saline (PBS), pooled, and centrifuged at 1,000 x g for 10 min. The parasite pellets were washed twice, counted, and suspended in PBS at a concentration of 2 x 107 cells/ml.
Antigens and immunological tests. Indirect immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA) were carried out as previously described (38). Both tests determined the presence or absence of anti-T. gondii immunoglobulin G (IgG) antibodies. Serum samples were used in serial dilutions and assayed in duplicate. For IF, the tachyzoites were incubated in 2% buffered formalin for 30 min at 37°C, washed twice in PBS at 1,000 x g for 10 min, and fixed on glass slides. The sera were diluted from 1:4 to 1:4,096, and the cutoff was considered to be 1:16. For ELISA, T. gondii antigen was obtained by sonication. The optical density (OD) cutoff was 0.143 at 492-nm wavelength. The starting serum dilution was 1:500, and it was assayed up to 1:128,000. Intermediate titers were considered to be between 1:32 and 1:512 in IF and between 1:500 and 1:2,000 in ELISA. High titers were considered to be up to 1:1,024 in IF and up to 1:4,000 in ELISA. The cutoff of both reactions was determined after testing 100 reactive and 200 nonreactive sera (data not shown). The Toxoplasma-specific IgG avidity assay was performed as described before (25, 28). The basic test used was ELISA. The differences were that (i) each serum sample was analyzed in two fourfold titration rows at a dilution of 1:500 and (ii) after 1 hour of incubation at 37°C, the first row was washed three times with 250 μl of 6 M urea in phosphate-buffered saline containing 0.05% Tween 20 in order to remove low-avidity antibodies from their binding sites. The control row was washed three times with buffer without urea. The IgG avidity index was calculated as the following ratio: (OD values under dissociative conditions)/(OD values of untreated control without urea) x 100. A low avidity index (15%) represented the predictive value for an infection of less than 5 months, and an index between 15 and 30% represented the predictive value for an infection of more than 5 months. A high avidity index (over 30%) determined a chronic infection.
DNA purification. The blood samples were centrifuged and washed with phosphate-buffered saline at 3,000 x g for 10 min. The supernatants with plasma were discarded. In order to lyse the erythrocytes, the packed cells were mixed with a 3x volume of a buffer containing 150 mM ammonium chlorate, 1 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.3; incubated for 15 min at room temperature with mild shaking; and centrifuged for 10 min at 3,000 x g. The pellet, containing only the cells with nucleus, were digested with proteinase K (100 μg/ml) in 50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 2% sodium dodecyl sulfate and incubated for 30 min at 56°C. DNA was extracted by the phenol-chloroform-isoamyl alcohol method and precipitated with isopropanol (35). After being washed with 70% ethanol for 10 min at 5,000 x g, the DNA pellet was dissolved in ultrapure water containing RNase at 20 μg/ml. For positive controls, DNA was extracted from tachyzoites of infected mice. The DNA concentrations were determined as the OD at 260 nm.
PCR. The amplifications were carried out with a kit from Amersham-Pharmacia-Biotech (Little Chalfont, Buckinghamshire, England) as described before (38). The primer pair used was B22 and B23, which amplified a 115-base-pair sequence in a specific repetitive region of the B1 gene (5, 6). Each amplification run contained two negative controls (ultrapure water and a negative DNA for toxoplasmosis) and one positive control. After the thermal cycles, the amplicons were electrophoresed in a 2% agarose gel and stained with ethidium bromide. The DNA fragments were visualized under UV illumination (35).
Statistical analysis. Predictive values, accuracy, sensitivity, specificity, and chi-square tests were performed with SPSS 10.0 software.
RESULTS
The results of the serological and molecular tests are shown in Table 1. In group I, 58 (91%) of the 64 patients with cerebral toxoplasmosis had detectable Toxoplasma-specific antibodies. T. gondii DNA was detected by PCR in 51 (79.7%) of 64 blood samples. In group II, 72 (56%) of 128 AIDS patients without cerebral toxoplasmosis had Toxoplasma-specific IgG antibodies. Three of the 128 patients were found to be PCR positive. Two of the three were Toxoplasma antibody positive. Positive and negative predictive values for PCR were 94.4% and 90.6%, respectively, with 91.7% accuracy. In both groups, all patients with Toxoplasma-specific antibodies presented high avidity indexes. Figure 1 presents data on the levels of antibody detection in both groups. Patients with cerebral toxoplasmosis (89%) presented higher titers of anti-T. gondii IgG antibodies than patients with other diseases (57%) (P < 0.001).
DISCUSSION
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, 11, 15, 29, 30, 36, 38). 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, 16, 17, 24, 31, 32). In addition, methodologies have been developed using different sets of primers for different DNA targets and small numbers of samples (4, 38). 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, 20, 22, 38). 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, 27, 34). 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, 18). 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, 18). 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, 34).
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.
ACKNOWLEDGMENTS
We thank M. C. D. S. Fink and C. S. Pannuti from Virology Laboratory, Instituto de Medicina Tropical, USP, for carrying out the JC virus diagnosis.
This work was supported by a grant from Fundaao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-03/12307-1). F.A.C. and J.E.V. contributed equally to this work and were supported by fellowships from "Coordenaao de Aperfeioamento de Pessoal de Nível Superior (CAPES) do Ministerio da Educaao do Brazil".
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