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Research Article

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Rafael T. Mesquita, Ângela P. Ziegler, Roberto M. Hiramoto, Jose E. Vidal, Vera L. Pereira-Chioccola

  • 1Laboratório de Parasitologia, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil
  • 2Departamento de Neurologia, Instituto de Infectologia Emílio Ribas, Sao Paulo, SP, Brazil
  • 3Serviço de Extensão ao Atendimento de Pacientes HIV/AIDS, Divisão de Moléstias Infecciosas e Parasitárias, Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, SP, Brazil
  • Correspondence
    Vera L. Pereira-Chioccola
    pchioccola{at}ial.sp.gov.br

    • Journal of Medical Microbiology 2010; 59(6):641–647 · https://doi.org/10.1099/jmm.0.016261-0

    View at publisher PubMed

    Abstract

    Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis. The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe. One was B1Tg, which amplified a sequence from the B1 gene. The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were: positive results were observed in 33.6 % (50) patients. The sensitivities were 98 % for cnPCR (B22/B23), and 86 and 98 % for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66.4 % patients. The specificities were 97 % for cnPCR and qrtPCR (B1Tg), and 88.8 % for RETg. These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers. However, B1Tg PCR had good specificity, but lower sensitivity. Among the patients, 20 had blood and CSF collected simultaneously. Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive); (iii) positive molecular diagnosis with CSF and negative with blood; and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients. Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples. qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

    INTRODUCTION

    Toxoplasma gondii is a widely distributed apicomplexan parasite of great medical importance infecting all mammalian cells. During acute infection, T. gondii differentiates into tissue cysts that reside in the muscles and brain. Most primary infections are asymptomatic, but some symptomatic chronic forms exist as ocular and disseminated infections (Dubey, 1996, 1998; Montoya & Liesenfeld, 2004; Hill et al., 2005). However, symptomatic forms are frequent when primary infection occurs during pregnancy, which can result in severe neonatal malformations and ocular complications in the fetus. In human immunodeficiency virus (HIV)-infected patients, low levels of CD4+ T cells allow reactivation of the latent infection, parasite proliferation and result in symptomatic disease such as cerebral toxoplasmosis, a life-threatening condition without timely diagnosis and treatment (Luft & Remington, 1992; Cohen, 1999; Montoya & Liesenfeld, 2004; Hoffmann, 2005).

    Cerebral toxoplasmosis remains a prevalent disorder of the central nervous system, particularly among severely immunosuppressed HIV-infected patients in the absence of antibiotic prophylaxis (Antinori et al., 2004). Its incidence among HIV-infected individuals directly correlates with the prevalence of anti-T. gondii antibodies among the general population. It represents the most common cerebral mass lesion in AIDS patients and is the third-most-frequent condition associated with AIDS in Brazil, still accounting for high mortality and morbidity, despite free access to highly active antiretroviral treatment (HAART) (Vidal et al., 2005).

    The use of molecular diagnosis is particularly appropriate for such patients, since it does not depend on the immunological status of the host. Molecular diagnosis based on conventional PCR (cnPCR) has become an indispensable tool for toxoplasmosis diagnosis (Bretagne, 2003; Vidal et al., 2004; Colombo et al., 2005; Bastien et al., 2008). However, we have reported some difficulties in its use in laboratory practice. Parasite levels in blood and, principally, cerebrospinal fluid (CSF) are very poor in some patients. Consequently, a reduced amplification of cnPCR products is seen in the agarose gels resulting in false readings due to difficulty in interpreting the cnPCR product.

    In recent years, real-time quantitative PCR (qrtPCR) has been introduced for molecular diagnosis of diverse diseases and has been used frequently in medical centres (Espy et al., 2006; Bastien et al., 2008). Several studies have demonstrated the applicability of qrtPCR for toxoplasmosis diagnosis, and others compared the different targets (Costa et al., 2000; Buchbinder et al., 2003; Hierl et al., 2004; Edvinsson et al., 2006; Brenier-Pinchart et al., 2007). Among them, two targets are more frequently used because of their high sensitivity and specificity. One is the 529 bp sequence, which has 200–300 copies in the genome of T. gondii (RE) (Homan et al., 2000). The other is the B1 gene that has 35 copies in the genome and is conserved in different parasite strains (B1) (Burg et al., 1989). The sensitivity, accuracy and comparison of the targets, RE and B1 gene, for qrtPCR have been well analysed (Costa et al., 2000; Hierl et al., 2004; Reischl et al., 2003; Chabbert et al., 2004; Cassaing et al., 2006; Calderaro et al., 2006; Edvinsson et al., 2006). The majority of these studies aimed to compare the sensitivities of both DNA regions were conducted using European clinical samples; however, Brazilian samples are poorly studied. Recently, we have shown that targets for both B1 and RE in cnPCR were highly sensitive for T. gondii determination although B22/B23 (for B1) was the best (Pereira-Chioccola et al., 2009). In order to investigate the capacity of qrtPCR for determining the molecular diagnosis of cerebral toxoplasmosis, we have reported here the evaluation of qrtPCR of blood and CSF samples from AIDS patients in Brazil. We investigated two primer sets from regions highly repetitive in the T. gondii genome (B1 and RE), and the results allowed for the discussion of the different cerebral toxoplasmosis diagnoses that occur in laboratory practice

    METHODS

    Clinical samples.

    This prospective study was conducted for 2 years (June 2007 to May 2009). Samples received by our laboratory from patients suspected of having cerebral toxoplasmosis were tested during routine diagnosis. From each patient, 3 ml CSF and/or 5 ml peripheral blood with EDTA were collected for DNA extraction. Sera for serological diagnosis were obtained from 5 ml blood. No patient had received HAART before the blood sample collection. The tests included cnPCR using the primer pair B22/B23, which amplified a 115 bp sequence of B1 gene (Burg et al., 1989) and indirect immunofluorescence (IF). Simultaneously two qrtPCR protocols were tested. Sensitivity and specificity were evaluated from the analysis of DNA from 149 AIDS patients (98 blood and 51 CSF samples). Cerebral toxoplasmosis was confirmed in 50 patients (36 blood and 14 CSF samples) by clinical and radiological diagnosis. The case definition for toxoplasmosis included the following criteria: (i) progressive neurological deficits; (ii) contrast-enhancing mass lesion(s) on computed tomography scans; (iii) a successful response within 2 weeks to specific treatment (Portegies et al., 2004; Vidal et al., 2005). Other diseases such as cryptococcal meningoencephalitis, progressive multifocal leukoencephalopathy, HIV-associated cognitive motor disorder, pulmonary tuberculosis, bacterial pneumonia, cerebral tuberculosis, chronic renal deficiency, hepatitis C, cytomegalovirus encephalitis, epilepsy, histoplasmosis and oral candidiasis were confirmed in 99 patients (62 blood and 37 CSF samples). The ethics committees of both institutions approved this study.

    T. gondii DNA.

    The RH strain was grown and maintained in Swiss mice by intraperitoneal inoculation. Tachyzoites were harvested from the peritoneal cavities in PBS, pooled, and then centrifuged and washed twice, with centrifugation at 2000 g for 10 min. The parasite pellets were used for DNA extraction. For IF antigens, the parasite pellets were washed twice, counted and resuspended in PBS at a concentration of 2×107 cells ml−1. The tachyzoites were incubated in 2 % buffered formalin for 30 min at 37 °C, washed twice in PBS, with centrifugation at 1000 g for 10 min and then fixed on glass slides.

    IF.

    IF was carried out as previously described (Colombo et al., 2005) and was used to determine the presence or absence of anti-T. gondii IgG and IgM antibodies. Serial dilutions of the samples were used and samples were assayed in duplicate. The sera were diluted from 1 : 4 to 1 : 4096, and the cut-off was considered to be 1 : 16. The CSF samples were used and diluted to 1 : 64. Dilutions more than 1 : 4 were considered reactive.

    DNA purification.

    DNA from blood samples was extracted using a PureLink genomic DNA kit (Invitrogen) according to the manufacturer's instructions. Previously, blood samples were centrifuged at 2500 g for 10 min and washed with PBS. The supernatants with plasma were discarded. In order to lyse the erythrocytes, the packed cells were mixed with a three times 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 under mild shaking and centrifuged for 10 min at 3000 g. The blood pellets, containing only nuclei cells were digested with proteinase K (20 μg ml−1) in 50 mM Tris/HCl, 25 mM EDTA, pH 8.0, 2 % SDS and incubated for 30 min at 56 °C. DNA pellets were dissolved in ultrapure water. As a positive control, DNA was extracted from the tachyzoite pellets using the kit. DNA from CSF samples was extracted as described previously (Vidal et al., 2004). Samples were centrifuged for 10 min at 3000 g. CSF supernatants were used in serological tests for anti-T. gondii antibody determination. After washing the packed cells twice in PBS, whole cells were lysed by incubation for 5 min at 100 °C in 50 μl ultrapure water. DNA purity was determined by the ratio of the optical density at 260 and 280 nm in a NanoDrop ND100 (Thermo Scientific) spectrophotometer.

    Primer selection.

    The primer pairs used for cnPCR were: (1) B22/B23 (5′-AACGGGCGAGTAGCACCTGAGGAGA-3′ and 5′-TGGGTCTACGTCGATGGCATGACAAC-3′), which amplified a 115 bp sequence from a specific repetitive region of the B1 gene (Burg et al., 1989); (2) β1/β2 (5′-ACCACCAACTTCATCCACGTTCACC-3′ and 5′-CTTCTGACACAACTGTGTTCACTAGC-3′) (Lee et al., 2001), which amplified a 140 bp fragment of the human β-globulin gene, and was used to assay all samples to control the course of extraction and check for PCR inhibitors. The reactions using the human marker were run simultaneously with the same temperature protocol for the B22/B23 primer and in the same PCR machine. For qrtPCR, two primer sets were selected. One directed to B1: B1Tg-F (forward) 5′-CAAGCAGCGTATTGTCGAGTAGAT-3′; B1Tg-R (reverse) 5′-GCGTCTCTTTCATTCCCACATTTT-3′; and B1Tg-TM [TaqMan probe, 6-carboxyfluorescein (FAM) dye-labelled] 5′-FAM-CAGAAAGGAACTGCATCCGTT-3′). The second was directed to RE: RETg-F (forward) 5′-GGCGAGGGTGAGGATGAG-3′); RETg-R (reverse) 5′-CATCTTCTCCCTCTCCGACTCT-3′; and RETg-M (TaqMan probe, FAM dye-labelled) 5′-FAM-TCGCTTCCCAACCACG-3′). Both primer sets had NFQ (non-fluorescent quencher) as a reporter quencher and the location of the sequences in the T. gondii genome is shown in Fig. 1⇓. The primer design was carried out with Primer Express software from Applied Biosystems.

    Figure image not available in archive
    Fig. 1.

    Details of the primer sets used in qrtPCR. (a) T. gondii B1 gene partial sequence 2214 bp (GenBank accession no. AF 179871). The B1Tg primer sequences are located between nucleotides 664 and 747. The forward (B1Tg-F) and reverse (B1Tg-R) primers are shown in bold and underlined. The location of the FAM dye-labelled TaqMan probe (B1Tg-M) is shown in bold, italic and boxed text. (b) T. gondii repeat region linear 529 bp DNA (GenBank accession no. AF146527). RETg is located between nucleotides 270 and 334. The forward (RETg-F) and reverse (RETg -R) primers are shown in bold and underlined. The location of the FAM dye-labelled TaqMan probe (RETg-M) is shown in bold, italic and boxed text.

    cnPCR.

    The reactions were carried out in a LongGene thermal cycler in a final volume of 25 μl. The DNA samples (5 μl for blood or 10 μl for CSF), or the controls, and 25 pmol each primer were added to reagents from a kit purchased from Promega (GoTaq Green master mix). The PCR mix (12.5 μl) was composed of 1 unit Taq DNA polymerase, 10 mM Tris/HCl pH 8.5, 50 mM KCl, 1.5 mM MgCl2 and 200 mM each dNTP. The amplification run contained two negative controls (ultrapure water and DNA from a sample from a toxoplasmosis-negative patient) and one positive control (DNA extracted from the RH strain). The thermal cycling conditions were: 1 initial denaturation cycle at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 62 °C for 1 min and extension at 72 °C for 1 min. The procedure was completed with a final extension cycle at 72 °C for 5 min. The PCR products were electrophoresed in 2 % agarose gel and stained with ethidium bromide. The stained DNA fragments were visualized under UV illumination. The images were analysed by a MiniBIS gel imager and documentation system (BioSystematica). The size of fragments was determined based on comparison with a 100 bp ladder.

    qrtPCR.

    The reactions were performed with an Applied Biosystems 7500 real-time PCR system in a final volume of 20 μl. The clinical samples or control DNA (3 μl) were added to a reaction mixture containing 10 μl 2×TaqMan Universal PCR master mix, and 1 μl ‘assay mix’ that included (i) the forward primer (18 μM), (ii) the reverse primer (18 μM) and (iii) the TaqMan FAM dye-labelled (5 μM) MGB (minor groove binder) probe. Amplification runs contained two negative controls and one positive control, as used for cnPCR. The thermal cycling conditions included a 2 min 50 °C step for optimal AmpliErase UNG activity and denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Reactions were previously standardized using serial T. gondii DNA dilutions as template DNA. The cycle threshold value, indicative of the quantity of target gene at which the fluorescence exceeds a preset threshold, was automatically determined by the equipment.

    Data analysis.

    All divergent results were repeated at least twice. Clinical and laboratory (IF and cnPCR) diagnoses were used to establish sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs), and were calculated as: (i) per cent of sensitivity – ratio of true positives/true positives+false negatives×100; (ii) per cent of specificity – ratio of true negatives/true negatives+false positives×100; (iii) PPV – ratio of true positives/(true positives+false positives); and (iv) NPV – ratio of true negatives/(true negatives+false negatives).

    RESULTS

    The objective of this study was to investigate two primer sets for qrtPCR and to compare them with cnPCR in routine molecular diagnosis. We used the B22/B23 primer set in cnPCR, which has been highly evaluated in our laboratory (Vidal et al., 2004; Colombo et al., 2005). For qrtPCR we chose two primer sets from two described genes. One was the B1Tg set, which amplified a (partial) sequence from B1 gene (GenBank accession number AF179871) (Burg et al., 1989) with 35 copies in the genome. The other was the RETg set, which amplified a sequence from the 529 bp sequence (GenBank accession number AF146527) (Homan et al., 2000) that has 200–300 copies in the genome (Fig. 1⇑).

    The overall cnPCR and qrtPCR results of the 149 DNA samples were evaluated to determine the sensitivity, specificity, PPV and NPV for each individual primer set. Among the 98 DNA samples extracted from blood, positive results and sensitivity were observed in 35 (97.2 %), 32 (88.9 %) and 35 (97.2 %) samples for B22/B23, B1Tg and RETg, respectively. The sensitivities for the 51 CSF samples were 78.6 % (11) for B1Tg, and 100 % (14) for B22/B23 and RETg. When the 149 samples were analysed together, the sensitivities were 98.0 % (49) for B22/B23 and RETg, and 86.0 % (43) for B1Tg. According to the specificity, in blood, the reactions using the primer sets B22/B23, B1Tg and RETg were negative in 61 (98.4 %), 61 (98.4 %) and 55 (88.7 %) samples, respectively. The results for the CSF samples were 35 (97.2 %) in B22/B23 and RETg, and 33 (91.7 %) in B1Tg. The specificities of all 149 samples were 97.0 % (96) for B22/B23 and B1Tg, and 88.8 % (88) for RETg. Despite the high sensitivity of RETg (98 %), its specificity (88.8 %) and NPV (0.900) were lower than B22/B23 and B1Tg (0.970). However, B1Tg had the lowest sensitivity (86.0 %) and PPV (0.877). Table 1⇓ shows the results of each individual primer set in detail.

    Table 1.

    Molecular diagnosis for cerebral toxoplasmosis in 149 AIDS patients employing primer sets B22/B23 (cnPCR), and B1Tg and RETg (qrtPCR): sensitivity, specificity, PPVs and NPVs

    The PPV, NPV, sensitivity and specificity for each primer set were determined considering the clinical diagnosis described in Methods.

    Among the patients, 20 had blood and CSF collected simultaneously. Thus, their results permitted us to analyse and compare the molecular, serological and clinical information of the same patient. Nine patients were diagnosed as having cerebral toxoplasmosis but the laboratorial diagnosis differed, and four situations can be seen in Table 2⇓. The first and second cases had negative molecular diagnosis for CSF and positive for blood. IF titres can be variable in CSF, as shown for case 1 (negative) and case 2 (positive). Thus a negative serological profile does not exclude the presence of the active infection, principally when the serological diagnosis is made using CSF, since its value in CSF is limited because the sensitivity and specificity are around 60 to 70 % (Collazos, 2003). The third case had a positive molecular diagnosis for CSF and a negative diagnosis for blood. The fourth case had a positive molecular diagnosis for both samples. IF titres in sera and CSF can be variable, high as in case 3, or low as in case 4.

    Table 2.

    Clinical and laboratory diagnosis determined in patients with AIDS and cerebral toxoplasmosis (cases 1 to 4) or other opportunistic infections (cases 5 to 8)

    Blood and CSF samples were collected simultaneously.

    The other 11 patients had other neurological opportunistic infections and cases 5, 6, 7, 8 illustrate the negative. Although all samples had negative molecular diagnosis, IF titres in sera and CSF were variable: negative (case 5), high (case 6) or low (case 7) titres. Negative IF for CSF and positive for sera were also shown (case 8).

    DISCUSSION

    In recent years, great strides have been made in developing laboratory techniques for diagnosing infectious diseases including toxoplasmosis. Nevertheless, the diagnosis of cerebral toxoplasmosis in HIV-infected patients still presents difficulties. In different clinical centres, specific treatment is usually initiated according to a presumptive diagnosis, which is based on clinical and radiological findings (Luft & Remington, 1992; Vidal et al., 2005). However, the importance of additional laboratory methods, principally molecular ones, is incontestable. Thus, the evaluation of such methods for diagnosis seems to be important. As a result, several European studies have demonstrated qrtPCR applicability in toxoplasmosis diagnosis (Costa et al., 2000; Buchbinder et al., 2003; Hierl et al., 2004; Edvinsson et al., 2006; Brenier-Pinchart et al., 2007). As similar findings may be expected for Brazilian patients, the present study investigated the diagnostic value of qrtPCR, in this population, using primer sets constructed to amplify products of the B1 and RE regions known to be highly repetitive in the T. gondii genome.

    As a laboratory control, we used cnPCR (with B22/B23 primers), which is rated highly and has been used routinely for molecular diagnosis in our laboratory (Vidal et al., 2004; Colombo et al., 2005). For qrtPCR evaluation, we analysed the B1Tg primer set that amplified a sequence from the B1 gene partial sequence (GenBank accession number AF179871) (Burg et al., 1989), and RETg that amplified part of the 529 bp sequence (GenBank accession number AF146527) (Homan et al., 2000). The primer sets chosen were joined to the procedure chequerboard as DNA extraction, cnPCR and qrtPCR, aiming at optimal working conditions. In order to improve the sensitivity and specificity, the clinical samples were processed rapidly within 48 h of collection to prevent Taq polymerase inhibition. Many components found in biological samples such as blood and CSF inhibit Taq polymerase, modifying PCR results (Al-Soud & Rådström, 1998). As the amount of human chromosomal DNA and the pathogen can vary from sample to sample, and interfere in the results, all samples were quantified after extraction. Therefore, the DNA extraction and PCR inhibitors were evaluated by a β1–β2 marker that amplified a PCR fragment of human β-globulin gene. Positive amplifications showed that no substance present in the DNA samples inhibited the reaction. The clinical centres were informed to collect blood or CSF from the suspected patients before or during the first 3 days of the specific therapy (Colombo et al., 2005). Previous studies reported that anti-toxoplasmic therapy decreases diagnostic sensitivity, especially if samples were collected after the first week of treatment (Cingolani et al., 1996; Gianotti et al., 1997).

    Molecular diagnosis using CSF or peripheral blood samples is a useful tool for early minimally invasive diagnosis of cerebral toxoplasmosis (Bretagne, 2003; Bastien, 2002; Colombo et al., 2005). However, results should be always interpreted in association with serological, clinical and radiological information (Pereira-Chioccola et al., 2009).

    This prospective study analysed a considerable number of clinical samples (149 patients with confirmed clinical and radiological diagnosis). Among them, 20 patients had two samples collected simultaneously, one from blood and another from CSF, allowing for better understanding of the different scenarios of laboratory and clinical diagnosis. This small group of patients serves to illustrate different situations. The results of the patients with cerebral toxoplasmosis allowed for concluding that divergent situations can be observed in laboratory and clinical practice of this opportunistic infection. Some patients may have a negative molecular diagnosis for CSF and positive for blood, and the anti-T. gondii antibodies may be variable. Others can present with a positive molecular diagnosis for CSF and a negative one for blood; or positive molecular diagnosis from both samples with the frequent presence of high titres of anti-T. gondii antibodies. However, a small proportion of these patients had negative serological diagnoses (Meira et al., 2008). According to a recent Brazilian study, patients with four or more brain lesions present greater positivity in molecular diagnosis (Correia et al., 2010).

    The patients with other opportunistic diseases had negative molecular diagnosis for both clinical samples despite the fact that some of them presented anti-T. gondii antibodies for sera or CSF. The analysis of the differential diagnosis of expansive brain lesions in HIV patients is extremely significant because it determines the effectiveness of specific treatment. The majority of the samples analysed in this study were negative (66.4 %). cnPCR employing the B22/B23 primer set presents a high specificity, as repeatedly shown before, including for Brazilian samples (Bretagne, 2003; Vidal et al., 2004; Colombo et al., 2005; Okay et al., 2009; Correia et al., 2010), and has been used in different laboratories. The same high specificity was observed in qrtPCR using the B1Tgt set whose target region is the B1 gene as for B22/B23. These data confirmed those from other studies (Raffi et al., 1997; Bretagne, 2003; Reischl et al., 2003; Nagy et al., 2006; Okay et al., 2009; Correia et al., 2010), but RETg, whose target region is the 529 bp fragment, presented lower specificity (88.8 %) and NPV (0.908) than two other sets from the B1 gene.

    Among all studied samples, 33.6 % (50) were positive. cnPCR sensitivity was 98.0 %, as shown before in samples from our laboratory (Colombo et al., 2005; Vidal et al., 2005) and others (Contini et al., 2005; Okay et al., 2009). A similar high sensitivity was shown when the RETg set was used. These data were extensively confirmed by published studies (Homan et al., 2000; Costa et al., 2000; Calderaro et al., 2006; Cassaing et al., 2006). As RETg PCR amplified a region in the T. gondii genome 10 times more repetitive than the B1 gene, the arithmetic mean of the samples cycle threshold value was lower (27.11) than that shown for B1Tg (32.52) (data not shown). In addition, B1Tg had the lowest sensitivity (86.0 %), as has been shown in other studies (Bretagne, 2003; Reischl et al., 2003; Cassaing et al., 2006; Correia et al., 2010).

    As this study analysed a considerable number of samples under the same laboratory conditions, only varying the primer sets and technology (cnPCR and qrtPCR), we can conclude that RETg PCR was highly sensitive as it amplified a repeat region with many copies. However, its specificity was lower than that of the other markers. Around 10 % of the negative samples can present positive results when this primer is used (NPV=0.900), principally in DNA extracted from blood samples. Similar results were shown recently by Wahab et al. (2010). However, B1Tg had good specificity, but lower sensitivity (86 %) compared with the other markers. Negative results were seen in approximately 12 % of the samples from patients with cerebral toxoplasmosis (PPV=0.877).

    From our point of view, even though the number of copies of the DNA target in the genome of the micro-organism is an essential factor that affects sensitivity (Bastien et al., 2008), the specificity is also extremely important. qrtPCR allows for rapid identification of T. gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

    Acknowledgments

    This study was supported by grants from the FAPESP (Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo, Brazil) proc-08/09311-0. R. T. M. was supported by fellowships from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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