This retrospective and prospective study evaluated clinical samples from 807 patients. Instituto Adolfo Lutz (São Paulo, Brazil) received these samples from January 2008 through November 2016 for molecular diagnosis of toxoplasmosis. Clinical samples originated from patients with known final clinical diagnosis were chosen. The clinical samples analyzed during this period were as follows: 173 (2008–2010), 204 (2011–2013), 138 (2014–2015), and 292 (2016). These clinical samples sent to the laboratory included CSF (302), BL with EDTA (439), AF (47), and formalin-fixed paraffin-embedded (FFPE) tissues sectioned in 4-μm-thick (19) (AU). For serological tests, BL samples were collected in tubes with no anticoagulants. Clinical samples for PCR and serology were sent to the laboratory within 48h after collection, and immediately processed. In the retrospective part of this study, the routine diagnosis of 515 clinical samples collected until 2015 included conventional PCR (cPCR) and enzyme-linked immunosorbent assay (ELISA). In the prospective study, the 292 clinical samples sent to the laboratory in 2016 were analyzed by qPCR using the primers sets REP-529 and B1.

The samples evaluated in this study were from symptomatic patients with the following clinical diagnosis of toxoplasmosis: cerebral (404 HIV-patients), ocular (193 with ocular alterations), congenital infection by transplacental route (125 newborns), gestational infection (66 pregnant women), and disseminated (19 brain autopsies from deceased HIV-patients).

HIV-infected patients with suspicion of cerebral toxoplasmosis presented progressive neurological deficits and contrast-enhancing mass lesion(s) on computed tomography or magnetic resonance imaging. These cases were included in “definition for cerebral toxoplasmosis” previously described.6,15 Those who died from severe disseminated toxoplasmosis had positive serology and immunohistochemistry for toxoplasmosis of at least two organs. Both group of patients were admitted and treated at the Instituto de Infectologia Emilio Ribas by Dr Jose E. Vidal's group. Patients with suspicion of ocular toxoplasmosis had ocular alterations, such as toxoplasmic retinochoroiditis scars or retinal exudative lesions. They were admitted and treated at Ambulatorio de Oftalmologia, by Dr Fábio B. Frederico's group. Pregnant women with suspicion of acute toxoplasmosis infection were admitted and treated at the High Risk Antenatal Care and Fetal Medicine Service. After enrolling in a high-risk pregnancy clinic, pregnant women were routinely screened for TORSCH (Toxoplasmosis, Rubella, Syphilis, Cytomegalovirus, Hepatitis and HIV).36,37 Those clinically, epidemiologically suspected of toxoplasmosis and having positive IgM or low IgG avidity for anti-T. gondii antibodies underwent amniocentesis for PCR testing in amniotic fluid. Before collection pregnant women were informed of the procedure and required to sign informed consent forms. The newborns of these mothers with suspected or confirmed fetal infection were admitted and treated at the High Risk Pediatric Service and Fetal Medicine Service. Mothers and newborns were treated by Dr Lígia CJF Spegiorin's group.

T. gondii lysate antigen and ELISA were performed exactly as described before.19,38,39

BL samples (5mL) were prepared as described before.28 Plasma samples were separated by centrifugation and used in serological diagnosis. Pellets were mixed with three times the volume of a buffer containing 150-mM ammonium chlorate, 1-mM potassium bicarbonate, 0.1-mM EDTA, pH 7.3, incubated for 15min at room temperature under mild shaking and centrifuged (2500g/10min). AF samples (10mL) were centrifuged (2500g/10min) and the supernatant was discarded. Blood pellets, containing only nuclei cells, AF packed cells and crushed tachyzoites (for control and standard curves) were digested for 10min at 56°C with proteinase K (20μg) in 200μL of AL buffer (Qiagen). AU fragments (5 sections of 4-μm-thick FFPE) were dissolved in xylene (1mL), incubated for 30sec, centrifuged twice (8000g/1min) and supernatants were removed. Pellets were mixed with 1mL ethanol, centrifuged (8000g/1min) and incubated for 10min, at room temperature for complete xylene evaporation.40

DNA molecules were extracted from BL, AF, and tachyzoites by QIAamp DNA Mini Kit (Qiagen); and from AU, by QIAamp DNA FFPE Tissue kit (Qiagen). The protocols were followed according to the manufacturer's instructions in a Robotic workstation for automated purification of DNA (QIAcube, Qiagen). DNA molecules from CSF samples (3mL) were extracted as described before.41 DNA concentrations and purity were determined by the ratio of O.D. at 260 and 280nm in NanoDrop ND1000. For use in PCR, samples with high DNA concentrations (as some BL and AU) were diluted with ultra-pure water until concentration 100ng/μL.

T. gondii was identified by cPCR using the primer pair B22-B23,31 designed to amplify a 115-bp amplicon from the B1 gene as target. The reactions were run following the same conditions described elsewhere.42 Absence of PCR inhibitors was verified using a housekeeping gene that amplified a 140bp fragment of the human β-globulin gene.43 Each amplification run contained two negative controls (ultra-pure water and negative DNA for toxoplasmosis) and one positive (DNA extracted from tachyzoites). After thermal cycles, PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide and visualized under UV illumination.

The performance of two different primer sets for qPCR was analyzed. The first (B1) amplified a 71-bp fragment of the B1 gene as template,29,35 which has 35 copies in the genome and is conserved in different parasite strains.31 The design included the forward (5′-GAAAGCCATGAGGCACTCCA-3′) and reverse (5′-TTCACCCGGACCGTTTAGC-3′) primers; and a hybridization probe (5′-CGGGCGAGTAGCACCTGAGGAGATACA-3′) labeled with FAM (6-carboxyfluorescein) and BHQ1 (Black Hole Quencher 1) at the 5′ and 3′ ends, respectively.

The second (REP-529) amplified a 112-bp of the highly repetitive sequence REP-529 (Genbank AF487550),23,29 which has 200–300copies in T. gondii genome.30 The design included the forward (5′-AGAGACACCGGAATGCGATCT-3′) and reverse (5′-TTCGTCCAAGCCTCCGACT-3′) primers; and the hybridization probe (5′-TCGTGGTGATGGCGGAGAGAATTGA-3′) labeled with FAM and BHQ1. The design and location in the gene of each primer pair is described in Fig. 1. Both molecular marker sequences were previously standardized in our laboratory using serial dilutions of DNA from tachyzoites (RH strain) as template. To confirm the absence of PCR inhibitors, a primer set to a Eukaryotic 18S rRNA gene (GenBank accession code X03205.1), purchased from Applied Biosystems was used as housekeeping gene. The reactions were performed in final volume of 20μL. DNA samples (3μL of DNA until 100ng/μL), DNA control (5ng/μL) or DNA for standard curve (3μL for each point) were added to reaction mixture containing 10μL of 2X TaqMan Universal PCR Master Mix. Next, 1.25μL of “Assay Mix” (18μM of forward and reverse primers and 5μM of the hydrolysis probe). Amplifications were performed in an Applied Biosystems 7500 Real-time PCR System using the following thermal profile: 2min, 50°C, and 95°C for 10min. Next, 40 cycles were performed at 95°C for 15sec and 60° C for 1min.

Fig. 1.

Design and genes location of primer sets used in this study. (A) cPCR (B22-B23), (B) qPCR B1, and (C) qPCR REP-529. The analyses were done in https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctg_time=1486921082&job_key=1N4LxE9DQutl1VjQVbB84i-rbdACuHbNAw&CheckStatus=Check for B22/B23; https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctg_time=1486924834&job_key=l51I5ugp5YHCu3W-eN5RjALFQL4v1lujLg for (B1), and https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctg_time=1487009304&job_key=ycMWuJUKmKK_mAidBf0sr3_mPZ1S9SaAUw&CheckStatus=Check for REP-529. The schemes were based on Lin et al.46

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Two T. gondii standard curves using REP-529 and B1 primer sets were constructed in nine concentrations of DNA obtained from 1×107 tachyzoites (in triplicate). Initial concentration (1×107 tachyzoites) was 35ng/μL and serial dilutions ranged up to 10−1 tachyzoites (3.5fg). The cycle threshold values (CT) were plotted as mean (triplicate) against the standard curve values to determine the detection limit of both primer sets. Parasite concentrations were determined after the calculation of the linear regression equation (y=ax+b), where y=CT; a=curve slope (slope); x=parasite number; and b=where the curve intersects y-axis (y intercept).44

In each PCR run, a blank control was used consisting of DNA-free water plus the PCR mix. Separate rooms were used for (i) DNA extraction; (ii) PCR mix and primer preparation; (iii) adding DNA from clinical samples and positive control; and (iv) post-PCR agarose gel electrophoresis analysis in case of the cPCR. DNA samples were assayed in triplicate and, at least twice to determine reproducibility. The quality of DNA samples stocked for nine years and retrospectively re-analyzed with qPCR was confirmed by the positivity using the 18S primer set.

The discordant results in qPCR were re-analyzed, using the same protocols, at least twice. Sensitivities and specificities of both primer sets in clinical samples were determined considering the true diagnosis (clinical and laboratory diagnoses) and were calculated as: (i) percent of sensitivity=ratio of true positives/true positives+false negatives×100; (ii) percent of specificity=ratio of true negatives/(true negatives+false positives)×100. Reproducibility calculations were done exactly as described before.45 The percentages of concordant indices were calculated as the following ratio: (number of concordant results)/(number total of samples)×100. Linear regressions were constructed from the standard curves for REP-529 and B1 and CT mean comparison of both primer pairs. Both curves were statistically analyzed using an unequal-variance t-test based on a critical value of p≤0.05. All calculations were run in GraphPad Prism 6.0 software.

The first step was to evaluate the reportable range of the reaction that was calculated using DNA extract from tachyzoites (RH strain) using the REP-529 (Fig. 2A) and B1 (Fig. 2B). The resulting standard curve showed the detection limit of each curve. R2=0.9923 for REP-529 and 0.9985 for B1 showed high linearity among the variables.46 The minimum detection limit (in CT) was 39.28 (corresponding to one parasite) for REP-529, higher than the 37.18 (corresponding to 100 parasites) for B1.

Fig. 2.

Standard curve of T. gondii, tachyzoites using REP-529 (A) and B1 (B) primer sets, respectively using the hydrolysis probe FAM dye-labeled. Results are shown as mean cycle threshold (CT) obtained from triplicate of each DNA concentration. Standard curve analysis was done in 10-fold serial dilutions of DNA extracted from tachyzoites, at initial concentration of 35ng/μL (1×107 tachyzoites). For REP-529, R2=0.9923 and B1, R2=0.9985.

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The first 515 DNA samples (collected until 2015) had results of clinical diagnosis, ELISA and cPCR (B22-B23). These DNA samples were used to test REP-529 and B1 in qPCR, and results are shown in Table 1. DNA samples of 122 patients had negative serological, molecular, and clinical diagnosis (without toxoplasmosis). REP-529 and B1 provided specificities of 99.2% and 100%, respectively. All samples were negative when considering results of at least one primer set.

Table 1 also shows PCR results of 393 clinical samples with positive ELISA. Positive IgG antibodies anti-T. gondii are not necessarily indicative of a symptomatic infection, whereas detection of tachyzoite DNA in biological fluids as cerebral, ocular, congenital or disseminating forms can characterize a symptomatic infection.19,40,42,47,48 Thus, sensitivities of REP-529 and B1 were 95.9% and 83.6%, respectively were calculated considering cPCR and clinical diagnosis (146 samples, which included 94 patients with cerebral toxoplasmosis, 10 newborns with congenital infection, 21 patients with ocular toxoplasmosis, six pregnant women with toxoplasmosis, and 15 postmortem patients with severe disseminated toxoplasmosis) (Table 2). REP-529 amplified 140/146 samples with average CT of 31.71±0.36. B1 amplified 122/146, with average CT of 32.12±0.38.

In the prospective study (2016), a total of 292 clinical samples were analyzed by REP-529 and B1. As shown in Table 2, among 807 clinical samples, 217 (26.89%) had positive PCR, at least one primer set and a confirmed clinical diagnosis. These included: (i) 30.94% (125/404) patients with cerebral toxoplasmosis; (ii) 12.80% (16/125) newborns with congenital alterations; (iii) 18.18% (12/66) of pregnant women with acute toxoplasmosis with T. gondii DNA in AF or BL; (iv) 23.83% (46/193) patients with ocular alterations and T. gondii DNA in BL; and (v) 94.74% (18/19) postmortem patients with severe disseminated toxoplasmosis.

Table 3 describes the performance of primer sets and clinical characteristics of 217 positive samples. REP-529 amplified T. gondii DNA in 97.23% (211/217) of positive samples, and B1 were positive only in 78.80% (171/217) of them.

Although average CT of REP-529 is lower than for B1, the CT with both primer sets were not statistically different in positive samples (Table 4).

The concordance index among the primer sets was calculated based on the positive results. The total number of samples was based on the results of 515 clinical samples with positive (146) and negative (369) results in cPCR (B22-B23) until 2015, and 807 clinical samples with positive (217) and negative (590) results in qPCR (REP529 and B1) (2008–2016). As shown in Table 5, concordance of results among the three primer sets was 83.56%. Concordance between B22-B23 and REP-529 was 95.89%; between B22-B23 and B1 was 83.56%; and between REP-529 and B1, 83.87%.