Seroepidemiological analysis of toxoplasmosis in college students

Background Toxoplasmosis is a zoonosis caused by an obligate intracellular parasite, Toxoplasma gondii, which affects warm-blooded animals including humans. Its prevalence rates usually vary in different regions of the planet. Methods In this study, an analysis of the seroprevalence of toxoplasmosis among Brazilian students was proposed by means of IgG specific antibodies detection. The presence of anti-Toxoplasma gondii antibodies by indirect fluorescent antibody test (IFAT) was also evaluated in order to compare it with enzyme-linked immunosorbent assay (ELISA) and to assess the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine dihydrochloride chromogens. Results The IFAT method showed a seroprevalence of 22.3%. These results were similar to those obtained by ELISA (24.1%). The seroprevalence was directly estimated from the IgG avidity, which showed that in a sample of 112 students, three of them had acute infection, an incidence of 1.6% in the studied population. Conclusion In this study, the use of different chromogenic substrates in immunoenzymatic ELISA assays did not display different sensitivity in the detection of T. gondii-reagent serum. The extrapolation of results to this population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows us to emphasize the importance of careful and well prepared studies to identify risk factors for toxoplasmosis, to adopt preventive measures and to offer guidance to at-risk populations about the disease.

Three suspicious cases of toxoplasmosis were reported in São Paulo State University (UNESP), in Assis, São Paulo state, Brazil (22°39′ 42″ S, 50°24′ 44″ W) stirring up a series of controversies and discussions. The first one refers to clinical likelihood of the disease in college students. However, no serological test was conducted to uphold the diagnosis. The second one is the relation between these cases was the increased cat population. And, finally, a student who had an eye affliction and tested positive for T. gondii.
Since these reports and suppositions were based on inconsistent diagnostic assertions and subjective causal links, without the due clinical evaluation and laboratory diagnostic evidence, a project was devised to find out the seroprevalence and incidence of toxoplasmosis in a sample of UNESP students, by means of serological tests for IgG specific antibodies detection. In addition, the study also verified the presence of anti-T. gondii antibodies in students by IFAT, in order to compare IFAT and ELISA techniques for the toxoplasmosis diagnosis, and to evaluate the use of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and o-phenylenediamine dihydrochloride (OPD) chromogens in the ELISA test for the toxoplasmosis diagnosis.

Methods
The project was carried out at the Laboratory of Human

Characteristics of the area and population under study
Assis is located in western São Paulo state, Brazil, 434 km away from the state capital and comprises a population of 100.911 inhabitants. The annual average temperature is 21.37°C ( Figure 1) [38].
Serological tests were carried out in 112 UNESP freshmen of Assis campus, within an age range from 17 to 25 years old. The selection of students was random (by a raffle) and the sample size was proportional to the number of students in each course (Table 1). Freshman students answered a questionnaire, in order to establish the likelihood of epidemiological correlations, risk factors and contact with cats before being accepted into the university.

Collection of blood and serum samples
The serological information of the students was recorded in individual files and kept confidential. Two blood samples were collected from each voluntary subject of the research. The first one was immediately after the students arrived for the beginning of the 2009 school year and the second one, six months after the first sample had been collected. A 5-mL blood sample was collected from each student, by brachial venous puncture. Meticulous aseptic cautions were taken for blood collection and for the protection of those in charge of it. The Figure 1 Assis is located in the west region of São Paulo state, Brazil, 434 km away from the state capital and comprises a population of 100.911 inhabitants [38]. The annual average temperature is 21.37°C. samples were sent to the Laboratory of Human Parasitology and Immunology for blood processing, plasma separation and storage for later serological analysis methods. Each serum sample was obtained from the whole blood, after centrifugation at 350 × g, for 15 minutes. The resulting supernatant was stored at −20°C.

Indirect Fluorescent Antibody Test (IFAT)
The indirect fluorescent antibody test (IFAT) technique for the screening of anti-T. gondii antibodies, of the IgG class, was carried out according to the methodology described by Camargo [23]. Human anti-IgG serum produced from rabbit inoculations was employed conjugated with fluorescein isothiocyanate (Sigma Chemical Corp., USA). For each serum sample, dilutions at 1:16, 1:64, 1:256, 1:1024 and 1:14096 were prepared. All reactions had a previously known positive and negative control serum. The reading of the plates was made in a fluorescence microscope (Nikon™, Japan). Dilutions greater than or equal to 1:16A were considered positive results.

ELISA immunoenzymatic test
Anti-T. gondii IgG antibodies were determined by the methodology of Camargo [24] and Uchôa et al. [39].
Plates with flat-bottom wells (Corning®, USA) sensitized with 1 μg/mL of T. gondii soluble antigen extract were used for OPD ELISA; and those sensitized with 10 μg/ mL of T. gondii soluble antigen extract were used for ABTS ELISA. Plates were sensitized with antigen by incubation at 4°C for 18 hours, with 100 μL of antigen solution per well. After washing, plates were blocked by incubation for one hour at 37°C with 250 μL of PBS-T per cavity (0.05% of Tween 20 in phosphate-buffered saline solution -PBS) containing 3 g of skimmed milk powder (Molico®, Nestlé, Brazil). Afterward, plates were washed five times in PBS-T for five-minute periods.
Plates containing serum with human-peroxydase-conjugated anti-IgG dilutions (Sigma®, USA) were incubated for the test in stove for one hour. Following both incubations, plates were washed three times in PBS-T for five minutes. For OPD ELISA and ABTS ELISA, in both the first analysis and the one six months after, the cutoff point was obtained from the mean of results from negative control sera evaluated by IFAT plus two standard deviations.

IgG avidity
The same procedure was used for IgG analysis through in-house ELISA, according to the methodology described by Hedman et al. [40]. The plate was coated by the addition, for 24 hours at 4°C, of 100 μL of T. gondii protein antigen to the wells at 1 μg/mL concentration, in an 8 M urea chaotropic (Sigma®) solution in PBS. After blocking possible free sites, the wells received 100 μL of PBS-T diluted serum samples (1/100, 1/200, 1/400, 1/800). Plates were incubated for one hour at 37°C and washed five times in PBS-T. For each dilution, two wells received additional ten-minute incubation at 37°C with 6 M urea neutral chaotropic solution (Sigma®) in PBS-T. Wells containing the control solutions were kept in PBS-T. Afterwards, the antibody development was carried out with the use of human anti-IgG peroxydase conjugate (Sigma®). The estimation of effective titer of each serum was carried out by means of the isolated dilutions of each sample in a log-log linear regression model. The titers of total or high avidity chaotropic-resistant antibodies were achieved by means of log-log regression, with the use of the values able to generate 1.0 absorbance in ELISA (Log = 0). Avidity (AVT) was determined by the percentage of chaotropic resistant titers. Samples with AVT values higher than 30% were considered as high avidity. The optical density (OD) was determined by the reading at 492 nm in a plate spectrophotometer.

Statistical analysis
The correlation rate was used for the analysis among the interspecific titers and the analysis of association among the positive results and variables related to risk factors (gender, direct contact with soil, contact with cats, consumption of raw or undercooked meat and raw vegetables). Values were submitted to the chi-square analysis, with the help of the Epi Info version 6.04 statistics software [41]. A reliable 95% range was considered and p-value < 0.05 was considered statistically significant. In order to check the agreement among ELISA and OPD or ABTS chromogen and IFAT diagnosis tests, the results were reviewed by the kappa test (K), and by sensitivity, specificity, positive predictive value and negative predictive value. Statistical comparisons were made using the GraphPad Prism version 5.00 software for Windows (Graphpad Software, USA).

Ethics committee approval
The present study was approved by the Research Ethics

Results
Serum samples from 112 students were submitted to anti-T. gondii IgG antibody screening by means of IFAT and ELISA with OPD or ABTS chromogen substrate. The correlation between the results obtained in the immunoenzymatic and immunofluorescence assays may be seen in Table 2, in which two discordant results can be noted, two samples that tested positive by OPD ELISA were negative by IFAT whereas two other samples that were positive by ABTS ELISA tested negative by IFAT.
According to IFAT, the usual standard, the seroprevalence was 22.3% (95% CI: 14.5-28.7), similar to the one found in two imunoenzymatic tests. The OPD ELISA test showed a seroprevalence of 24.1% (95% CI: 16.2-32.0), the same found in the ABTS ELISA.
The confirmation and comparison of IgG serological test results by IFAT and ELISA with OPD and ABTS chromogen substrate methods are shown in Table 2. There is a high agreement among the three methods. The comparison of IFAT with OPD and ABTS ELISA test showed a higher agreement than the one found among the immunoenzymatic tests alone (kappa = 0.9499) with 100% of sensitivity (95% CI: 97.6%-100), specificity of 97.7% (95% CI: 94.2-99.1), positive predictive value of 92.5% (95% CI: 88.6-93.8), and negative predictive value of 100% (95% CI: 96.7-100).
The use of different chromogenic substrates in the immunoenzymatic ELISA assays did not show different sensitivity for detecting T. gondii-reagent serum. The ELISA test with OPD chromogenic substrate with plates sensitized with 1 μg of antigen revealed 27 positive sera, the same result observed in the ELISA IgG test with ABTS chromogenic substrate and plates sensitized with 10 μg of antigen.
After six months, new blood samples were examined, by means of IFAT and ELISA with OPD and ABTS chromogen substrates, in order to check the presence of IgG anti-T. gondii antibodies. The analyses of 112 students showed that three who tested negative in the first time, six months after were seropositive by IFAT, OPD ELISA and ABTS ELISA. The other serum samples showed the same results of first analysis, with exception of four samples: two were negative by IFAT and OPD ELISA and positive by ABTS ELISA whereas the other two were negative by IFAT and ABTS ELISA and positive by OPD ELISA. The three seroconverted samples were submitted to IgG avidity test. Students showed low IgG avidity, indicating recently acquired infection (Table 3).
In the present study, the seroprevalence of toxoplasmosis was 22.3% and 25% in the first and second evaluation, respectively. The incidence was directly estimated from the IgG avidity, which showed that, from the 112 students, three had results suggesting acute infection with an incidence of 1.6% (95% CI: 0.6-4.7%), which  allows projecting an annual incidence of 3.2% in the studied population. Table 4 shows the distributions of the variables according to T. gondii infection frequency. No differences were observed when variables taken into account were: gender (χ2 = 0.23, p > 0.05), contact with the cats of the university (χ2 = 1.4; p > 0.05), consumption of raw vegetables (χ2 = 0.11; p > 0.05), and the direct contact with soil (χ2 = 0.58, p > 0.05). The variables contact with cats outside the university (χ2 = 4.2; p < 0.05) and ingestion of raw or undercooked meat (χ2 = 5.0, p < 0.05) were the statistically significant.

Discussion
The screening of IgG anti-T. gondii antibodies by IFAT showed a 22.3% prevalence in students. The seroprevalence data achieved by IFAT serological method were confirmed by the immunoenzymatic ELISA assay with OPD and ABTS chromogenic substrate (24.1%). The tests had highly coherent results, an unusual fact for assays with different antigens such as IFAT and hemagglutination [24]. Therefore, as proposed by Tanyuksel et al. [42] and Reis et al. [43], the avidity test was employed to identify cases suggesting recent infection. The results enabled the projection of an annual seroconversion of 3.2%. Although the avidity test may help to explain the Toxoplasma gondii acute infection diagnosis, some patients have shown persistent low avidity antibodies against the lysate of T. gondii antigenic cells for several months [44,45]. On the other hand, IgG antibody maturity may occur in different periods, and it will set an analytical and interpretive trend of the results [46,47].
Another aspect that may interfere in seroprevalence results of a given sample refers to the chromogen used in immunoenzymatic assays. In addition, when comparing OPD chromogen sensitivity with ABTS, it is possible to find that OPD chromogen is more sensitive and detects antibodies expressing higher optical densities as previously reported [48]. Thus, we used a higher concentration of antigen to sensitize the plate in ELISA with ABTS due to lower detection sensitivity of this chromogen.
In this study, ELISA test with IgG antibodies, ABTS or OPD chromogen, demonstrated the same sensitivity and specificity for toxoplasmosis reagent sera (Table 2). However, it is important to emphasize that values could be the result of the amount of antigen employed in the sensitization of plates, since 10 μg was used in the ELISA test with ABTS chromogen and, in the ELISA test with OPD, only 1 μg was used. This difference in antigen amount may have favored the convergence of results of toxoplasmosis seroprevalence in immunoenzymatic assays.
A careful evaluation of serological tests may result in useful data for the prevention and institution of protocols for the detection of groups at risk, including the IgG avidity test. Transversal studies, as simple as this one, may help in a correct analysis of the results and, accordingly, collaborate for proposals and development of strategies to facilitate the interpretation of diagnosis, and this may result in more reasonable prophylactic measures for controlling this infection.
Soil and consumption of food contaminated with oocysts are considered, in some regions, the major sources of infection by T. gondii [49]. However, in the sample of the present study, the relation between these variables  and the frequency of seropositive results was not statistically significant. On the other hand, the variable contact with cats outside the university possibly had a more important role, based on a higher frequency of seropositive results among those who had contact with those animals (76.0%) and those who were used to consume raw or undercooked meat (92.0%). Such differences are considered statistically significant, in agreement with previous studies, in which frequent ingestion of raw or undercooked meat and contact with oocysts of infected felines are considered the main risk factors for T. gondii infection [49][50][51][52].
According to the current findings, the cat population within the university is not a risk factor for T. gondii infection. Therefore, one may conclude that the most relevant risk factors for toxoplasmosis were contact with cats outside UNESP and eating raw or uncooked meat ( Table 4).
The extrapolation of these results to the general population must be carefully considered, since the investigation was conducted on a reduced sample. However, it allows stating the importance of careful and well prepared studies, not only to identify risk factors for toxoplasmosis, but also to adopt preventive measures and offer guidance to at-risk populations. Thus, the implementation of simple cross-sectional studies may provide important epidemiological contribution and establish consistent causal associations, avoiding thereby the development of hypotheses that do not support rigorous questioning.