Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 http://www.jvat.org/content/21/1/1 RESEARCH Open Access Seroepidemiological analysis of toxoplasmosis in college students Jaqueline Polizeli Rodrigues4, Fernando Frei1, Italmar Teodorico Navarro2, Luciana Pereira Silva1, Monica Yonashiro Marcelino3, Heitor Franco de Andrade-Junior4, Carolina Arruda de Faria5, Marislene Santos1 and João Tadeu Ribeiro-Paes1,6* Abstract 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. Keywords: Toxoplasma gondii, Toxoplasmosis, Enzyme-linked immunosorbent assay, Indirect fluorescent antibody test, Seroprevalence, Epidemiology Background Toxoplasma gondii is a protozoan parasite that is respon- sible for toxoplasmosis in warm-blooded animals [1-7]. The infection is spread mainly through contact with oocysts eliminated in feces of infected cats, ingestion of contaminated raw or undercooked meat and congenital infection [1,8-14]. The environmental contamination by this protozoa demands attention since it may trigger other routs of transmission and can lead to outbreaks, such as the waterborne epidemic in Santa Isabel do Ivaí, Paraná state, Brazil (23° 0′ 19″ S, 53° 11′ 16″ W) [15]. * Correspondence: jtrpaes@yahoo.com.br 1Department of Biological Sciences, (UNESP), Assis, São Paulo State, Brazil 6Laboratório de Genética e Terapia Celular (GenTe CeL), Departamento de Ciências Biológicas, Universidade Estadual Paulista (UNESP), Campus de Assis, Av. Dom Antônio, 2100, Assis, SP CEP 19.806-900, Brazil Full list of author information is available at the end of the article © 2015 Rodrigues et al.; licensee BioMed Cent Commons Attribution License (http://creativec reproduction in any medium, provided the or Dedication waiver (http://creativecommons.or unless otherwise stated. Human infection is usually asymptomatic. The main clinical signs after the onset of the disease include lymphadenitis, fever, asteny, and myalgia. Encephalitis, meningoencephalitis, ocular lesions, septic syndrome, myo- carditis or hepatitis may be found occasionally [1,3,16,17]. Symptoms are manifested mainly in immunocompromised people and newborns [18-21]. Recently the diagnosis of toxoplasmosis has been draw- ing close attention. Several serological techniques have been applied and have shown good sensitivity, specificity, and are quickly carried out. Among different diagnostic techniques, the following are noteworthy: Sabin and Feldman technique, indirect fluorescent antibody test (IFAT), complement fixation (CF), hemagglutination (HA), enzyme-linked immunosorbent assay (ELISA) and immu- nosorbent agglutination assay (ISAGA) [22-27]. ral. This is an Open Access article distributed under the terms of the Creative ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and iginal work is properly credited. The Creative Commons Public Domain g/publicdomain/zero/1.0/) applies to the data made available in this article, mailto:jtrpaes@yahoo.com.br http://creativecommons.org/licenses/by/4.0 http://creativecommons.org/publicdomain/zero/1.0/ 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. Table 1 Distribution of new students per course in 2009 in UNESP campus of Assis, and sample size of subjects who provided biological material (blood) for serological analysis Course Freshman Blood samples Biology 40 20 Biotechnological engineering 40 17 History 40 19 Psychology 90 29 Languages 80 27 Total 290 112 Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 2 of 7 http://www.jvat.org/content/21/1/1 Seroprevalence indexes have varied in different coun- tries and regions of the planet [28]. Serological research showed rates of 6.7% in Korea, 12.3% in China, 22.5%, in the US adult population, 23.9% in Nigeria, 38.8% in Spain, 46% in Tanzania and 47% in rural zones of the France [29-35]. A study with anti-T. gondii antibodies carried out in the Republic of Benin revealed a seropre- valence of 87.7% [36]. In different regions in Brazil, the seropositive rate varied between 37% and 91% [37,4]. 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 be- tween these cases was the increased cat population. And, finally, a student who had an eye affliction and tested posi- tive 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 anti- bodies 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-ethylbenzo- thiazoline-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 Parasitology and Immunology of São Paulo State Uni- versity (UNESP), in Assis, with the cooperation of the Laboratory of Zoonoses and Public Health of the Depart- ment of Preventive Veterinary Medicine, State University of Londrina (UEL), in Londrina, Paraná state, Brazil (23° 19′ 39.5″ S, 51° 11′ 56.5″ W) and of the Laboratory of Protozoonoses of the Institute of Tropical Medicine of São Paulo, University of São Paulo (USP), São Paulo, São Paulo state, Brazil (23° 33′ 18.5″ S, 46° 40′ 18.2″ W). 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 fresh- men 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 likeli- hood of epidemiological correlations, risk factors and con- tact with cats before being accepted into the university. Collection of blood and serum samples The serological information of the students was recor- ded 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 sam- ple had been collected. A 5-mL blood sample was col- lected from each student, by brachial venous puncture. Meticulous aseptic cautions were taken for blood collec- tion and for the protection of those in charge of it. The Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 3 of 7 http://www.jvat.org/content/21/1/1 samples were sent to the Laboratory of Human Parasit- ology and Immunology for blood processing, plasma sep- aration 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 result- ing 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 de- scribed by Camargo [23]. Human anti-IgG serum pro- duced 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 fluores- cence 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-conju- gated 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. The color reaction was carried out subsequently in the dark for 15 minutes at room temperature with 100 μL of the OPD chromogenic solution (o-phenylenediamine 0.05%, Sigma® + citric acid 1% +Na2HPO4 1.45% in H2O, addition of 10 μL de H2O2 30% for each 20 mL of the so- lution) and was quenched by the addition of 25 μL of 2 M sulfuric acid solution. For the OPD chromogenic solution, the optical density (OD) was determined by the reading at 492 nm, and for the ABTS chromogenic solution, by the reading at 414 nm and 612 nm, in a plate spectropho- tometer (700 Plus Spectrophotometer, Fendo, Brazil). 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 de- scribed 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 percent- age 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, con- sumption of raw or undercooked meat and raw vegeta- bles). 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 pre- dictive 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 Committee of Marília Medical School (FAMEMA – Marília, SP, Brazil) under protocol n. 449/08. It included Figure 2 Quantitative reactivity and linear regression analysis of IgG anti-T. gondii in 112 serum samples from university students using ELISA with chromogen substrate OPD and ABTS. Interruptions in the axes show the thresholds of positivity of the assays (ELISA OPD cutoff = 0.486 and ELISA ABTS cutoff = 0.239). Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 4 of 7 http://www.jvat.org/content/21/1/1 only patients who authorized the collection of blood samples through free informed consent according to the Brazilian National Committee of Ethics in Research (CONEP), resolution 236/96, National Health Council, Brazilian Ministry of Health. 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 sam- ples that were positive by ABTS ELISA tested negative by IFAT. The comparison between OPD and ABTS ELISA en- zymatic assays showed high agreement (kappa = 0.9024), 92.5% sensitivity (95% confidence interval – CI: 90.5-96.3) and 97.6% specificity (95% CI: 93.2-97.8) and the positive predictive values were 92.5% (95% CI: 90.6-96.3) and negative predictive values 97.6% (95% CI: 93.2-97.8) (Figure 2). According to IFAT, the usual standard, the seropreva- lence 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 Table 2 Results of anti-T. gondii in serum samples of 112 UNESP students by means of IFAT (reference test), ELISA with OPD or ABTS chromogen substrate Assay Result IFAT IgG Positive Negative ELISA IgG Positive 25 2 OPD Negative 0 85 K = 0.949 ELISA IgG Positive 25 2 ABTS Negative 0 85 K = 0.949 ABTS: 2.2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), CI: confidence interval, ELISA: enzyme-linked immunosorbent assay, IFAT: indirect fluorescent antibody assay, IgG: immunoglobulin G, K: kappa index, OPD: O-phenylenediamine dihydrochloride. 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 toxoplas- mosis was 22.3% and 25% in the first and second evalu- ation, 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 Table 3 Serological profile of three UNESP students in Assis six months after the first analysis Age (years) IgGa ELISA OPD (OD) IgGb ELISA ABTS (OD) IFAT IgG AVT (%) Interpretationc Serological profile 23 0.491 0.069 Positive 28.90% Low avidity Acute phase 20 2.440 0.646 Positive 27.41% Low avidity Acute phase 21 3.372 0.851 Positive 29.65% Low avidity Acute phase acutoff: 0.427; bcutoff: 0.054; cIgG low avidity < 30%. AVT: avidity of titer, ELISA: enzyme-linked immunosorbent assay, IgG: immunoglobulin G, OD: optical density. Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 5 of 7 http://www.jvat.org/content/21/1/1 allows projecting an annual incidence of 3.2% in the studied population. Table 4 shows the distributions of the variables ac- cording 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 vege- tables (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 seropreva- lence 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 as- says 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%. Al- though the avidity test may help to explain the Toxo- plasma 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]. Table 4 Anti-T. gondii IgG antibody levels in students by mea factors for toxoplasmosis Risk factors Total Anti- Gender % of males (n/total) 40.1% (45/112) Direct contact with soil (n/total) 1.8% (2/112) Contact with the cats of the university (n/total) 4.4% (5/112) Contact with cats outside the university (n/total) 26.9% (29/112) Eating raw or undercooked meat (n/total) 33.0% (37/112) Raw vegetables consumption (n/total) 94.6% (106/112) χ2: chi square test; NS: not significant (p > 0.05). Another aspect that may interfere in seroprevalence results of a given sample refers to the chromogen used in immunoenzymatic assays. In addition, when compar- ing OPD chromogen sensitivity with ABTS, it is possible to find that OPD chromogen is more sensitive and de- tects antibodies expressing higher optical densities as previously reported [48]. Thus, we used a higher concen- tration 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 re- sults of toxoplasmosis seroprevalence in immunoenzy- matic assays. A careful evaluation of serological tests may result in useful data for the prevention and institution of proto- cols 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, ac- cordingly, collaborate for proposals and development of strategies to facilitate the interpretation of diagnosis, and this may result in more reasonable prophylactic mea- sures for controlling this infection. Soil and consumption of food contaminated with oo- cysts 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 ns of indirect fluorescent antibody test (IFAT), and risk T. gondii IgG positive Anti-T. gondii IgG negative χ2 (p values) 36.0% (9/25) 41.3% (36/87) 0.23 (NS) 0% (0/25) 2.2% (2/87) 0.58 (NS) 0% (0/25) 5.7% (5/87) 1.4 (NS) 76.0% (23/25) 11.4% (10/87) 4.2 (p < 0.05) 92.0% (23/25) 16.0% (14/87) 5.0 (p < 0.05) 96.0% (24/25) 94.2% (82/87) 0.11 (NS) Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 6 of 7 http://www.jvat.org/content/21/1/1 and the frequency of seropositive results was not statisti- cally significant. On the other hand, the variable contact with cats outside the university possibly had a more im- portant 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 consid- ered statistically significant, in agreement with previous studies, in which frequent ingestion of raw or under- cooked meat and contact with oocysts of infected felines are considered the main risk factors for T. gondii infec- tion [49-52]. Concerning gender, no significant differences were noted, which is in agreement with Souza [53], Daguer et al. [54] and Sharif et al. [55]. Conclusions In the present study, the use of 2,2′-azinobis(3-ethylbenzo- thiazoline-6-sulfonate) (ABTS) and O-phenylenediamine dihydrochloride (OPD) chromogens in immunoenzymatic ELISA assay with adequate quantities of T. gondii antigen for each substrate showed the same sensitivity for detection of toxoplasmosis reagent sera (Table 2). 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 popu- lation must be carefully considered, since the investiga- tion 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 epidemio- logical contribution and establish consistent causal associ- ations, avoiding thereby the development of hypotheses that do not support rigorous questioning. Competing interests The authors declare that they have no competing interests. Authors’ contributions JTRP, ITN and JPR conceived the project and experimental design. JPR, MS and MYM contributed to the execution and data analysis of the project. FF, ITN, LPS, HFAJ and CAF performed statistical analysis and discussion of the data. All the authors contributed to data analysis, writing, and revision of the manuscript. All authors read and approved the final manuscript. Acknowledgments The authors would like to thank the Fundação para o Desenvolvimento da Unesp (Fundunesp), the Municipal Government of Assis (SP, Brazil) and CIVAP/Saúde for the financial support. MYM received a grant from the Coordenação para Aperfeiçoamento de Pessoal de Nivel Superior(CAPES) and CAF was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors would also like to thank Roselaine Pereira Alvim Cardoso, Fernanda Evers, Fernando Nakanishi Hamada and Irani Alves for the technical support during the experiments. Author details 1Department of Biological Sciences, (UNESP), Assis, São Paulo State, Brazil. 2Department of Veterinary Medicine, Londrina State University, Londrina, Paraná State, Brazil. 3Graduate Program in Biotechnology, University of São Paulo (USP)/Butantan Institute/Institute for Technological Research (IPT), São Paulo, São Paulo State, Brazil. 4Institute of Tropical Medicine of São Paulo, University of São Paulo (USP), São Paulo, São Paulo State, Brazil. 5Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, São Paulo State, Brazil. 6Laboratório de Genética e Terapia Celular (GenTe CeL), Departamento de Ciências Biológicas, Universidade Estadual Paulista (UNESP), Campus de Assis, Av. Dom Antônio, 2100, Assis, SP CEP 19.806-900, Brazil. Received: 16 June 2014 Accepted: 17 December 2014 Published: 5 January 2015 References 1. Tenter AM, Heckeroth AR, Weis LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000;30(12–13):1217–58. 2. Marques JM, Da Silva DV, Correia NAB, Velásquez LG, Da Silva RC, Langoni H, et al. Prevalence and risk factors for human toxoplasmosis in a rural community. J Venom Anim Toxins Incl Trop Dis. 2008;14(4):673–84 [http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992008 000400010] 3. Langoni H. Zoonoses and human beings. J Venom Anim Toxins Incl Trop Dis. 2004;10(2):111 [http://www.scielo.br/scielo.php?pid=S1678- 91992004000200001&script=sci_arttext] 4. Vasconcelos CGC. Occupational zoonoses: Serum-epidemiological survey of veterinary students and analysis of risk of Leptospirosis, Brucellosis and Toxoplasmosis. J Venom Anim Toxins Incl Trop Dis. 2004;10(2):192 [http://www.jvat.org.br/full/j-2004/volume_10/number_2/thesis2_10_2_ 2004.htm] 5. Muraro LS, Caramori Júnior JG, Amendoeira MRR, Pereira JA, Filho JXO, Vicente RT, et al. Seroprevalence of Toxoplasma gondii infection in swine matrices in Nova Mutum and Diamantino, Mato Grosso. Bras Rev Bras Parasitol Vet. 2010;23(4):254–5. 6. Vilela SMO, Silva JSA, Pinheiro Junior JW, Moraes EPBX, Saukas TN, Gondim LFP, et al. Sparrows (Passer domesticus L.) as intermediary hosts of Toxoplasma gondii in poultry farms from the “agreste” region of Pernambuco, Brazil. Pesq Vet Bras. 2011;31(2):169–72. 7. Dubey JP, Lago EG, Gennari SM, Su C, Jones JL. Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology. 2012;139(11):1375–424. 8. Benenson MW, Takafuji ET, Lemon SM, Greenup RL, Sulzer AJ. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N Engl J Med. 1982;307(11):666–9. 9. Coutinho SG, Lobo R, Dutra G. Isolation of Toxoplasma from the soil during an outbreak of toxoplasmosis in a rural area in Brazil. J Parasitol. 1982;68(5):866–8. 10. Dubey JP, Towle A, editors. Toxoplasmosis in sheep: a review and annotated bibliography. St. Albans, England: Commonwealth Institute of Parasitology; 1986. 11. Bahia-Oliveira LM, Jones JL, Azevedo-Silva J, Alves CC, Oréfice F, Addiss DG. Highly endemic, waterborne toxoplasmosis in north Rio de Janeiro state. Bras Emerg Infect Dis. 2003;9(1):55–62. 12. Silva RC, Souza LC, Langoni H, Tanaka EM, Lima VY, Silva AV. Risk factors and presence of antibodies to Toxoplasma gondii in dogs from the coast of São Paulo State. Bras Pesq Vet Bras. 2010;30(2):161–6. 13. Hill D, Coss C, Dubey JP, Wroblewski K, Sautter M, Hosten T, et al. Identification of a sporozoite-specific antigen from Toxoplasma gondii. J Parasitol. 2011;97(2):328–37. 14. Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev. 2012;25(2):264–96. 15. de Moura L, Bahia-Oliveira LM, Wada MY, Jones JL, Tuboi SH, Carmo EH, et al. Waterborne toxoplasmosis, Brazil, from field to gene. Emerg Infect Dis. 2006;12(2):326–9. 16. Silva LA, Vieira RS, Serafini LN, Carlotti Junior CG, Figueiredo JFC. Toxoplasmose do sistema nervoso central em paciente sem evidência de imunossupressão: relato de caso. Rev Soc Bras Med Trop. 2001;34(5):487–90. http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992008000400010 http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992008000400010 http://www.scielo.br/scielo.php?pid=S1678-91992004000200001&script=sci_arttext http://www.scielo.br/scielo.php?pid=S1678-91992004000200001&script=sci_arttext http://www.jvat.org.br/full/j-2004/volume_10/number_2/thesis2_10_2_2004.htm http://www.jvat.org.br/full/j-2004/volume_10/number_2/thesis2_10_2_2004.htm Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2015, 21:1 Page 7 of 7 http://www.jvat.org/content/21/1/1 17. Kaye A. Toxoplasmosis: diagnosis, treatment, and prevention in congenitally exposed infants. J Pediatr Health Care. 2011;25(6):355–64. 18. Almeida RAMB, Silva GF, Llanos JC, Winckler CC, Gomes MRB, Biagioni DS, et al. Cytomegalovirus and Toxoplasma gondii seroprevalence in a Brazilian liver transplant waiting list. J Venom Anim Toxins Incl Trop Dis. 2007;13(4):881–4 [http://www.scielo.br/scielo.php?pid=S1678- 91992007000400016&script=sci_arttext] 19. Fan CK, Su KE, Chung WC, Tsai YJ, Chiou HY, Lin CF, et al. Seroprevalence of Toxoplasma gondii antibodies among Atayal aboriginal people and their hunting dogs in northeastern Taiwan. Jpn J Med Sci Biol. 1998;51(1):35–42. 20. Zajdenweber M, Muccioli C, Belfort Jr R. Acometimento ocular em pacientes com AIDS e toxoplasmose do sistema nervoso central - antes e depois do HAART. Arq Bras Oftalmol. 2005;68(6):773–5. 21. Kung DH, Hubenthal EA, Kwan JY, Shelburne SA, Goodman JC, Kass JS. Toxoplasmosis myelopathy and myopathy in an AIDS patient: a case of immune reconstitution inflammatory syndrome? Neurologist. 2011;17(1):49–51. 22. Sabin AB, Feldman HA. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science. 1948;108(2815):660–3. 23. Camargo ME. Introdução às técnicas de imunofluorescência. Rev Bras Patol Clín. 1974;10(1):143–71. 24. Camargo ME. Alguns aspectos atuais do diagnóstico de laboratório da toxoplasmose. An Acad Nac Med. 1995;155:236–9. 25. Jacobs J, Lunde MN. A hemagglutination test for toxoplasmosis. J Parasitol. 1957;43(3):308–14. 26. Machín Sánchez R, Cruz Castillo F, Prividal Grana J. Comparación de ELISA con las técnicas de inmunofluorescencia indirecta y fijación del complemento para el diagnóstico de la toxoplasmosis. Rev Cuba Med Trop. 1985;37(3):269–77. 27. Ashburn D, Joss AW, Pennington TH, Ho-Yen DO. Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis. J Clin Pathol. 1995;48(1):64–9. 28. Furtado JM, Smith JR, Belfort Jr R, Gattey D, Winthrop KL. Toxoplasmosis: a global threat. J Glob Infect Dis. 2011;3(3):281–4. 29. Shin DW, Cha DY, Hua QJ, Cha GH, Lee YH. Seroprevalence of Toxoplasma gondii infection and characteristics of seropositive patients in general hospitals in Daejeon, Korea. Kor J Parasitol. 2009;47(2):125–30. 30. Xiao Y, Yin J, Jiang N, Xiang M, Hao L, Lu H, et al. Seroepidemiology of human Toxoplasma gondii infection in China. BMC Infect Dis. 2010;10:4. doi:10.1186/1471-2334-10-4. 31. Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, McAuley JB. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol. 2001;154(4):357–65. 32. Kamani J, Mani AU, Egwu GO, Kumshe HA. Seroprevalence of human infection with Toxoplasma gondii and the associated risk factors, in Maiduguri, Borno state, Nigeria. Ann Trop Med Parasitol. 2009;103(4):317–21. 33. Jaqueti J, Hernández-Garcia R, Nicolás D, Martínez DH, Navarro FG. Serologia frente a Toxoplasma gondii en mujere gestantes. Evolución de tasas de prevalência a lo largo de cuatro años. Rev Clin Esp. 1991;188(6):278–9. 34. Swai ES, Schoonman L. Seroprevalence of Toxoplasma gondii infection amongst residents of Tanga district in north-east Tanzania. Tanzan J Health Res. 2009;11(4):205–9. 35. Fromont EG, Riche B, Rabilloud M. Toxoplasma seroprevalence in a rural population in France: detection of a household effect. BMC Infect Dis. 2009;9:76. doi:10.1186/1471-2334-9-76. 36. Rodier MH, Berthonneau J, Bourgoin A, Giraudeau G, Agius G, Burucoa C, et al. Seroprevalences of Toxoplasma, malaria, rubella, cytomegalovirus, HIV and treponemal infections among pregnant women in Cotonou, Republic of Benin. Acta Trop. 1995;59(4):271–7. 37. Secretaria de Vigilância em Saúde. Surto de Toxoplasmose adquirida, Anápolis -GO, fevereiro de 2006. Brasília: Ministério da Saúde; 2007 [http://www.saude.gov.br/svs] 38. Instituto Brasileiro de Geografia e Estatística (IBGE). Censo Populacional 2010. [http://censo2010.ibge.gov.br/]. Accessed on Octuber 10 2014. 39. Uchôa CMA, Duarte R, Laurentino-Silva V, Alexandre GMC, Ferreira HG, Amendoeira MRR. Padronização de ensaio imunoenzimático para pesquisa de anticorpos das classes IgM e IgG anti-Toxoplasma gondii e comparação com a técnica de imunofluorescência indireta. Rev Soc Bras Med Trop. 1999;32(6):661–9. 40. Hedman K, Lappalainen M, Seppäiä I, Mäkelä O. Recent primary toxoplasma infection indicated by a low avidity of specific IgG. J Infect Dis. 1989;159(4):736–41. 41. Dean AG. Microcomputers and the future of epidemiology. Public Health Rep. 1994;109(3):439–41. 42. Tanyuksel M, Guney C, Araz E, Saracli MA, Doganci L. Performance of the immunoglobulin G avidity and enzyme immunoassay IgG/IgM screening tests for differentiation of the clinical spectrum of toxoplasmosis. J Microbiol. 2004;42(3):211–15. 43. Reis MM, Tessaro MM, D’Azevedo PA. Toxoplasma-IgM and IgG-avidity in single samples from areas with a high infection rate can determine the risk of mother-to-child transmission. Rev Inst Med Trop Sao Paulo. 2006;48(2):93–8. 44. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965–76. 45. Piergili Fioretti D. Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals. Parassitologia. 2004;46(1–2):177–81 [Article in Italian]. 46. Montoya JG, Liesenfeld O, Kinney S, Press C, Remington JS. VIDAS test for avidity of Toxoplasma-specific immunoglobulin G for confimatory testing of pregnant women. J Clin Microbiol. 2002;40(7):2504–8. 47. Coura JR, editor. Dinâmica das doenças infecciosas e parasitárias. Volume IIth ed. Rio de Janeiro: Guanabara Koogan; 2005. 48. KPL Technical service Report. Comparison of ABTS, TMB, and OPD peroxidase substrate systems. [http://www.kpl.com/docs/techdocs/ML108.PDF] 49. Spalding SM, Amendoeira MR, Klein CH, Ribeiro LC. Serological screening and toxoplasmosis exposure factors among pregnant women in South of Brazil. Rev Soc Bras Med Trop. 2005;38(2):173–7. 50. Navarro IT, Vidotto O, Giraldi N, Freire R. Estudo da resistência do Toxoplasma gondii ao efeito do cloreto de sódio e condimentos em linguiça frescal de suínos. OPAS. 1992;112:138–43. 51. Amendoeira MRR, Costa T, Spalding SM. Toxoplasma gondii Nicolle & Manceaux, 1909 (Apicomplexa: Sarcocystidae) e a Toxoplasmose. Rev Souza Marques. 1999;1(1):15–35. 52. Elmore SA, Jones JL, Conrad PA, Patton S, Lindsay DS, Dubey JP. Toxoplasma gondii: epidemiology, feline clinical aspects, and prevention. Trends Parasitol. 2010;26(4):190–6. 53. Souza WJS. Epidemiologia da toxoplasmose: avaliação sorológica de suínos e trabalhadores em abatedouros na mesorregião do Grande Rio de Janeiro. PhD thesis. Universidade Federal Rural do Rio de Janeiro, Toxicology Department; 1995. 54. Daguer H, Vicente RT, Costa T, Virmond MP, Hamann W, Amendoeira MRR. Soroprevalência de anticorpos anti-Toxoplasma gondii em bovinos e funcionários de matadouros da microrregião de Pato Branco, Paraná. Brasil. Ciênc Rural. 2004;34(4):1133–7. 55. Sharif M, Daryani A, Barzegar G, Nasrolahei M. A seroepidemiological survey for toxoplasmosis among schoolchildren of Sari, Northern Ira. Trop Biomed. 2010;27(2):220–5. doi:10.1186/1678-9199-21-1 Cite this article as: Rodrigues et al.: Seroepidemiological analysis of toxoplasmosis in college students. Journal of Venomous Animals and Toxins including Tropical Diseases 2015 21:1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.scielo.br/scielo.php?pid=S1678-91992007000400016&script=sci_arttext http://www.scielo.br/scielo.php?pid=S1678-91992007000400016&script=sci_arttext http://www.saude.gov.br/svs http://censo2010.ibge.gov.br/ http://www.kpl.com/docs/techdocs/ML108.PDF Abstract Background Methods Results Conclusion Background Methods Characteristics of the area and population under study Collection of blood and serum samples Indirect Fluorescent Antibody Test (IFAT) ELISA immunoenzymatic test IgG avidity Statistical analysis Ethics committee approval Results Discussion Conclusions Competing interests Authors’ contributions Acknowledgments Author details References << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /PageByPage /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails true /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /NoConversion /DestinationProfileName () /DestinationProfileSelector /NA /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure true /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles true /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [595.440 793.440] >> setpagedevice