Introduction Toxoplasmic retinochoroiditis appears to be more common in Brazil than in Europe or North America and more severe. It is a leading cause of blindness in Brazil[1] but not in Europe or North America.[2],[3] These differences are not adequately explained by high rates of postnatal or congenital infection in Brazil, as similar rates of infection have been observed in France and Eastern Europe.[4]–[8] Once infected, population-based studies of adolescents and adults, most of whom have postnatally acquired infection, report the risk of retinochoroiditis to vary from 2% in North Eastern Brazil to 25% in Southern Brazil.[4], [9]–[11] No comparable studies have been done in Europe or North America but case series report risks of 0.3% to 1% in adults in the year or two after acquisition of infection.[12]–[14] The fact that retinochoroiditis results in more and larger lesions in South America than in Europe or North America is well accepted by clinicians but differences could reflect delayed presentation due to poor access to health care, referral bias, acquisition of infection from oocysts rather than tissue cysts, or exposure to a higher parasite load or to infection earlier in childhood in Brazil.[15] No published studies have directly compared ocular sequelae of toxoplasmic infection in Brazil with Europe or North America. Differences in the ocular sequelae of toxoplasmosis have been attributed to recent findings that distinctly different populations of Toxoplasma gondii exist in South America compared with Europe and North America.[16],[17] Strains in Brazil appear to be more virulent, and have been identified in several patients with severe ocular disease.[18],[19] In-vitro studies suggest that strain virulence affects triggering of the immune response, tissue penetration and the ability to encyst. As treatment is effective only during the tachyzoite phase, prior to encystment, this raises the possibility that the response to anti-toxoplasma treatment may differ between strains.[20],[21] Possible clinical and policy implications of these findings could be the development of targeted treatment and preventive strategies depending on the prevailing parasite genotype. Our aim was to inform clinical practice and policy, by quantifying differences in ocular sequelae after toxoplasmic infection in Brazil compared with Europe. We directly compared cohorts of children with congenital toxoplasmosis identified by neonatal or prenatal screening. This approach minimized differences in the route and timing of infection as all fetuses were infected by transplacental transmission of tachyzoites. Health care access was also similar as screening was applied to all births, and we prospectively followed up children using the same protocol in both continents. Methods Study population We prospectively recruited and followed up a cohort of live-born children with congenital toxoplasmosis identified between 1996 and 2003 by neonatal or delivery screening in 2 Brazilian centers, by neonatal screening in 3 European centers (Sweden, Denmark, and Poland), and by prenatal screening in 10 European centers (7 in France, 2 in Italy, 1 in Austria). We also included children identified in a prospective national Danish neonatal screening study between 1992 and 1996.[22] Details of screening methods and treatment in the European centers have been reported elsewhere.[22]–[25] In all centers, we excluded any mother or infant who was not first identified by universal screening and who could have been referred for testing because of symptomatology. The diagnosis of congenital toxoplasmosis was based on persistence of specific IgG antibodies after 11.5 months of age.[23],[24] In Brazil, neonatal screening was based on testing of the neonatal Guthrie card blood spot using an IgM immunoassay (VIDAS, Biomerieux) in Campos Dos Goytacazes, and a commercial capture IgM fluorometric enzyme immunoassay (Labsystems) in Porto Alegre. For screening of mothers at delivery, an IgM immunoassay (VIDAS, BioMérieux) was used.[26],[27] In Campos Dos Goytacazes, neonatal screening was offered to approximately 25% of the total births (about 9,000 births per year) who delivered on two days each week at public sector hospitals within the area. Study infants were recruited between 1999 and 2001. In Porto Alegre, all patients were enrolled who had congenital toxoplasmosis identified between 1998 and 2003 by routine neonatal (n = 17) or delivery (n = 5) screening and who started follow-up in the Congenital Infections Clinic of Sao Lucas Hospital before 2 months of life (3 patients followed up elsewhere were excluded). Twelve of these 22 children were followed up in the public sector and 10 in the private sector. Postnatal treatment was prescribed for 12 months. Pyrimethamine (1 mg/kg/day) and sulphadiazine (100 mg/kg/day) given for the first six months were changed for the subsequent six months to alternating spiramycin and pyrimethamine-sulphonamide in Campos Dos Goytacazes and to a lower dose of pyrimethamine (1 mg/kg/3 times per week) with sulphonamide (100 mg/kg/day) in Porto Alegre. Ophthalmic assessments We used a standard questionnaire to record findings at routine ophthalmoscopic examinations before 4 months, at 12 months of age, and annually thereafter for all children in the cohort. Clinicians were asked to dilate the pupil and use indirect ophthalmoscopy. They used a standard proforma to record whether the retina was adequately visualized, describe the site of lesions using a diagram and text, and estimate lesion size in multiples of the optic disc diameter. We defined a recurrence as a new lesion that was detected for the first time more than one week after a previous adequate visualization of the retina. Analyses of lesion size were based on the size of the largest lesion at each new occurrence. Multiple lesions were based on the total number of separate lesions detectable at the last examination. We assumed that 2 included children (in Brazil and Europe) had retinochoroiditis although microphthalmia and severe vitreal opacities prevented adequate examination. One of us (MRS) categorized each eye according to whether visual impairment ( 4 years (number) 1.1 (10) 0.52 (6) Predicted visual impairment || Anticipated visual impairment in best eye 1 (7) 4 (12) 5 (10) 11 (37) Anticipated visual impairment(% affected eyes) 7 (30) 13 (28) 20 (28) 27 (87) * Three children from Brazil were included in a previous study by Neto et al.[27] † Compared with Europe prenatal ‡ Compared with Europe total § Excludes two children (1 Europe prenatal and 1 Brazil) with presumed retinochoroiditis, microphthalmia and vitreal opacities in whom recurrence could not be assessed. || Based on ophthalmologist assessment of retinal diagrams. Criteria for impairment is acuity <6/12. Five eyes without diagrams assumed to be unimpaired based on reported site of lesions. ¶ Mean size of the largest lesion at each new lesion occurrence measured in disc diameters. Europe datasets combined because of small numbers IQR = interquartile range Recurrence Retinochoroidal lesion recurred at an earlier age in Brazil than in Europe (p = 0.0406; Table 1). By 4 years of age, the probability of a second lesion among children with a first lesion was 43% in Brazil compared with 29% in Europe (Table 1). The risk of multiple recurrences was also greater in Brazil (hazard ratio for the time from birth to multiple lesions: 3.44; 95%CI: 2.23, 5.32). There was no significant difference in the age at recurrence between European prenatal and neonatal screening centers (p = 0.1740) (Table 1). Site of lesions Children in Brazil were more likely than those in Europe to have retinochoroidal lesions that affected the posterior pole and to have visual impairment predicted by the ophthalmologist assessing the retinal diagrams (p<0.001; Table 1). Relatively more children in Brazil than Europe had multiple lesions (p<0.0001). Size of lesions The size of the largest lesion was recorded for 62% (74/119) of first or recurrent lesions. Figures 2 A to C depict the size of the largest lesion for each lesion occurrence according to age at detection. The overall mean size was greater in Brazil than in Europe (p<0.0001, Table 1). In both continents, lesion size decreased with the age at detection (p = 0.0007). 10.1371/journal.pntd.0000277.g002 Figure 2 Size measured in disc diameters (y axis) and age (years, x axis) of the first (black), second (blue) and third (red) and fourth (green) newly detected retinochoroidal lesions in (A) Brazil, and (B) European Neonatal Centers and (C) European Prenatal Centers. Discussion Children with congenital toxoplasmosis in Brazil developed retinochoroiditis earlier than children in Europe, had multiple lesions more frequently, and had larger lesions that were more likely to affect the posterior pole and hence to threaten vision. Lesions appeared to diminish in size with age at detection. This is the first study to compare toxoplasmic retinochoroiditis in concurrent cohorts from Brazil and Europe. Strengths of the study include prospective enrollment and follow up of children in the same way in both continents. A potential source of bias is the fact that Brazilian ophthalmologists have more experience with toxoplasmic retinochoroiditis than their European counterparts and used indirect ophthalmoscopy for virtually all examinations whereas only 54% of examinations were done this way in Europe (73% of European children had at least one indirect examination). Such detection bias is unlikely to explain the very large differences between Brazil and Europe, which persisted for posterior pole lesions, which all ophthalmologists should be able to detect regardless of ophthalmoscopy method. There are three possible confounding factors that could have contributed to the differences in the risk of retinochoroiditis between Brazil and Europe. Firstly, two-thirds of children in the European cohort received prenatal treatment. We have reported elsewhere that prenatal treatment did not statistically significantly reduce the risk of retinochoroiditis, and similar findings have been reported by others.[23], [31]–[33] Moreover, the differences between Brazil and Europe persisted when we compared neonatal screening centers in Brazil and Europe. The fact that postnatal treatment was started later in Brazil than in European neonatal screening centers would not affect the higher prevalence of lesions at birth in Brazil. Secondly, the mothers of children in Brazil would have seroconverted on average later in pregnancy than those identified by prenatal screening in Europe because IgM screening favors detection of children infected in the latter half of pregnancy.[34] Given the weak association between early gestational age at maternal seroconversion and the risk of retinochoroiditis, our results would be biased in favor of underestimating differences in the risk of retinochoroiditis in Brazil compared with Europe.[23],[31],[32] Thirdly, termination of fetuses with ocular disease in the prenatal centers could have reduced the risk of ocular disease in Europe. This seems unlikely as the number of pregnancy terminations for toxoplasmosis in European prenatal screening centers was very low: 17 out of 1327 women had terminations for toxoplasmosis, of which 9 were infected and very few (n = 6) had evidence of clinical manifestations at autopsy.[35] Differences between cohorts in calendar time and ethnicity are unlikely to account for the large differences observed between Europe and Brazil. Previous analyses based on European data have shown weak effects of calendar time, with a decreased risk of lesions in later years. This source of bias would lead to an underestimate of the difference between the two continents. Ethnic variation is unlikely to explain the very large differences observed as epidemiological studies have shown that the prevalence of toxoplasmic infection and ocular disease in populations is associated with country of birth and region of early childhood, rather than ethnicity.[36],[37] Differences between Brazil and Europe are unlikely to be due to anomalies in the regions included in the cohort. Within Europe, increasing latitude has been weakly associated with a diminished risk of clinical manifestations, but the European cohort was representative of all latitudes across western Europe with children recruited from southern, central and northern Europe. The Brazilian cohort was representative of the mix of public and private provision of health care in Brazil where 20% of the population have private health care but our findings differed from a previous cohort study of children with congenital toxoplasmosis in Brazil, which found that the risk of clinical manifestations (intracranial and/or ocular lesions) in the first year of life (17%; 8/47) was very similar to Europe.[27],[31] This low rate of ocular disease in the study by Neto et al may reflect inadequate follow up, a reduced risk in more affluent socioeconomic groups, or regional variation in the incidence of exposure to toxoplasmosis and risk of ocular disease in Brazil.[4],[6],[38] The study by Neto et al was based on children from all regions of Brazil whose families could afford private health care, whereas the present study was based in two endemic areas and only one-third of the children had private health care. However, we found no evidence that the risk of retinochoroiditis differed according to public (12/20) or private sector patients (8/10). Incidence rates of congenital toxoplasmosis in Porto Alegre are also similar in the private and public sectors (private 4.4/10,000 live births; 95%CI : 1.3–9.6, in 1998–2003; public 6.0/10,000; 95%CI: 2.4–12.5, in 2002; personal communication EG Lago, Porto Alegre). We suggest that the increased frequency and severity of ocular disease in Brazil compared with Europe is due to exposure to more virulent strains of T.gondii in Brazil. Type 1 and atypical strains appear to be associated with more severe ocular disease[18],[19] compared with type II strains, which predominate in Europe and North America.[16], [17], [38]–[41] The increased virulence of strains in Brazil compared with Europe may reflect more frequent sexual reproduction in the life-cycle of T.gondii in Brazil and human exposure to recombinant strains due to more frequent acquisition of infection via oocysts in Brazil.[42] Recent in-vitro studies suggest that the more virulent strains have a higher rate of growth, a remarkable ability to migrate across epithelial barriers to penetrate host tissue,[43] and can down regulate interleukin 12 more efficiently than the type II strain, which could impair the child's protective immune response.[20],[21],[44] The finding that the size of lesions diminished with time since infection has not been reported previously. Cautious interpretation is required as detection bias could explain this finding if small lesions are more readily detected as ophthalmoscopy becomes easier with age. Alternatively, lesions may be walled off more effectively as the host immune response matures or the organism load may diminish as bradyzoite cysts involute. Ophthalmologist predictions for visual impairment should also be regarded with caution. These were used as a surrogate measure as children in Brazil did not have visual acuity measures. However, limited accuracy in ophthalmologist prediction (85% specific and 59% sensitive for visual impairment of <6/18 Snellen),[45] is unlikely to explain the very large differences in predicted visual impairment observed between Brazil and Europe. Further research is required to determine whether virulence factors are associated with prolongation of the tachyzoite phase, which could create a longer therapeutic window before tissue cyst formation when anti-toxoplasmic treatment might be effective. Prospective cohort studies in Europe have shown no evidence for a protective effect of prenatal treatment on ocular disease and data from the EMSCOT study found no effect of postnatal treatment, although the power to detect an effect was limited.[31]–[33] Clearly postnatal treatment was not highly effective in 13/20 children in the Brazil cohort, who developed 17 new lesions while on treatment for at least two weeks (as did 15/50 children with18 lesions in the European cohort). Randomized controlled trials are needed most urgently in South America to determine the effectiveness of postnatal treatment for congenital toxoplasmosis and hence, whether neonatal screening is worthwhile. Extrapolation of results on treatment effectiveness across continents may not be justified in view of the possibility that pharmacodynamics might differ according to parasite genotype. Conclusions In Brazil, congenital toxoplasmosis resulted in more frequent and more severe ocular disease than in Europe. There is indirect evidence that these differences may be related to the predominance of virulent genotypes of the T.gondii parasite in Brazil. Randomized controlled trials are urgently needed in South America to determine treatment efficacy and the clinical effectiveness of neonatal screening. Supporting Information Alternative Language Article S1 Translation of the Article into Portuguese by Eleonor G. Lago (0.34 MB DOC) Click here for additional data file.
