**5. Immune privileged status and cytokine responses are key factors in toxoplasmic retinochoroiditis**

The pathogenesis of OT is directly linked to the anatomical characteristics of the eye resulting in an immune privileged status. The presence of the hemato-retinal barrier and the

absence of lymphatic vessels limits the passage of inflammatory cells and lymphocytes and of antibodies and complement components [34]. In addition, the ocular characteristics of the distribution and the functions of antigen presenting cells are also of importance. For example, corneal epithelium is deprived of Langerhans cells and the dendritic cells of the ciliary epithelium are not activated by GM-CSF and do not stimulate T lymphocytes [35, 36]. There is a low expression of classical MHC class IA molecules which reduce the lytic activity of CD8+ lymphocytes usually stimulated by the MHC I molecules. MHC II molecules are not expressed in the eye, which limits CD4+ lymphocyte activation. Increased expression of surface molecules like CD46, CD55 and CD59 will also inhibit complement activation [37]. A local production of immunosuppressive cytokines, such as TGF-β, limits B and Th1 lymphocyte activation but activates Th2 lymphocytes [34, 38, 39]. Finally, retinal cells express surface molecules involved in apoptosis such as TNF- Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL). FasL interacts with FasR (Fas receptor) carried by the inflammatory cells, inducing their apoptosis. This would control the entry of Fas-expressing lymphoid cells and limit the alteration of ocular cells by these cells [40, 41]. Whereas the mechanisms that underlie retinal damage in OT are yet not fully understood, the immune response might directly affect the pathogenesis of toxoplasmic retinochoroiditis and some cytokines have been shown to be fundamental to either control or block a protective response against *T. gondii* in experimental models. As early as 1998, Gazzinelli & Denkers [42] stated that initiating a strong T-cell-mediated immunity was crucial in the immune defense against *T. gondii*. High levels of gamma interferon (IFNγ) were induced by the parasite during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFNγ synthesis. They also stated that "while part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokinemediated immunopathologic changes may also contribute to disease progression". In animal experiments, many authors have described that IFNγ and TNFα, which enhance macrophage activation and induce production of other cytokines such as IL-12, give rise to a type Th1 immune response that plays a crucial role in parasite control [43]. These two cytokines could play a major role in immunological responses that control parasite proliferation by induction of indoleamine 2,3-dioxygenase production in retinal pigment epithelial cells [44]. Moreover, Gazzinelli et al. [45] observed that compared to control animals, mice treated with IFNγ or TNFα antagonists or antibodies against T cells (CD4 and CD8), showed more severe lesions characterized by exacerbated ocular damage and increased parasite detection in the eye. Conversely, a shift to a Th2 immune response with production of anti-inflammatory cytokines including IL-10, TGF-β and IL-4 promoted parasite survival, and was required to maintain immune privilege in the eye and prevent immune tissue destruction [46]. IFNγ and TNFα are also inhibitors of parasite replication in retinal pigment epithelial cells [47]. In humans, the participation of inflammatory mediators in physiopathology of OT is not yet clear. Nevertheless, a study by Yamamoto et al. [48] showed that asymptomatic patients secreted significantly more IL-12 and IFNγ in response to *T. gondii* antigens than patients with ocular damage. Conversely, acquired OT was associated with high levels of IL-1 and TNFα. They also observed that in comparison with Risk Factors, Pathogenesis and Diagnosis of Ocular Toxoplasmosis 133

non-infected subjects, IL-2 and IFNγ production by peripheral blood mononuclear cells in response to *T. gondii* was decreased in subjects with congenital infection, suggesting a status of parasite tolerance. Ongkosuwito et al. [49] measured the levels of six chemokines directly in aqueous humor samples from patients presenting with viral or toxoplasmic uveitis. Interestingly, IL-6 titers in patients with OT correlated with the degree of activity of toxoplasmic chorioretinitis. This cytokine is now described as essential in Th17 differentiation and Th17 cells are involved in inflammatory and autoimmune uveitis, supporting the hypothesis that the host immune response takes part in ocular damage [50]. The expansion of IL-17 producing cells in human OT has been demonstrated by Lahmar et al [51] who monitored cytokine patterns in serum and aqueous humor of subjects suffering from OT, infectious or non-infectious uveitis and cataract. High levels of IL-17 were reported in aqueous humor samples from 70 % patients presenting OT. Similar findings were also reported in patients suffering from other ocular inflammatory diseases showing that inflammatory processes could play a major role in the establishment of ocular damage in the chronic stage of OT. Due to large inter-individual variations of cytokine levels within each group of patients, no correlation was found between cytokine titers and clinical presentation. In addition, increased levels of pro-inflammatory mediators MCP-1, IL-8 and IL-6 were found in intraocular fluid samples from OT, but these variations were not specific for toxoplasmic uveitis [51]. IL-12 enhances TNF production and synthesis was higher in OT than in other ocular diseases in accordance with the importance of the Th1 response in mouse models. The Th2 cytokines (Il-4, IL-5, IL-10), which counterbalance inflammatory processes, were up-regulated and consequently the authors were unable to define the respective roles of Th1 and Th2 responses in the pathogenesis of human OT. As observed in experimental autoimmune uveitis, it is now proposed that eye damage may be induced by pathogenic responses mediated by Th-17 cells producing TNFα [52]. Conversely, host hypersensitivity pathways in the eye might be counterbalanced by IL-27 secretion upregulated by IFNγ from Th1 cells [52]. A possible association between polymorphisms in cytokine genes and OT was searched for in patients. Specific IL1, IL10 and IFNγ alleles were preferentially found in patients with OT. No such association was found with TNFα gene polymorphisms [53-56]. A putative summary of the role of the different cytokines and T cells in defense against the parasite but also in the occurrence of tissue lesions is

**6. Diagnosis is based on clinical signs and some selected biological** 

The diagnosis is usually based on ophthalmological examination showing unilateral, whitish, fuzzy-edged, round, focal lesions surrounded by retinal edema (figure 2). Cells are found in the vitreous, particularly overlying the active lesion. In the area surrounding the active retinitis, one may see hemorrhage, as well as sheathing of the retinal blood vessels. Fluorescein angiography of the active lesion demonstrates early blockage with subsequent leakage of the lesion. Cells in the anterior chamber may also be noted and may appear to be either a granulomatous or non granulomatous uveitis. The discovery of healed pigmented

summarized in figure 1.

**assays** 

non-infected subjects, IL-2 and IFNγ production by peripheral blood mononuclear cells in response to *T. gondii* was decreased in subjects with congenital infection, suggesting a status of parasite tolerance. Ongkosuwito et al. [49] measured the levels of six chemokines directly in aqueous humor samples from patients presenting with viral or toxoplasmic uveitis. Interestingly, IL-6 titers in patients with OT correlated with the degree of activity of toxoplasmic chorioretinitis. This cytokine is now described as essential in Th17 differentiation and Th17 cells are involved in inflammatory and autoimmune uveitis, supporting the hypothesis that the host immune response takes part in ocular damage [50]. The expansion of IL-17 producing cells in human OT has been demonstrated by Lahmar et al [51] who monitored cytokine patterns in serum and aqueous humor of subjects suffering from OT, infectious or non-infectious uveitis and cataract. High levels of IL-17 were reported in aqueous humor samples from 70 % patients presenting OT. Similar findings were also reported in patients suffering from other ocular inflammatory diseases showing that inflammatory processes could play a major role in the establishment of ocular damage in the chronic stage of OT. Due to large inter-individual variations of cytokine levels within each group of patients, no correlation was found between cytokine titers and clinical presentation. In addition, increased levels of pro-inflammatory mediators MCP-1, IL-8 and IL-6 were found in intraocular fluid samples from OT, but these variations were not specific for toxoplasmic uveitis [51]. IL-12 enhances TNF production and synthesis was higher in OT than in other ocular diseases in accordance with the importance of the Th1 response in mouse models. The Th2 cytokines (Il-4, IL-5, IL-10), which counterbalance inflammatory processes, were up-regulated and consequently the authors were unable to define the respective roles of Th1 and Th2 responses in the pathogenesis of human OT. As observed in experimental autoimmune uveitis, it is now proposed that eye damage may be induced by pathogenic responses mediated by Th-17 cells producing TNFα [52]. Conversely, host hypersensitivity pathways in the eye might be counterbalanced by IL-27 secretion upregulated by IFNγ from Th1 cells [52]. A possible association between polymorphisms in cytokine genes and OT was searched for in patients. Specific IL1, IL10 and IFNγ alleles were preferentially found in patients with OT. No such association was found with TNFα gene polymorphisms [53-56]. A putative summary of the role of the different cytokines and T cells in defense against the parasite but also in the occurrence of tissue lesions is summarized in figure 1.

132 Toxoplasmosis – Recent Advances

absence of lymphatic vessels limits the passage of inflammatory cells and lymphocytes and of antibodies and complement components [34]. In addition, the ocular characteristics of the distribution and the functions of antigen presenting cells are also of importance. For example, corneal epithelium is deprived of Langerhans cells and the dendritic cells of the ciliary epithelium are not activated by GM-CSF and do not stimulate T lymphocytes [35, 36]. There is a low expression of classical MHC class IA molecules which reduce the lytic activity of CD8+ lymphocytes usually stimulated by the MHC I molecules. MHC II molecules are not expressed in the eye, which limits CD4+ lymphocyte activation. Increased expression of surface molecules like CD46, CD55 and CD59 will also inhibit complement activation [37]. A local production of immunosuppressive cytokines, such as TGF-β, limits B and Th1 lymphocyte activation but activates Th2 lymphocytes [34, 38, 39]. Finally, retinal cells express surface molecules involved in apoptosis such as TNF- Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL). FasL interacts with FasR (Fas receptor) carried by the inflammatory cells, inducing their apoptosis. This would control the entry of Fas-expressing lymphoid cells and limit the alteration of ocular cells by these cells [40, 41]. Whereas the mechanisms that underlie retinal damage in OT are yet not fully understood, the immune response might directly affect the pathogenesis of toxoplasmic retinochoroiditis and some cytokines have been shown to be fundamental to either control or block a protective response against *T. gondii* in experimental models. As early as 1998, Gazzinelli & Denkers [42] stated that initiating a strong T-cell-mediated immunity was crucial in the immune defense against *T. gondii*. High levels of gamma interferon (IFNγ) were induced by the parasite during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFNγ synthesis. They also stated that "while part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokinemediated immunopathologic changes may also contribute to disease progression". In animal experiments, many authors have described that IFNγ and TNFα, which enhance macrophage activation and induce production of other cytokines such as IL-12, give rise to a type Th1 immune response that plays a crucial role in parasite control [43]. These two cytokines could play a major role in immunological responses that control parasite proliferation by induction of indoleamine 2,3-dioxygenase production in retinal pigment epithelial cells [44]. Moreover, Gazzinelli et al. [45] observed that compared to control animals, mice treated with IFNγ or TNFα antagonists or antibodies against T cells (CD4 and CD8), showed more severe lesions characterized by exacerbated ocular damage and increased parasite detection in the eye. Conversely, a shift to a Th2 immune response with production of anti-inflammatory cytokines including IL-10, TGF-β and IL-4 promoted parasite survival, and was required to maintain immune privilege in the eye and prevent immune tissue destruction [46]. IFNγ and TNFα are also inhibitors of parasite replication in retinal pigment epithelial cells [47]. In humans, the participation of inflammatory mediators in physiopathology of OT is not yet clear. Nevertheless, a study by Yamamoto et al. [48] showed that asymptomatic patients secreted significantly more IL-12 and IFNγ in response to *T. gondii* antigens than patients with ocular damage. Conversely, acquired OT was associated with high levels of IL-1 and TNFα. They also observed that in comparison with

#### **6. Diagnosis is based on clinical signs and some selected biological assays**

The diagnosis is usually based on ophthalmological examination showing unilateral, whitish, fuzzy-edged, round, focal lesions surrounded by retinal edema (figure 2). Cells are found in the vitreous, particularly overlying the active lesion. In the area surrounding the active retinitis, one may see hemorrhage, as well as sheathing of the retinal blood vessels. Fluorescein angiography of the active lesion demonstrates early blockage with subsequent leakage of the lesion. Cells in the anterior chamber may also be noted and may appear to be either a granulomatous or non granulomatous uveitis. The discovery of healed pigmented

Risk Factors, Pathogenesis and Diagnosis of Ocular Toxoplasmosis 135

retinochoroidal scars facilitates the diagnosis [57, 58]. OT is also confirmed by a favorable clinical response to specific therapy. However, diagnosis and treatment can be delayed in patients with atypical lesions (unusual and complicated forms) or patients showing an inadequate response to antimicrobial therapy as particularly observed in elderly or immunocompromised patients [13, 17]. In such cases, rapid identification of the causative agent requires aqueous humor sampling by anterior chamber paracentesis [59]. Laboratory diagnosis is based on the comparison of antibody profiles in ocular fluid and serum samples in order to detect intraocular specific antibody synthesis, based on the Goldmann-Witmer coefficient (GWC) or on the observation of qualitative differences between eye fluid and serum by immunoblotting (IB) [60]. The GWC is based on the comparison of the levels of specific antibodies to total immunoglobulin in both aqueous humor and serum. Recent studies have shown the usefulness of PCR applied to aqueous humor, in combination with serologic tests, for the diagnosis of OT [61-68]. However, although this combined approach improves diagnostic sensitivity, the volume of the ocular fluid sample may not be adequate for PCR, IB, and GWC. We showed [68] that a combination of all three methods had a 85% sensitivity and a 93% specificity for the diagnosis of atypical or extensive toxoplasmic retinochoroiditis. The sensitivity of GWC alone for atypical uveitis (based mainly on aqueous humor samples) ranges from 39% to 93% [60, 63, 65, 67, 69-71]. Discrepancies could be explained by differences in (i) the interval between symptom onset and paracentesis, (ii) the characteristics of the uveitis (typical or atypical), (iii) underlying immunological status, and (iv), the chosen GWC positivity threshold, which ranges from 2 to 8 in the literature. The specificity of the GWC is usually high if the retinal barrier has not been impaired. IB on aqueous humor has sensitivities ranging from 50 to 81% for the diagnosis of atypical [60, 65] and typical [66, 68, 69] OT. Apparently the sensitivity of IB increases with the length of the interval between onset of symptoms and paracentesis. The sensitivity of real-time PCR ranges from 36 % to 55% [63, 67, 68]. The sensitivity was higher with a real-time PCR assay targeting the *T. gondii* repeat element of 529 base pairs [68] than with real-time PCR targeting the B1 gene (40% and 36% respectively). Real-time PCR has been shown to be more sensitive on a variety of samples when the 529-bp repeat element rather than the B1 gene was used as a target [71]. In contrast to the IB and GWC results, the results of PCR are not influenced by the interval between symptom onset and paracentesis. The total size of acute retinal foci is larger in PCR-positive patients [64, 67, 68]. PCR seems more informative than the GWC and IB for immunocompromised patients [62, 64]. The rate of detection of specific intraocular antibodies seems related to the interval between symptom onset and paracentesis. Early sampling is often associated with negative GWC results and with low IB sensitivity. The sensitivity of the GWC increases when sampling is performed at least 10 days after symptom onset, and IB was positive for 72% of cases 30 days after symptom onset [68]. Several studies have examined the influence of this interval on GWC results. Fardeau et al. [64] reported that the GWC was useless during the first 2 weeks but that its sensitivity increased sharply when anterior chamber puncture was performed between the 3rd and 8th week after symptom onset. Garweg et al. [69] showed that GWC sensitivity increased from 57% to 70% when puncture was performed at 6 weeks instead of 3 weeks. As stated above, PCR sensitivity was not influenced by this interval. Combining the three biological

**Figure 1.** T cells and cytokines involved in ocular toxoplasmosis and parasite destruction

**Figure 2.** Eye fundus aspects of ocular toxoplasmosis. a: active lesion; b, d: scar; c: active lesion and scars (source A.P. Brézin and Anofel)

retinochoroidal scars facilitates the diagnosis [57, 58]. OT is also confirmed by a favorable clinical response to specific therapy. However, diagnosis and treatment can be delayed in patients with atypical lesions (unusual and complicated forms) or patients showing an inadequate response to antimicrobial therapy as particularly observed in elderly or immunocompromised patients [13, 17]. In such cases, rapid identification of the causative agent requires aqueous humor sampling by anterior chamber paracentesis [59]. Laboratory diagnosis is based on the comparison of antibody profiles in ocular fluid and serum samples in order to detect intraocular specific antibody synthesis, based on the Goldmann-Witmer coefficient (GWC) or on the observation of qualitative differences between eye fluid and serum by immunoblotting (IB) [60]. The GWC is based on the comparison of the levels of specific antibodies to total immunoglobulin in both aqueous humor and serum. Recent studies have shown the usefulness of PCR applied to aqueous humor, in combination with serologic tests, for the diagnosis of OT [61-68]. However, although this combined approach improves diagnostic sensitivity, the volume of the ocular fluid sample may not be adequate for PCR, IB, and GWC. We showed [68] that a combination of all three methods had a 85% sensitivity and a 93% specificity for the diagnosis of atypical or extensive toxoplasmic retinochoroiditis. The sensitivity of GWC alone for atypical uveitis (based mainly on aqueous humor samples) ranges from 39% to 93% [60, 63, 65, 67, 69-71]. Discrepancies could be explained by differences in (i) the interval between symptom onset and paracentesis, (ii) the characteristics of the uveitis (typical or atypical), (iii) underlying immunological status, and (iv), the chosen GWC positivity threshold, which ranges from 2 to 8 in the literature. The specificity of the GWC is usually high if the retinal barrier has not been impaired. IB on aqueous humor has sensitivities ranging from 50 to 81% for the diagnosis of atypical [60, 65] and typical [66, 68, 69] OT. Apparently the sensitivity of IB increases with the length of the interval between onset of symptoms and paracentesis. The sensitivity of real-time PCR ranges from 36 % to 55% [63, 67, 68]. The sensitivity was higher with a real-time PCR assay targeting the *T. gondii* repeat element of 529 base pairs [68] than with real-time PCR targeting the B1 gene (40% and 36% respectively). Real-time PCR has been shown to be more sensitive on a variety of samples when the 529-bp repeat element rather than the B1 gene was used as a target [71]. In contrast to the IB and GWC results, the results of PCR are not influenced by the interval between symptom onset and paracentesis. The total size of acute retinal foci is larger in PCR-positive patients [64, 67, 68]. PCR seems more informative than the GWC and IB for immunocompromised patients [62, 64]. The rate of detection of specific intraocular antibodies seems related to the interval between symptom onset and paracentesis. Early sampling is often associated with negative GWC results and with low IB sensitivity. The sensitivity of the GWC increases when sampling is performed at least 10 days after symptom onset, and IB was positive for 72% of cases 30 days after symptom onset [68]. Several studies have examined the influence of this interval on GWC results. Fardeau et al. [64] reported that the GWC was useless during the first 2 weeks but that its sensitivity increased sharply when anterior chamber puncture was performed between the 3rd and 8th week after symptom onset. Garweg et al. [69] showed that GWC sensitivity increased from 57% to 70% when puncture was performed at 6 weeks instead of 3 weeks. As stated above, PCR sensitivity was not influenced by this interval. Combining the three biological

134 Toxoplasmosis – Recent Advances

**Figure 1.** T cells and cytokines involved in ocular toxoplasmosis and parasite destruction

**Figure 2.** Eye fundus aspects of ocular toxoplasmosis. a: active lesion; b, d: scar; c: active lesion and

scars (source A.P. Brézin and Anofel)

techniques increases the sensitivity and the specificity but sometimes the volume size of the sample is so small that it is not possible to perform all three. On the basis of the presented results, we propose an algorithm for choosing the test with the best sensitivity according to ophthalmologic findings and delay after onset of the disease (figure 3). When paracentesis is performed during the 10 days following symptom onset, real-time PCR is most suitable, especially if the patient is immunocompromised or if the total size of the foci is large (> 2 optic disc diameters). Beyond 10 days, the best choice is the GWC if old scars are present and/or if the reaction in the anterior chamber is mild to severe, or PCR if the total size of foci is large (>2 optic disc diameters); IB should be preferred when paracentesis is performed more than 30 days after symptom onset.

Risk Factors, Pathogenesis and Diagnosis of Ocular Toxoplasmosis 137

localizations and this is now possible because the recommended association pyrimethamine/azythromycine is better tolerated and has a better compliance than pyrimethamine associated to sulfadiazine [76, 77]. Corticosteroids (prednisone at 0.5 to 1mg/kg/d) are constantly administered for several weeks, except for immunocompromised patients [73, 78]. Pyrimethamine in adults is used at 100mg/d for several days then decreased at 50mg/d. It should be associated with sulfadiazine at 75mg/kg/d divided in 4 doses or better with azythromycine 250 mg/d. The total length of the treatment will be of 3 to 6 weeks, sometimes more, depending on the initial size of the lesion. In patients intolerant to treatment, clindamycine at 450-600 mg/d should be associated [79]. The treatment of congenital retinochoroiditis in newborns is based on sulfadiazine (50mg/kg/d in 2 doses) associated with pyrimethamine at 1mg/kg/d for 6 to 12 months. Fifteen mg folinic acid is given every 3 days. The prophylaxis of congenitally acquired OT is based on national programs of prevention of CT (e.g. France, Austria) but their efficiency is discussed [80-82] and depends on the local epidemiology and virulence of strains [83]. Peyron et al. [84] stated that "treating CT has little effect on the quality of life and visual function of the affected individuals". However, Kieffer et al. [10] showed that a period exceeding 8 weeks between maternal infection and the beginning of treatment was a risk factor for retinochoroiditis; therefore emphasizing the need to prevent and treat CT. Evidence for the effectiveness of prenatal or postnatal treatment for CT is still needed. Randomised controlled trials and cohort studies are in progress to provide information on prognosis, especially disability [85]. There is no radical prevention of acquired toxoplasmosis besides hygienic rules in preparing meals. Eating well done or deeply frozen meat should be particularly recommended in regions where highly pathogenic isolates are prevalent. In HIV patients, drug prevention of toxoplasmosis has been successfully used for years and is now less needed since the use of efficient HAART.

Acquired or CT can be complicated by OT. The diagnosis relies on clinical aspects, responses to specific treatment and results of biological assays. The incidence and the prevalence of this complication are both difficult to establish precisely and depend on the parasite prevalence in the general population, and are affected by different factors such as type of exposure to the parasite, genetic background of the different parasites and the host, and the type of immune response elicited by the parasite. Prevention of CT (though still discussed), and a rapid specific treatment of acquired cases could be the key measures to avoid severe visual impairment but evaluation of these procedures is urgently needed.

, Hana Talabani, Florence Leslé and Hélène Yera *Service de Parasitologie-Mycologie, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris,* 

**8. Conclusions** 

**Author details** 

 \*

Jean Dupouy-Camet\*

Corresponding Author

*Université Paris Descartes, Paris, France* 

**Figure 3.** Algorithm for the biological diagnosis of ocular toxoplasmosis (in severely immunosuppressed patients a negative serology does not exclude OT which can be then confirmed by PCR)
