Murine Model of Primary Acquired Ocular Toxoplasmosis: Fluorescein Angiography and Multiplex Immune Mediator Profiles in the Aqueous Humor.

Purpose: To establish a murine model of primary acquired ocular toxoplasmosis (OT) and to investigate the immune mediator profiles in the aqueous humor (AH). Methods: C57BL/6 mice were perorally infected with Toxoplasma gondii. The ocular fundus was observed, and fluorescein angiography (FA) was performed. The AH, cerebrospinal fluid (CSF), and serum were collected before infection and at 28 days post-infection (dpi); the immune mediator levels in these samples were analyzed using multiplex bead assay. Results: Fundus imaging revealed soft retinochoroidal lesions at 14 dpi; many of these lesions became harder by 28 dpi. FA abnormalities, such as leakage from retinal vessels and dilation and tortuosity of the retinal veins, were observed at 14 dpi. Nearly all these abnormalities resolved spontaneously at 28 dpi. In the AH, interferon-γ, interleukin (IL)-1α, IL-1ß, IL-6, IL-10, IL-12(p40), IL-12(p70), CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß, CCL5/RANTES, and CXCL1/KC levels increased after infection. All these molecules except IL-1α, IL-4, and IL-13 showed almost the same postinfection patterns in the CSF as they did in the AH. The tumor necrosis factor α, IL-4, and IL-5 levels in the AH and CSF of the T. gondii-infected mice were lower than those in the serum. The postinfection IL-1α, IL-6, CCL2/MCP-1, CCL4/MIP-1ß, and granulocyte colony-stimulating factor levels in the AH were significantly higher than those in the CSF and serum. Conclusions: A murine model of primary acquired OT induced via the natural infection route was established. This OT model allows detailed ophthalmologic, histopathologic, and immunologic evaluations of human OT. Investigation of AH immune modulators provides new insight into OT immunopathogenesis.

Clinical manifestations and visual outcomes associated with ocular toxoplasmosis in a Brazilian population.

Ocular toxoplasmosis is the leading cause of posterior uveitis worldwide. We conducted an observational study of 262 consecutive individuals (n = 344 eyes) with ocular toxoplasmosis who were followed over a 34-month period. Most subjects were T. gondii IgG + /IgM- (n = 242; 92.4%; 317 eyes), and 140 eyes (40.7%) had active lesions. For eyes in which retinal lesions were active at recruitment and best-corrected visual acuity (BCVA) could be measured (n = 133), 21.0% (n = 28) remained blind (BCVA below 20/400) after inflammation resolved. In these eyes, atypical ocular toxoplasmosis (OR 4.99; 95% CI 1.14-22.85; p = 0.0330), macular lesion (OR 9.95; 95% CI 2.45-47.15; p = 0.0019) and any complication (OR 10.26; 95% CI 3.82-30.67; p < 0.0001) were associated with BCVA below 20/200. For eyes with only inactive lesions at recruitment and BCVA measured (n = 178), 28.1% (n = 50) were blind. In these eyes, having at least one lesion larger than one disc-diameter (OR 6.30; 95% CI 2.28-22.46; p = 0.0013) and macular lesion (OR 5.69; 95% CI 2.53-13.54; p < 0.0001) were associated with BCVA below 20/200. Older age (OR 1.02; 95% CI 1.00-1.05; p = 0.0493) and active disease at presentation (OR 4.74; 95% CI 1.95-12.91; p = 0.0011) were associated with recurrences. Additional clinical attention should be directed towards patients with risk factors for poor visual outcome.

Optical Coherence Tomography Angiography Analysis of Retinal and Choroidal Vascular Networks during Acute, Relapsing, and Quiescent Stages of Macular Toxoplasma Retinochoroiditis.

PURPOSE: To highlight the advantages of optical coherence tomography angiography (OCTA) in delineating the morphological features of the retinal and choroidal vascular network during acute, relapsing, and quiescent stages of macular toxoplasma retinochoroiditis. METHODS: This prospective study included patients presenting with both active and quiescent ocular toxoplasmoses. OCTA was obtained to diagnose and follow the subsequent vascular network changes at diagnosis and six months after acute presentation. RESULTS: Twenty-three eyes of 23 patients were included. In active lesions, OCTA showed extensive, well-delineated areas of intense hyposignal and perifoveal capillary arcade disruption in the parafoveal superficial capillary plexus (pSCP) and less extensive hyposignal in the parafoveal deep capillary plexus (pDCP). Signals of decreased deep capillary density and disorganization were also seen in the choroid. In nonactive lesions, OCTA demonstrated a homogenous and equally attenuated grayish hyposignal of the pSCP and pDCP and a partial restoration of the nonperfused choroidal areas. CONCLUSION: OCTA is a useful technique for vascular network analysis in toxoplasma retinochoroiditis. It allows the visualization of the different network changes and behaviors during the different stages of the infection.

Case Report: Acute Vision Loss in a Young Returning Traveler with Dengue Fever.

Ocular complications are rare in patients with dengue fever, but may cause permanent loss of vision. We present the case of a 29-year-old German woman who developed severe acute vision loss because of dengue-associated maculopathy after traveling to Vietnam and Cambodia. Initially, the optical coherence tomography showed detachment of the retinal pigment epithelium, a central shift in the retinal pigmentation and intraretinal cysts. The patient was hospitalized and treated with a short course of intravenous prednisolone. Vision improved, and the patient showed full recovery at 9 months after the onset. This case highlights the importance of awareness and adequate management for ocular involvement in patients with dengue fever, including travelers.

Histological and Behavioral Toxicity of Tributyltin in the Tropical Guppy Poecilia vivipara.

The tropical estuarine guppy Poecilia vivipara was used to address fish early life stage toxicity caused by the antifouling contaminant tributyltin. Six-day-old P. vivipara were exposed for 7 d to control water and waterborne tributyltin at 15.8, 83.8, 716, and 818 ng tin (Sn) L-1 . After exposure, swimming, feeding, growth, and eye histological endpoints were evaluated. Histopathological analysis of the retinal pigment epithelium (RPE) indicated alterations in pigment positioning at all tributyltin concentrations. A dose-dependent increase in photoreceptor layer disorganization and iris melanin hyperpigmentation was verified, and high frequencies of RPE invaginations and iris epithelial cell atrophy were observed even at the lowest exposure concentration of 15.8 ng Sn L-1 . At the highest exposure level (818 ng Sn L-1 ) fish also presented reductions in swimming speed, swimming resistance, daily capture of Artemia nauplii, and growth in weight of 85, 60, 33, and 56% relative to controls, respectively. This association between retinal histopathology and reduced swimming and foraging behavior can reduce recruitment to the adult population. Environ Toxicol Chem 2020;39:1953-1963. © 2020 SETAC.

Chorio-retinal toxoplasmosis: treatment outcomes, lesion evolution and long-term follow-up in a single tertiary center.

BACKGROUND: Ocular toxoplasmosis is a common cause of ocular inflammation worldwide. The aim of this study is to characterize the clinical outcomes and lesion evolution of patients with ocular toxoplasmosis and to compare the primary and reactivation subgroups. METHODS: A retrospective population-based cohort study at one uveitis-specialized tertiary referral center. Patients presenting with active ocular toxoplasmosis between the years 2007-2016 were included. Primary ocular toxoplasmosis and reactivations were compared. RESULTS: Included were 22 patients, 64% female with a mean age of 29 ± 18 years, 59% (n = 13) were primary, 9% (n = 2) congenital and 32% (n = 7) reactivations. Visual acuity improved from 0.38 ± 0.44 to 0.20 ± 0.27 LogMAR (P = 0.026) after a mean of 37 ± 33 months. Initial lesion size was 2.38 ± 1.1 optic disc areas, reducing to 1.56 ± 1.24 following 2 months (34% reduction, P = 0.028) and to 1.17 ± 0.87 disc areas following one year (51% reduction, P = 0.012). Patients with macula-threatening lesions had worse visual acuity (0.50 ± 0.46 vs. 0.05 ± 0.07 LogMAR, P = 0.047). Primary and reactivation subgroups had similar presentations, visual outcomes and recurrence rates (all P > 0.05). CONCLUSIONS: In this population, primary ocular toxoplasmosis was the most common presentation. Lesion size reduced during the initial months with limited change thereafter and a third of cases recurred. Macula-threatening lesions were associated with worse visual acuity, and no significant differences were seen between the primary and reactivation subgroups.

Model Systems for Studying Mechanisms of Ocular Toxoplasmosis.

The most common human disease caused by infection with Toxoplasma gondii is ocular toxoplasmosis, which typically is manifest as recurrent attacks of necrotizing retinal inflammation with subsequent scarring. The multilayered retina contains specialized cell populations, including endothelial cells, epithelial cells, neurons and supporting cells, all of which may be involved in this condition. In vitro investigations of basic mechanisms operating in human ocular toxoplasmosis use cellular and molecular methods that are common to the study of many pathological processes, and the novel aspect of this research is the use of human retinal cell subsets. Most in vivo research on ocular toxoplasmosis is conducted in the laboratory mouse. Experimental models involve local or systemic inoculation of parasites to induce acute disease, or sequential systemic and local parasite inoculations to trigger recurrent disease. We present methods for in vitro and in vivo studies of ocular toxoplasmosis, including dissection of the human eye, and culture and infection of differentiated cell populations from the retina, as well as induction of mouse ocular toxoplasmosis by intraocular, or sequential systemic and intraocular, inoculations, and imaging of toxoplasmic retinal lesions.

CD40 in Endothelial Cells Restricts Neural Tissue Invasion by Toxoplasma gondii.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.

Type I ROP16 regulates retinal inflammatory responses during ocular toxoplasmosis.

Ocular toxoplasmosis (OT), mostly retinochorioditis, is a major feature of infection with the protozoan parasite Toxoplasma gondii. The pathophysiology of this infection is still largely elusive; especially mouse models are not yet well developed. In contrast, numerous in vitro studies showed the highly Toxoplasma strain dependent nature of the host-parasite interactions. Some distinct polymorphic virulence factors were characterized, notably the rhoptry protein ROP16. Here, we studied the strain-dependent pathophysiology in our OT mouse model. Besides of two wild type strains of the canonical I (RH, virulent) and II (PRU, avirulent) types, we used genetically engineered parasites, RHΔROP16 and PRU ROP16-I, expressing the type I allele of this virulence factor. We analyzed retinal integrity, parasite proliferation and retinal expression of cytokines. PRU parasites behaved much more virulently in the presence of a type I ROP16. In contrast, knockout of ROP16 in the RH strain led to a decrease of intraocular proliferation, but no difference in retinal pathology. Cytokine quantification in aqueous humor showed strong production of Th1 and inflammatory markers following infection with the two strains containing the ROP16-I allele. In strong contrast, immunofluorescence images showed that actual expression of most cytokines in retinal cells is rapidly suppressed by type I strain infection, with or without the involvement of its homologous ROP16 allele. This demonstrates the particular immune privileged situation of the retina, which is also revealed by the fact that parasite proliferation is nearly exclusively observed outside the retina. In summary, we further developed a promising OT mouse model and demonstrated the specific pathology in retinal tissues.

Isolation of viable Toxoplasma gondii from organs and Brazilian commercial meat cuts of experimentally infected pigs.

The present study evaluated the distribution and viability of Toxoplasma gondii tissue cysts in the organs and Brazilian commercial cuts of experimentally infected pigs. The pigs were infected with 3 × 103 oocysts of the T. gondii isolate TgCkBr57 (Type BrII). Mouse bioassays were performed on the brain, retina, tongue, diaphragm, and heart as well as the following muscle cuts: loin (longissimus), coppa (longissimus, spinalis dorsi, rhomboideus), tenderloin (psoas major), outside flat (biceps femoris), topside (semimembranosus), and top sirloin (gluteus medius). Toxoplasma gondii was isolated from the coppa, heart, diaphragm, and tongue of three pigs; from the tenderloin, outside flat, and brain of two pigs; and from the top sirloin and loin of one pig. Thus, the viability of T. gondii cysts was observed in all of the organs and cuts evaluated (except for the topside and retina), demonstrating the broad distribution of this parasite in pig organs and commercial meat cuts, and the importance of this species as a source of human infection.

Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii.

Little is known about strategies used by pathogens to facilitate CNS invasion. Toxoplasma gondii reaches the CNS by circulating in blood within leukocytes or as extracellular tachyzoites. T. gondii induces EGFR signaling in vitro during invasion of mammalian cells. We examined the effects of endothelial cell EGFR on CNS invasion. Transgenic mice whose endothelial cells expressed a dominant negative (DN) EGFR (inhibits EGFR signaling) exhibited diminished parasite load and histopathology in the brain and retina after T. gondii infection. I.V. administration of infected leukocytes or extracellular tachyzoites led to reduced parasite loads in mice with DN EGFR. This was not explained by enhanced immunity or reduced leukocyte recruitment. Endothelial cell infection is key for CNS invasion. Parasite foci in brain endothelial cells were reduced by DN EGFR. DN EGFR in these cells led to recruitment of the autophagy protein LC3 around T. gondii and spontaneous parasite killing dependent on the autophagy protein ULK1 and lysosomal enzymes. The autophagy inhibitor 3-MA prevented DN EGFR mice from exhibiting reduced CNS invasion. Altogether, EGFR is a novel regulator of T. gondii invasion of neural tissue, enhancing invasion likely by promoting survival of the parasite within endothelial cells.

Subretinal Cysticercosis Extraction With Bimanual, 3-D, Heads-Up-Assisted Pars Plana Vitrectomy: Clinicopathological Correlation and Surgical Technique.

A 16-year-old male presented with blurred vision in the right eye after recent travel to Nicaragua. Funduscopic examination revealed subretinal cysticercosis superior to the optic nerve. The cyst was drained and excised using a bimanual, three-dimensional, heads-up-assisted pars plana vitrectomy without complications. Technical maneuvers for cyst extraction along with clinicopathological correlation are described. Postoperatively, the patient exhibits no signs of recurrence and has excellent vision. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:708-711.].

Toxoplasma retinitis following intravitreal injection of triamcinolone acetonide: A case report and review of literature.

The aim of this study was to report a case of atypical toxoplasma retinochoroiditis following intravitreal triamcinolone acetonide (IVTA) injection and to review the literature pertaining to toxoplasma retinochoroiditis following intravitreal injection of corticosteroid. Clinical data were collected from a 64-year-old male who developed toxoplasma retinitis 2 months after IVTA. A review of the literature was conducted to identify additional reports on similar cases. A 64-year-old male, known diabetic with nonproliferative diabetic retinopathy in both the eyes and optic atrophy in the left eye, presented with atypical retinitis inferior to the disc following IVTA. Real-time polymerase chain reaction and serology confirmed the toxoplasma etiology, and the patient was started on anti-toxoplasma therapy along with oral corticosteroid leading to regression of the lesion by 3 months. A high index of suspicion and proper microbiological diagnosis with appropriate antimicrobial therapy can aid in the management of toxoplasma retinochoroiditis following intravitreal injection of corticosteroid.

Can the route of Toxoplasma gondii infection affect the ophthalmic outcomes?

Ocular toxoplasmosis is the most common cause of retinochoroiditis worldwide in humans. Some studies highlighted the idea that ocular lesions differ according to the route of infection but none of them mimicked the natural route. The current study aimed to investigate the ophthalmic outcomes in congenital and oral routes of infection with Toxoplasma in experimental animals. Mice were divided into three groups; group I: congenital infection, group II: acquired oral infection and group III: non-infected. We used Me49 chronic low-virulence T. gondii strain. We found that retina is the most affected part in both modes of infections. However, the retinal changes are different and more pronounced in case of congenital infection. The congenitally infected mice showed retinal lesions e.g. total detachment of retinal pigment epithelium from the photoreceptor layer and irregular arrangement of retinal layers. More severe damage was observed in mice infected early in pregnancy. While the postnatal orally infected mice showed fewer changes. In conclusion, the routes of Toxoplasma infection affect the ophthalmic outcomes and this may be the case in human disease. Although both are vision threatening, it seems that the prognosis of postnatal acquired ocular toxoplasmosis is better than that of congenital disease.

Investigation of tissue cysts in the retina in a mouse model of ocular toxoplasmosis: distribution and interaction with glial cells.

The conversion of tachyzoites into bradyzoites is a way for Toxoplasma gondii to establish a chronic and asymptomatic infection and achieve lifelong persistence in the host. The bradyzoites form tissue cysts in the retina, but not much is known about the horizontal distribution of the cysts or their interactions with glial cells in the retina. A chronic ocular toxoplasmosis model was induced by per oral administration of T. gondii Me49 strain cysts to BALB/c mice. Two months after the infection, retinas were flat-mounted and immunostained to detect cysts, ganglion cells, Müller cells, astrocytes, and microglial cells, followed by observation under fluorescence and confocal microscope. The horizontal distribution showed a rather clustered pattern, but the clusters were not restricted to certain location of the retina. Axial distribution was confined to the inner retina, mostly in ganglion cell layer or the inner plexiform layer. Both ganglion cells, a type of retinal neurons, and Müller cells, predominant retinal glial cells, could harbor cysts. The cysts were spatially separated from astrocytes, the most abundant glial cells in the ganglion cell layer, while close spatial distribution of microglial cells was observed in two thirds of retinal cysts. In this study, we demonstrated that the retinal cysts were not evenly distributed horizontally and were confined to the inner retina axially. Both neurons and one type of glial cells could harbor cysts, and topographic analysis of other glial cells suggests role of microglial cells in chronic ocular toxoplasmosis.

Tissue-specific immunopathology during malaria infection.

Systemic inflammation mediated by Plasmodium parasites is central to malaria disease and its complications. Plasmodium parasites reside in erythrocytes and can theoretically reach all host tissues via the circulation. However, actual interactions between parasitized erythrocytes and host tissues, along with the consequent damage and pathological changes, are limited locally to specific tissue sites. Such tissue specificity of the parasite can alter the outcome of malaria disease, determining whether acute or chronic complications occur. Here, we give an overview of the recent progress that has been made in understanding tissue-specific immunopathology during Plasmodium infection. As knowledge on tissue-specific host-parasite interactions accumulates, better treatment modalities and targets may emerge for intervention in malaria disease.

Cerebral malaria induces electrophysiological and neurochemical impairment in mice retinal tissue: possible effect on glutathione and glutamatergic system.

BACKGROUND: Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has usually been associated with cognitive, behavioural and motor dysfunctions, being the retinopathy the most serious consequence resulting from the disease. The pathophysiological mechanisms underlying this complication remain incompletely understood. Several experimental models of CM have already been developed in order to clarify those mechanisms related to this syndrome. In this context, the present work has been performed to investigate which possible electrophysiological and neurochemistry alterations could be involved in the CM pathology. METHODS: Experimental CM was induced in Plasmodium berghei-infected male and female C57Bl/6 mice. The survival and neurological symptoms of CM were registered. Brains and retina were assayed for TNF levels and NOS2 expression. Electroretinography measurements were recorded to assessed a- and b-wave amplitudes and neurochemicals changes were evaluated by determination of glutamate and glutathione levels by HPLC. RESULTS: Susceptible C57Bl/6 mice infected with ≈ 106 parasitized red blood cells (P. berghei ANKA strain), showed a low parasitaemia, with evident clinical signs as: respiratory failure, ataxia, hemiplegia, and coma followed by animal death. In parallel to the clinical characterization of CM, the retinal electrophysiological analysis showed an intense decrease of a- and-b-wave amplitude associated to cone photoreceptor response only at the 7 days post-infection. Neurochemical results demonstrated that the disease led to a decrease in the glutathione levels with 2 days post inoculation. It was also demonstrated that the increase in the glutathione levels during the infection was followed by the increase in the 3H-glutamate uptake rate (4 and 7 days post-infection), suggesting that CM condition causes an up-regulation of the transporters systems. Furthermore, these findings also highlighted that the electrophysiological and neurochemical alterations occurs in a manner independent on the establishment of an inflammatory response, once tumour necrosis factor levels and inducible nitric oxide synthase expression were altered only in the cerebral tissue but not in the retina. CONCLUSIONS: In summary, these findings indicate for the first time that CM induces neurochemical and electrophysiological impairment in the mice retinal tissue, in a TNF-independent manner.