Fábio Batista Frederico

Contribution of laboratory methods in diagnosing clinically suspected ocular toxoplasmosis in Brazilian patients

This prospective study evaluated the value of laboratorial diagnosis in ocular toxoplasmosis analyzing peripheral blood samples from a group of Brazilian patients by immunologic and molecular methods. We analyzed blood samples from 184 immunocompetent patients with ocular disorders divided into 2 groups: Group I, composed of samples from 49 patients with ocular toxoplasmosis diagnosed by clinical features; Group II, samples from 135 patients with other ocular diseases. Samples were assayed by conventional polymerase chain reaction (cnPCR), real-time PCR (qPCR) for Toxoplasma gondii, indirect immunofluorescence reaction (IF), avidity test (crude tachyzoite lysate as antigen), and excreted-secreted tachyzoite proteins as antigen (ESA-ELISA). cnPCR and qPCR profiles were concordant in all samples. Positive PCR was shown in 40.8% of group I patients. The majority of the positive blood samples (75%) were taken from patients with toxoplasmic retinochoroiditis scars, and the others (25%), from patients with retinal exudative lesions. Despite that 86 of the 135 patients from Group II had asymptomatic toxoplasmosis, all DNA blood samples had negative PCR. Concordant results were shown in the data obtained by serologic methods. Around 24% of the patients with ocular toxoplasmosis had high antibody titers determined by ESA-ELISA and IF. Anti-ESA antibodies are shown principally in patients with active infection. Collectively, these data demonstrate the presence of tachyzoites in the blood of patients with chronic infection, supporting the idea of recurrent disease. Circulating parasites in blood of immunocompetent individuals may be associated with the reactivation of the ocular disease.

Risk factors for ocular toxoplasmosis in Brazil

SUMMARY The aim of this study was to investigate risk factors for ocular toxoplasmosis (OT) in patients who received medical attention at a public health service. Three hundred and forty-nine consecutive patients, treated in the Outpatient Eye Clinic of Hospital de Base, São José do Rio Preto, São Paulo state, Brazil, were enrolled in this study. After an eye examination, enzyme-linked immunosorbent assay (ELISA) was used to determine anti-Toxoplasma gondii antibodies. The results showed that 25·5% of the patients were seronegative and 74·5% were seropositive for IgG anti-T. gondii antibodies; of these 27·3% had OT and 72·7% had other ocular diseases (OOD). The presence of cats or dogs [odds ratio (OR) 2·22, 95% confidence interval (CI) 1·24–3·98, P = 0·009] and consumption of raw or undercooked meat (OR 1·77, 95% CI 1·05–2·98, P = 0·03) were associated with infection but not with the development of OT. Age (OT 48·2 ± 21·2 years vs. OOD: 69·5 ± 14·7 years, P < 0·0001) and the low level of schooling/literacy (OT vs. OOD: OR 0·414, 95% CI 0·2231–0·7692, P = 0·007) were associated with OT. The presence of dogs and cats as well as eating raw/undercooked meat increases the risk of infection, but is not associated with the development of OT.

Cerebral and ocular toxoplasmosis related with IFN-γ, TNF-α, and IL-10 levels.

This study analyzed the synthesis of Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), and Interleukin 10 (IL-10) in chronically infected patients which developed the symptomatic disease as cerebral or ocular toxoplasmosis. Blood from 61 individuals were divided into four groups: Cerebral toxoplasmosis/AIDS patients (CT/AIDS group) (n = 15), ocular toxoplasmosis patients (OT group) (n = 23), chronic toxoplasmosis individuals (CHR group) (n = 13) and healthy individuals (HI group) (n = 10). OT, CHR, and HI groups were human immunodeficiency virus (HIV) seronegative. The diagnosis was made by laboratorial (PCR and ELISA) and clinical subjects. For cytokine determination, peripheral blood mononuclear cells (PBMC) of each patient were isolated and stimulated in vitro with T. gondii antigen. IFN-γ, TNF-α, and IL-10 activities were determined by ELISA. Patients from CT/AIDS and OT groups had low levels of IFN-γ when were compared with those from CHR group. These data suggest the low resistance to develop ocular lesions by the low ability to produce IFN-γ against the parasite. The same patients, which developed ocular or cerebral toxoplasmosis had higher TNF-α levels than CHR individuals. High TNF-α synthesis contribute to the inflammatory response and damage of the choroid and retina in OT patients and in AIDS patients caused a high inflammatory response as the TNF-α synthesis is not affected since monocytes are the major source this cytokine in response to soluble T. gondii antigens. IL-10 levels were almost similar in CT/AIDS and OT patients but low when compared with CHR individuals. The deviation to Th2 immune response including the production of anti-inflammatory cytokines, such as IL-10 may promote the parasites survival causing the tissue immune destruction. IL-10 production in T. gondii-infected brains may support the persistence of parasites as down-regulating the intracerebral immune response. All these indicate that OT and CT/AIDS patients produced low levels of IL-10 (Th2 response) and IFN-γ (Th1 response). They produced high TNF-α suggesting a high inflammatory response triggered by the parasite.

MHC Class I Chain-Related Gene A Polymorphisms and Linkage Disequilibrium with HLA-B and HLA-C Alleles in Ocular Toxoplasmosis

This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05–4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22–0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population.

Ocular toxoplasmosis: susceptibility in respect to the genes encoding the KIR receptors and their HLA class I ligands

The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1+/C2++ KIR3DS1+/Bw4-80Ile+) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1−/KIR3DL1+/Bw4-80Ile+ combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis.

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL‐6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL‐10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF‐β (at P < .005) for NC group. mRNA levels for TNF‐α and IL‐12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL‐27 and IFN‐γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively.

Molecular diagnosis of symptomatic toxoplasmosis: a 9-year retrospective and prospective study in a referral laboratory in São Paulo, Brazil

Symptomatic forms of toxoplasmosis are a serious public health problem and occur in around 10-20% of the infected people. Aiming to improve the molecular diagnosis of symptomatic toxoplasmosis in Brazilian patients, this study evaluated the performance of real time PCR testing two primer sets (B1 and REP-529) in detecting Toxoplasma gondii DNA. The methodology was assayed in 807 clinical samples with known clinical diagnosis, ELISA, and conventional PCR results in a 9-year period. All samples were from patients with clinical suspicion of several features of toxoplasmosis. According to the minimum detection limit curve (in CT), REP-529 had greater sensitivity to detect T. gondii DNA than B1. Both primer sets were retrospectively evaluated using 515 DNA from different clinical samples. The 122 patients without toxoplasmosis provided high specificity (REP-529, 99.2% and B1, 100%). From the 393 samples with positive ELISA, 146 had clinical diagnosis of toxoplasmosis and positive conventional PCR. REP-529 and B1 sensitivities were 95.9% and 83.6%, respectively. Comparison of REP-529 and B1 performances was further analyzed prospectively in 292 samples. Thus, from a total of 807 DNA analyzed, 217 (26.89%) had positive PCR with, at least one primer set and symptomatic toxoplasmosis confirmed by clinical diagnosis. REP-529 was positive in 97.23%, whereas B1 amplified only 78.80%. After comparing several samples in a Brazilian referral laboratory, this study concluded that REP-529 primer set had better performance than B1 one. These observations were based after using cases with defined clinical diagnosis, ELISA, and conventional PCR.

Toxoplasmic retinochoroiditis caused by Toxoplasma gondii strain ToxoDB#65

Ocular toxoplasmosis, caused by Toxoplasma gondii, is the most common cause of inflammation in the back of the eye. Analysis of the infecting strain may provide information regarding disease behavior and recurrence. Here, we describe clinical and epidemiological data for toxoplasmic retinochoroiditis in two Brazilian women infected by T. gondii strain ToxoDB#65, living in an urban region of São Paulo State, Brazil.

Ocular toxoplasmosis with positive polymerase chain reaction in peripheral blood – report of two cases, São Paulo State, Brazil

Aims: To describe the use of polymerase chain reaction (PCR) in peripheral blood and demonstrate its importance in the clinical follow-up of patients with ocular toxoplasmosis. Case description: Two immunocompetent patients were clinically diagnosed with acute ocular toxoplasmosis. The routine clinical evaluation consisted of fundus examination using binocular indirect ophthalmoscopy, color fundus photography, fluorescein angiography, and spectral domain optical coherence tomography. The serological diagnosis was made by ELISA (IgM, IgG) and confirmed by ELFA (IgG, IgM). The molecular diagnosis was made by PCR in peripheral blood using the B1 gene of Toxoplasma gondii as marker. The younger patient was male, had previous lesion in the right eye, complained of low visual acuity in the left eye and was under treatment. The older patient was male, had retinal detachment, and presented with sudden loss of acuity in the right eye. The fundus examination revealed chorioretinal scar in the left eye. IgG was reactive, IgM was non-reactive, and PCR was positive in the peripheral blood of both patients. New blood samples were collected for serological and molecular monitoring and PCR remained positive in both cases. Six weeks after treatment with oral sulfadiazine and pyrimethamine, the PCR yielded negative results. Conclusions: The results show that T. gondii antigens may be found in peripheral blood during ocular reactivations and that PCR may be a good tool for the follow-up of patients with ocular toxoplasmosis.

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