Interleukin 17F Gene Polymorphism as a Potential Protective Factor in the Immunopathology of Ocular Toxoplasmosis.

Ocular toxoplasmosis (OT) is characterised by intraocular inflammation due to Toxoplasma gondii infection. Studies have found that interleukin 17 (IL-17) plays a central role in the pathology of OT. However, nucleotide variability in IL17 and interleukin 17 receptor (IL17R) genes has not been characterised in OT. As cytokine gene polymorphisms may influence the expression of these molecules, the aim of this study was to verify whether IL17A (rs2275913), IL17F (rs763780), IL17RA (rs4819554) and IL17RC (rs708567) polymorphisms are associated with OT in a Brazilian population. This study enrolled 214 patients seropositive for T. gondii (110 with OT and 104 without) and 107 controls. Polymorphisms were identified by PCR-restriction fragment length polymorphism analysis, validated by DNA sequencing with chi-square and multivariate analyses being used to assess possible associations between polymorphisms and OT. Logistic regression under the dominant model revealed a protection factor against OT of the C mutant allele of the IL17F (rs763780) polymorphism. The T/C-C/C genotypes were significantly more common in patients without OT compared to those with OT (p value = 0.0066) and controls (p value = 0.014). Findings from this study suggest that the IL17F polymorphism may have an influence in the immunopathology of OT in Brazilian individuals.

miRNA 511_5p is a potential biomarker for ocular toxoplasmosis.

BACKGROUND: Ocular toxoplasmosis (OT) is a frequent clinical manifestation due to infection by Toxoplasma gondii. It is characterized by an inflammatory process involving macrophages activated by pro-inflammatory cytokines. The expression of microRNAs takes place during the inflammatory process and, among them, miRNA 511 regulates the activation of macrophages. This study evaluated the expression of miRNA 511_5p in patients with OT and healthy controls. METHODS: A total of 361 patients from the Hospital de Base of Fundação Faculdade de Medicina de São José do Rio Preto were enrolled and divided into four groups: G1-patients with active ocular lesions and reagent serology for T. gondii; G2-patients with scars and reagent serology for T. gondii; G3-patients without ocular lesions or scars and reagent serology for T. gondii; G4-patients without ocular lesions or scars and non-reagent serology for T. gondii. All patients underwent clinical and laboratory evaluation to confirm the diagnosis of OT. Serology tests, RNA extraction and cDNA synthesis were performed. RESULTS: The miRNA 511_5p levels were compared among the groups. The G1 group showed a high blood plasma concentration of miRNA 511_5p (mean 22.34) compared with the G2 (4.65), G3 (8.91) and G4 (3.52) groups (p<0.0001). CONCLUSION: These data suggest that miRNA 511_5p has significant potential as a biomarker for OT.

Evaluation of Serological and Molecular Tests Used for the Identification of Toxoplasma gondii Infection in Patients Treated in an Ophthalmology Clinic of a Public Health Service in São Paulo State, Brazil.

Ocular toxoplasmosis is one of the most common complications caused by the infection with the parasite Toxoplasma gondii. The risk of developing eye lesions and impaired vision is considered higher in Brazil than other countries. The clinical diagnosis is difficult and the use of sensitive and specific laboratorial methods can aid to the correct diagnosis of this infection. We compared serological methods ELISA and ELFA, and molecular cPCR, Nested PCR and qPCR for the diagnosis of T. gondii infection in groups of patients clinically evaluated with ocular diseases non-toxoplasma related (G1 = 185) and with lesions caused by toxoplasmosis (G2 = 164) in an Ophthalmology clinic in Brazil. Results were compared by the Kappa index, and sensitivity (S), specificity (E), positive predictive value (PPV), and negative (NPV) were calculated. Serologic methods were in agreement with ELISA more sensitive and ELFA more specific to characterize the acute and chronic infections while molecular methods were discrepant where qPCR presented higher sensitivity, however, lower specificity when compared to cPCR and Nested 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.

CCR5 chemokine receptor gene polymorphisms in ocular toxoplasmosis.

CC chemokine receptor type 5 (CCR5) is a chemokine receptor that influences the immune response to infectious and parasitic diseases. This study aimed to determine whether the CCR5Δ32 and CCR5 59029 A/G polymorphisms are associated with the development of ocular toxoplasmosis in humans. Patients with positive serology for Toxoplasma gondii were analyzed and grouped as 'with ocular toxoplasmosis' (G1: n=160) or 'without ocular toxoplasmosis' (G2: n=160). A control group (G3) consisted of 160 individuals with negative serology. The characterization of the CCR5Δ32 and CCR5 59029 A/G polymorphisms was by PCR and by PCR-RFLP, respectively. The difference between the groups with respect to the mean age (G1: mean age: 47.3, SD±19.3, median: 46 [range: 18-95]; G2: mean age: 61.3, SD±13.7, median: 61 [range: 21-87]; G3: mean age: 38.8, SD±17.9, median: 34 [range: 18-80]) was statistically significant (G1 vs.G2: p-value <0.0001; t=7.21; DF=318; G1 vs.G3: p-value <0.0001; t=4.32; DF=318; G2 vs. G3: p-value <0.0001; t=9.62; DF=318). The Nagelkerke r2 value was 0.040. There were statistically significant differences for the CCR5/CCR5 (p-value=0.008; OR=0.261), AA (p-value=0.007; OR=2.974) and AG genotypes (p-value=0.018; OR=2.447) between G1 and G2. Individuals with the CCR5/CCR5 genotype and simultaneously the CCR5-59029 AA or AG genotypes have a greater risk of developing ocular toxoplasmosis (4% greater), which may be associated with a strong and persistent inflammatory response in ocular tissue.

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

ABSTRACT 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.

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.

WITHDRAWN: Selection of reference genes in five types of human tissues for normalization of gene expression studies in infectious diseases.

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WITHDRAWN: Selection of reference genes in five types of human tissuesfor normalization of gene expression studies in infectious diseases

Gene expression analyses based on messenger RNA (mRNA) expression require accurate datanormalization. When using endogenous reference genes, these have to be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context.The aim of this study was to identify reference genes that present more stable mRNA levels amongindividuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brainautopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP),beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MTATP6).Furthermore, validation of their stability and suitability as reference genes was determined bythe geNorm program. The results show that in PBMC and fresh skin biopsies, TBP and UBC wereidentified as the most stable, while in FFPE lung autopsies and skin biopsies, GAPDH and B2M, andin FFPE brain autopsies, GAPDH and UBC turned out to be the most stable...

A Brazilian report using serological and molecular diagnosis to monitoring acute ocular toxoplasmosis.

BACKGROUND: Toxoplasmosis was recently included as a neglected disease by the Center for Disease Control. Ocular toxoplasmosis is one clinical presentation of congenital or acquired infection. The laboratory diagnosis is being used worldwide to support the clinical diagnosis and imaging. The aim of this study was to evaluate the use of serology and molecular methods to monitor acute OT in immunocompetent patients during treatment. METHODS: Five immunocompetent patients were clinically diagnosed with acute OT. The clinical evaluation was performed by ophthalmologic examination using the Early Treatment Diabetic Retinopathy Study, best-corrected visual acuity, slit lamp biomicroscopy, fundoscopic examination with indirect binocular ophthalmoscopy color fundus photography, fluorescein angiography and spectral optical coherence tomography (OCT). Serology were performed by ELISA (IgA, IgM, IgG) and confirmed by ELFA (IgG, IgM). Molecular diagnoses were performed in peripheral blood by cPCR using the Toxoplasma gondii B1 gene as the marker. Follow-up exams were performed on day +15 and day +45. RESULTS: Only five non-immunocompromised male patients completed the follow up and their data were used for analysis. The mean age was 41.2 ± 11.3 years (median: 35; range 31-54 years). All of them were positive for IgG antibodies but with different profiles for IgM and IgA, as well as PCR. For all patients the OCT exam showed active lesions with the inner retinal layers being abnormally hyper-reflective with full-thickness disorganization of the retinal reflective layers, which assumed a blurred reflective appearance and the retina was thickened. CONCLUSIONS: The presence of IgA and IgM confirmed the acute infection and thus was in agreement with the clinical evaluation. Our results show the adopted treatment modified the serological profile of IgM antibodies and the PCR results, but not the IgG and IgA antibodies and that imaging is a good tool to follow-up patients.

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 with positive polymerase chain reaction in peripheral blood ­ report of two cases, São Paulo State, Brazil / Toxoplasmose ocular com reação em cadeia da polimerase positiva em sangue periférico ­ relato de dois casos, estado de São Paulo, Brasil

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 enzyme-linked immunosorbent assay (ELISA) and confirmed by enzyme-linked fluorescent assay (ELFA). 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. Conclusion: 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.

Objetivos: Descrever o uso da reação em cadeia da polimerase (PCR) no sangue periférico e demonstrar sua importância no acompanhamento clínico de pacientes com toxoplasmose ocular. Descrição dos casos: Dois pacientes imunocompetentes foram clinicamente diagnosticados com toxoplasmose ocular aguda. Rotineiramente, a avaliação clínica foi feita por fundoscopia com o uso de oftalmoscópio binocular indireto, retinografia colorida, angiografia fluorescente e tomografia de coerência óptica espectral. A sorologia foi realizada por ensaio imunoenzimático (ELISA) e confirmada por ensaio imunoenzimático fluorescente ELFA (IgG, IgM). O diagnóstico molecular foi realizado por PCR em sangue periférico usando o gene B1 de Toxoplasma gondii como marcador. O paciente mais jovem era do sexo masculino, apresentava lesão prévia no olho direito, queixa de baixa acuidade visual no olho esquerdo e estava sob tratamento. O paciente mais velho era do sexo masculino, apresentava descolamento de retina e súbita diminuição de visão no olho direito. A fundoscopia revelou cicatriz coriorretiniana no olho esquerdo. Ambos os pacientes tinham IgG reagente, IgM não reagente e PCR positivo em sangue periférico. Novas amostras de sangue foram coletadas para monitoramento sorológico e molecular e a PCR permaneceu positiva em ambos os casos. Seis semanas após o início do tratamento com sulfadiazina e pirimetamina oral, os resultados do PCR tornaram-se negativos. Conclusões: Os resultados mostram que antígenos de T. gondii podem ser encontrados em sangue periférico durante as reativações oculares e que a PCR parece ser uma boa ferramenta para o acompanhamento de pacientes com toxoplasmose ocular.