Detection of Toxoplasma gondii DNA in peripheral blood and aqueous humor of patients with Toxoplasmic active focal necrotizing retinochoroiditis using real-time PCR / Detecção de DNA do Toxoplasma gondii no sangue periférico e humor aquoso por PCR em tempo real

ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR) for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43) of the aqueous humor samples and 2.33% (1/43) of the peripheral blood samples; further, 16.27% (7/43) of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

RESUMO Objetivo: Analisar o uso do PCR em tempo real (qPCR) na detecção do DNA do T. gondii no sangue periférico e no humor aquoso de pacientes com lesões de retinocoroidite focal, ativa por toxoplasmose. Métodos: Cinquenta e cinco pacientes com uveite infecciosa foram incluídos neste estudo. Os pacientes foram atendidos entre 2009 a 2013, no Departamento de Oftalmologia e Ciências Visuais da Universidade Federal de São Paulo. Quarenta e três pacientes tiveram o diagnóstico de lesões de retinocoroidite focal, ativa por toxoplasmose e, os outros 12 tiveram o diagnóstico de uveíte infecciosa não toxoplásmica e, por isso foram usados como grupo controle. A técnica de qPCR foi utilizada na detecção de DNA do T. gondii em amostras de sangue periférico e humor aquoso. Resultados: O qPCR foi positivo para o DNA do T. gondii em 37,21% (16/43) das amostras de humor aquoso, 2,33% (1/43) nas amostras de sangue periférico e, 16,27% (7/43) em ambas amostras simultaneamente. Conclusão: O qPCR foi capaz de detectar o DNA do T. gondii em pacientes com lesões de retinocoroidite focal, ativa por Toxoplasmose, no sangue bem como, no humor aquoso, podendo ajudar no diagnostico.

Real-time PCR in infectious uveitis as an alternative diagnosis / PCR em tempo real em uveites infecciosas como um diagnóstico alternativo

PURPOSE: Uveitis is a major visual impairment disease affecting parts or the entire uveal tract and occasionally the sclera, the cornea or the optic nerve. The disease is a major cause of ocular morbidity and blindness in immunocompetent and immunocompromised patients. In this work we analyzed the sensitivity and specificity of real-time PCR to detect the etiological agent from blood, plasma, vitreous and aqueous humor and compared with the diagnostic hypothesis. METHODS: Twenty-seven patients (13 male) were studied and Real-time PCR method was used for the detection of herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Mycobacterium tuberculosis (TB) and Toxoplasma gondii (Toxo) in the aqueous humor as well as in the vitreous, blood and plasma. RESULTS: Our results showed the presence of Toxo, CMV, VZV or HSV-2 in 19.2 percent of aqueous humor samples, and in 30 percent of vitreous humor samples. In plasma and blood samples, only CMV was detected (11.1 percent and 3.7 percent, respectively). CONCLUSION: Real-time PCR was able to detect and to confirm diagnostic hypothesis in uveitis. Our data also confirms that vitreous humor is the best source for molecular diagnosis of infectious uveitis but indicates aqueous humor samples that are easier to obtain may also be appropriate to be tested by Real-time PCR.

OBJETIVO: Uveíte é a maior causa de doença ocular que afeta o trato uveal, e ocasionalmente a esclera, cornea e o nervo óptico. Esta doença é a maior causa de morbidade ocular e cegueira em pacientes imunocompetentes e imunossuprimidos. Neste trabalho nós analisamos a sensiblidade e especificidade do PCR em tempo real para detectar agentes etiológicos no sangue, plasma, humor vítreo e aquoso, e comparamos com a hipótese diagnóstica. MÉTODOS: Vinte e sete pacientes (13 homens) foram estudados e o método de PCR em tempo real foi usado para detectar o vírus da herpes simples 1 (HSV-1), vírus da herpes simples 2 (HSV-2), vírus varicella zoster (VZV), citomegalovírus (CMV), Mycobacterium tuberculosis (TB) e Toxoplama gondii (Toxo) no humor aquoso e vítreo, além do sangue e plasma. RESULTADOS: Nossos resultados mostraram a presença de Toxo, CMV, VZV ou HSV-2 em 19,2 por cento das amostras de humor aquoso, e em 30 por cento das amostras de humor vítreo. Nas amostras de plasma e sangue somente CMV foi detectado (11,1 por cento e 3,7 por cento, respectivamente). CONCLUSÃO: PCR em tempo real foi capaz de detectar e confirmar a hipótese diagnóstica em uveíte. Nossos dados confirmam que o humor vítreo é a melhor fonte para diagnóstico molecular de uveíte infecciosa, porém o humor aquoso também foi uma fonte importante de detecção, além de ser mais fácil de se obter.

Total antioxidant capacity in Eales’ disease, uveitis & cataract.

Background & objectives: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales’ disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. Methods: The TAC assay was done spectrophotometrically in the serum of Eales’ disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. Results: TAC was found to be significantly lower in Eales’ disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales’ disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). Interpretation & conclusions: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Low-dose cyclosporine treatment for sight-threatening uveitis: Efficacy, toxicity, and tolerance.

Aim: To ascertain the effectiveness, tolerability, and safety of low-dose cyclosporine in the management of sight-threatening uveitis. Materials and Methods: This was a retrospective clinical case series of patients using oral low-dose cyclosporine for the management of sight-threatening uveitis in the uvea clinic (UC). Patients receiving cyclosporine were identified from the clinic database. Main outcome measures were degree of intraocular inflammation, visual acuity and dose reduction of oral steroid for effectiveness and adverse symptoms, systemic hypertension, and raised serum creatinine for tolerability and safety. Results: Intraocular inflammation was improved or stable in 97% of patients, visual acuity was improved or stable in 91%, and oral steroid dosage was reduced in 73% (by half or more in 51%). Adverse symptoms were almost universal, the commonest being peripheral paresthesia/burning in 70% and fatigue in 67%. Significant systemic hypertension developed in 27% and raised creatinine in 30%, necessitating dose reduction. Cyclosporine was discontinued in 35%, being intolerable in 20% and ineffective in 15%. Conclusions: Cyclosporine was found to be effective in reducing inflammation and protecting vision in sight-threatening uveitis. It was safe with proper monitoring, including in children. It had a significant toxicity profile and a high incidence of adverse symptoms which required close supervision, and a prompt dose reduction or drug exchange.

Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease

PURPOSE: To study the applicability (sensitivity, specificity) of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV), herpes virus (HSV) and varicella zoster (VZV), Epstein-Barr virus (EBV), Mycobacterium sp and Toxoplasma gondii in the diagnosis of patients with or without AIDS, with presumably infectious uveitis, using serum, aqueous humor and vitreous humor samples. METHODS: Twenty individuals with uveitis of presumed infectious origin were evaluated. Sixteen of them had AIDS, four were immunocompetent individuals. We also evaluated 4 normal controls who underwent vitrectomy surgery. Clinical evaluation of the patients was performed together by three clinicians. PCR evaluations of the serum, aqueous, and vitreous humor were performed in a masked fashion by the laboratory staff. RESULTS: Twelve patients had a clinical diagnosis of CMV retinitis. Of these 6 (50 percent) had a positive PCR for CMV in the vitreous, three (25 percent) had a positive PCR for CMV in the serum, and none were positive in the aqueous. Five patients had a clinical diagnosis of acute retinal necrosis (ARN). Three (60 percent) of these had positive PCR for HSV/VZV in the vitreous. One of these patients had a positive PCR reaction for both EBV and HSV/VZV in the vitreous samples. One patient with cutaneous herpes zoster had a positive PCR reaction for HSV/VZV in the serum. Four patients had a presumed diagnosis of ocular toxoplasmosis, one patient (25 percent) had a positive PCR for Toxoplasma gondii in the serum, 3 (75 percent) had positive results in the aqueous, and 2 (50 percent) had positive results in the vitreous. One patient with presumed ocular tuberculosis had a positive PCR reaction both in the serum and in the vitreous samples. Finally, none of the four control individuals revealed any positive PCR reaction. CONCLUSION: PCR is an auxiliary diagnostic procedure that should be evaluated together with ophthalmological aspects...

OBJETIVOS: Avaliar a aplicabilidade (especificidade, sensibilidade) do teste da reação da cadeia de polimerase (PCR) na detecção de citomegalovírus (CMV), herpes vírus e varicela zoster (HSV, VZV), Epstein-Barr vírus (EBV), Mycobacterium sp e Toxoplasma gondii no diagnóstico de pacientes com ou sem AIDS, com uveíte infecciosa presumível, utilizando amostras de humor aquoso, humor vítreo e soro. MÉTODOS: Vinte pacientes com uveíte infecciosa presumível foram estudados. Dezesseis destes apresentavam AIDS e quatro eram imunocompetentes. Foram utilizados quatro pacientes como grupo controle que se submeteram a vitrectomia. A avaliação clínica foi feita conjuntamente com três oftalmologistas. O exame do PCR do soro, aquoso e vítreo foi feito sem o conhecimento da hipótese diagnóstica pela equipe do laboratório. RESULTADOS: Doze pacientes tinham o diagnóstico clínico de retinite por CMV. Deste subgrupo 6 (50 por cento) eram PCR positivo para CMV no vítreo, 3 (25 por cento) eram PCR positivos para CMV no soro e nenhum destes foi positivo no aquoso. Cinco pacientes tinham o diagnóstico clínico de necrose aguda de retina (ARN). Três (60 por cento) destes eram PCR positivos para HSV/VZV no vítreo. Um destes pacientes era PCR positivo tanto para EBV e HSV/VZV na amostra do vítreo. Um destes pacientes com herpes zoster cutâneo era PCR positivo para HSV/VZV no soro. Quatro pacientes tinham o diagnóstico de toxoplasmose ocular presumida, um paciente (25 por cento) era PCR positivo para Toxoplasma gondii no soro, 3 (75 por cento) eram positivos no aquoso e 2 (50 por cento) eram positivo no vítreo. Um paciente com tuberculose ocular presumível era PCR positivo tanto no soro quanto no vítreo. Nenhum dos pacientes do grupo controle era PCR positivo em qualquer amostra. CONCLUSÃO: O exame do PCR é procedimento diagnóstico auxiliar que deve ser utilizado conjuntamente com os aspectos clínicos.

Comparación de la eficacia de la prednisolona y la rimexolona en el tratamiento de iridociclitis aguda en pacientes HLA-B27 positivos / Efficacy of prednisolone and rimexolone in HLA-B27 positive patients with acute anterior uveitis

Objetivo: Comparar la eficacia y seguridad de las suspensiones oftálmicas de acetato de prednisolona al 1% y de rimexolona al 1%, en el tratamiento de la uveítis anterior aguda (UAA) en pacientes HLA B27+. Material y métodos: Se seleccionaron al azar 68 pacientes con UAA HLA -B27+ para tratamiento con acetato de prednisolona al 1% o rimexolona al 1%. En todos los pacientes la inflamación en cámara anterior era leve a moderada. La presión intraocular (PIO) y el grado de inflamación fueron evaluados semanalmentre durante seis semanas. Fue un estudio clínico prospectivo, aleatorio y doble ciego. Resultados: Al cuantificar las células en cámara anterior no se encontraron diferencias estadísticamente significativas entre uno y otro grupos. En el grupo de rimexolona el flare disminuyó desde la primera semana. En los grupos la presión intraocular se elevó con respecto a la basal desde la primera semana, la variación fue más significativa en el grupo de rimexolona. La PIO final fue menor en el grupo de rimexolona, siendo esta diferencia estadísticamente significativa . Conclusión: Para el tratamiento de la UAA HLA -B27+, leve a moderada, la rimexolona al 1% y la prednisolona al 1% tiene una eficiencia similar. En este estudio las variaciones de presión intraocular en los dos grupos no fueron clínicamente significativas.

Purpose: To compare the efficacy and safety of prednisolone acetate 1 % vs. rimexolone 1 % ophthalmic suspension in the treatment of acute anterior uveitis (AAU) in HLA-B27+ patients. Methods: Sixty-eight AAU HLA-B27+ patients were randomly selected for treatment with prednisolone acetate 1% or Rimexolone 1%. All patients showed mild to moderate anterior chamber inflammation. This was a prospective, randomized, double blind, clinical trial. Results: There was no statistically significant difference between both groups when anterior chamber cells were measured. In the rimexolone group, flare diminished since the first week. In both groups the intraocular pressure (IOP) raised since the first week; the increase washighly significant in the rimexolone group. Final intraocular pressure was higher in the prednisolone group. Conclusion: Rimexolone 1 % is as effective as prednisolone acetate 1% in the treatment of mild to moderate AAU HLA B27+. IOP increased in both groups, but this variation was not clinically significant.