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 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.

Evaluation of epithelial cell proliferating activity and fibroblast nuclear kariometry in recurrent pterygium treated with mitomycin C / Avaliação da atividade proliferativa das células epiteliais e da cariometria do núcleo dos fibroblastos em pterígios recidivados previamente tratados com a mitomicina C

PURPOSE: To evaluate the effectiveness of mitomycin C (MMC) in preventing recurrence of pterygium following conjunctival autograft transplantation (CAT). Ki-67 antigen to evaluate epithelial cell proliferation and fibroblast nuclear kariometry were used to assist treatment evaluation. METHODS: Twenty-nine patients with recurrent pterygium were divided into three groups: Group (G) 1 - CAT and placebo eyedrops (PED); G2 - CAT, 0.015 percent MMC subconjunctivally, and PED; G3 - CAT and 0.02 percent MMC eyedrops. Immunohistochemistry for the Ki-67 antigen and fibroblast nuclei kariometry were performed on the excised tissue, divided into nasal and temporal sides. Kariometry was evaluated in terms of volume (Vl) and area (Ar) using at least 50 cells/patient. RESULTS: The percentage of positive epithelial cells for the Ki-67 antigen on the nasal and temporal side after treatment of the three groups were: nasal (5.39 percent G1, 4.49 percent G2, and 3.88 percent G3); temporal (3.30 percent G1, 4.46 percent G2, 4.14 percent G3), did not show significant differences. Fibroblast nucleus kariometry was: nasal Vl (792.1 µ3 G1, 605.1 µ3 G2, and 549.9 µ3 G3) and Ar (100.58 µ2 G1, 83.13 µ2 G2, and 78.41 µ2 G3). The three groups showed significant differences: p=0.039 and p=0.035, for respectively Vl and Ar, on the nasal side. After a six month of treatment, the three groups presented the following recurrence rates: G1, 22.22 percent, G2, 18.18 percent and G3, 33.33 percent, respectively. CONCLUSION: MMC did not reduce the number of positive epithelial cells for the Ki-67 antigen in recurrent pterygium, but decreased fibroblast nucleus volume and area on the nasal side of the pterygia. The number of positive epithelial cells for the Ki-67 antigen seemed not to be related to pterygium recurrence observed over a six-month post-surgery period. The role of epithelial cell proliferation in pterygium recurrence should be evaluated by further studies.

OBJETIVO: Avaliar a eficácia da mitomicina C (MMC) na prevenção da recorrência quando previamente utilizada no transplante autólogo de conjuntiva (TAC). A avaliação da proliferação celular epitelial pelo antígeno Ki-67 e a cariometria do núcleo dos fibroblastos foram usados como auxiliares na avaliação do tratamento. MÉTODOS: Vinte e nove pacientes com pterígio recidivado foram divididos em três grupos: Grupo (G) 1-TAC e colírio placebo (PLA); G2-TAC, MMC 0,015 por cento subconjuntival e PLA; G3-TAC e colírio de MMC 0,02 por cento. A imuno-histoquímica foi realizada no tecido excisado para o antígeno Ki-67, como a cariometria dos núcleos dos fibroblastos (divididos em lado nasal e temporal). A cariometria dos núcleos dos fibroblastos foi avaliada de acordo com os seguintes parâmetros: volume (Vl) e área (Ar) em pelos menos 50 células por paciente. RESULTADOS: A porcentagem das células epiteliais positivas para o antígeno Ki-67 no lado nasal e temporal após o tratamento dos três grupos estudados foi: nasal (3,30 por cento G1, 4,49 por cento G2 e 3,38 por cento G3) e temporal (3,30 por cento G1, 4,46 por cento G2 e 4,14 por cento G3) não mostrando diferença significativa. A cariometria do núcleo dos fibroblastos foi: Vl nasal (792,1 µ3 G1, 605,1 µ3 G2, e 549,9 µ3 G3) e a Ar (100,58 µ2 G1, 83,13 µ2 G2, e 78,41 µ2 G3). Os três grupos mostraram uma diferença significativa p=0,039 e p=0,035, respectivamente do Vl e da Ar no lado nasal. Após seis meses de tratamento, os três grupos apresentaram a seguinte taxa de recidiva: 22,22 por cento G1, 18,18 por cento, G2 e 33,33 por cento G3 respectivamente. CONCLUSÃO: O uso da MMC não interferiu nas células epiteliais positivas para o antígeno Ki-67 no pterígio recidivado, mas acarretou diminuição do volume e área dos núcleos dos fibroblastos no lado nasal do pterígio. As células epiteliais positivas para o antígeno Ki-67 parecem não ter relação com a recidiva do pterígio após seis meses...