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.

Reference genes for studies in infectious parasitic diseases in five types of human tissues

Gene expression analyses based on messenger RNA (mRNA) expression require accurate data normalization. When using endogenous reference genes, these should be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context. This study was aimed to identify reference genes that have more stable mRNA levels among individuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brain autopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6 endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP), beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MT-ATP6). Furthermore, validation of their stabilities and performance as reference genes was determined by geNorm and NormFinder programs. The results show that the most stable genes for PBMC and fresh skin biopsies were TBP and UBC; in FFPE lung autopsies and skin biopsies were GAPDH and B2M; and in FFPE brain autopsies were GAPDH and UBC. In addition, 18S rRNA was the least stable of all genes analyzed. These data concluded that even genes constitutively expressed have transcript level variations in different tissues as well as storage and experimental conditions. These observations suggest that suitable reference genes should be selected for normalization of gene expression data analysis.

As análises de expressão gênica baseadas na expressão do RNA mensageiro (mRNA) requerem normalização precisa dos dados. Ao usar genes de referência endógenos, estes devem ser cuidadosamente validados. Os genes de referência validados variam muito, dependendo do tecido, subconjuntos de células e contexto experimental. Este estudo teve como objetivo identificar genes de referência que apresentam níveis de mRNA mais estáveis ​​entre indivíduos em células mononucleares do sangue periférico (PBMC); biópsias de pele fresca; autópsias pulmonares e cerebrais, bem como biópsias de pele fixadas em formalina e embebidas em parafina (FFPE). Portanto, 6 genes de referência endógenos foram avaliados por reação quantitativa em cadeia da polimerase em tempo real: rRNA 18S, gliceraldeído-3-fosfato desidrogenase (GAPDH), proteína de ligação à caixa TATA (TBP), beta-2-microbolina (B2M), ubiquitina C (UBC) e ATP sintase 6 mitocondrialmente codificada (MT-ATP6). Além disso, a validação de suas estabilidades e desempenho como genes de referência foi determinada pelos programas geNorm e NormFinder. Os resultados mostram que os genes mais estáveis ​​para PBMC e biópsias de pele fresca foram TBP e UBC; nas autópsias pulmonares de FFPE e biópsias de pele foram GAPDH e B2M; e nas autópsias cerebrais de FFPE foram GAPDH e UBC. Além disso, o 18S rRNA foi o menos estável de todos os genes analisados. Esses dados concluíram que mesmo os genes expressos constitutivamente apresentam variações no nível de transcrição em diferentes tecidos, bem como condições experimentais e de armazenamento. Essas observações sugerem que genes de referência adequados devem ser selecionados para normalização da análise dos dados de expressão gênica.

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.

The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association

OBJECTIVE: Adenosine deaminase acts on adenosine and deoxyadenosine metabolism and modulates the immune response. The adenosine deaminase G22A polymorphism (20q.11.33) influences the level of adenosine deaminase enzyme expression, which seems to play a key role in maintaining pregnancy. The adenosine deaminase 2 phenotype has been associated with a protective effect against recurrent spontaneous abortions in European Caucasian women. The aim of this study was to investigate whether the G22A polymorphism of the adenosine deaminase gene is associated with recurrent spontaneous abortions in Brazilian women. METHODS: A total of 311 women were recruited to form two groups: G1, with a history of recurrent spontaneous abortions (N = 129), and G2, without a history of abortions (N = 182). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism. Fisher's exact test and odds ratio values were used to compare the proportions of adenosine deaminase genotypes and alleles between women with and without a history of recurrent spontaneous abortion (p<0.05). The differences between mean values for categorical data were calculated using unpaired t tests. The Hardy-Weinberg equilibrium was assessed with a chi-square test. RESULTS: Statistically significant differences were identified for the frequencies of adenosine deaminase genotypes and alleles between the G1 and G2 groups when adjusted for maternal age. CONCLUSIONS:The results suggest that the adenosine deaminase *2 allele is associated with a low risk for recurrent spontaneous abortions, but this association is dependent on older age.