The Role of microRNAs in the Infection by T. gondii in Humans.

MicroRNAs are molecules belonging to an evolutionarily conserved family of small non-coding RNAs, which act on post-transcriptional gene regulation, causing messenger RNA (mRNA) degradation or inhibiting mRNA translation into proteins. These molecules represent potential biomarkers for diagnosis, non-invasive prognosis, and monitoring the development of the disease. Moreover, they may provide additional information on the pathophysiology of parasitic infections and guide strategies for treatment. The Apicomplexan parasite Toxoplasma gondii modifies the levels of microRNAs and mRNAs in infected host cells by modulating the innate and adaptive immune responses, facilitating its survival within the host. Some studies have shown that microRNAs are promising molecular markers for developing diagnostic tools for human toxoplasmosis. MicroRNAs can be detected in human specimens collected using non-invasive procedures. changes in the circulating host microRNAs have been associated with T. gondii infection in mice and ocular toxoplasmosis in humans. Besides, microRNAs can be amplified from samples using sensitive and molecular-specific approaches such as real-time PCR. This review presents recent findings of the role that microRNAs play during T. gondii infection and discuss their potential use of these small nuclei acid molecules to different approaches such as laboratory diagnosis, modulation of cell and tissue infected as other potential applications in human toxoplasmosis.

Detection of anti-Toxoplasma gondiiantibodies in wild free-living birds and mammals from the northwest region of São Paulo state, Brazil / Detecção de anticorpos anti-Toxoplasma gondiiem aves e mamíferos silvestres de vida livre da região noroeste do estado de São Paulo, Brasil

Toxoplasmosis is a protozoonosis caused by an obligate intracellular parasite named Toxoplasma gondii, which can infect humans and a large number of homeothermic animal species with worldwide distribution. The present study aimed to detect anti-T. gondii antibodies from serological samples of free-living wild animals from the northwest region of São Paulo state, Brazil. Thirty-two samples (eight from birds and 24 from mammals) were analyzed by the modified agglutination test (MAT) using 5 cut-off points for birds and 25 for mammals. Seropositivity was observed in 25% (2/8) of birds, including the species Rupornis magnirostris (roadside hawk) and Caracara plancus (southern caracara), and 29.2% (7/24) animals were seropositive among mammals, including one hoary fox (Lycalopex vetulus), two maned wolves (Chrysocyon brachyurus), one black howler monkey (Alouatta caraya), two crab-eating foxes (Cerdocyon thous) and one gray brocket deer (Mazama gouazoubira). The results obtained with the present study indicate the exposure to T. gondiiof free-living wild animals from the northwest region of São Paulo state and, therefore, that they probably play a role in the transmission and maintenance of T. gondii in the environment they inhabit. Thus, identification of the infection in several animal species in the region indicates the environmental contamination of the area. Studies of this nature may help to understand the importance of the prevention and control of this disease in Brazil.(AU)

A toxoplasmose é uma protozoonose causada por um parasita intracelular obrigatório denominado Toxoplasma gondii, que pode infectar os humanos e um vasto número de espécies animais homeotérmicas, apresentando distribuição mundial. O presente estudo objetivou a detecção de anticorpos anti-T. gondii a partir de amostras sorológicas de animais silvestres de vida livre da região noroeste do estado de São Paulo. Foram analisadas 32 amostras (oito de aves e 24 de mamíferos) por meio do teste de aglutinação modificado (MAT), utilizando ponto de corte 5 para as aves e 25 para os mamíferos. Soropositividade foi observada em 25% (2/8) das aves, incluindo as espécies Rupornis magnirostris (gavião-carijó) e Caracara plancus (carcará); entre os mamíferos, 29,2% (7/24) foram soropositivos incluindo uma raposa-do-campo (Lycalopex vetulus), dois lobos-guará (Chrysocyon brachyurus), um bugio-preto (Alouatta caraya), dois cachorros-do-mato (Cerdocyon thous) e um veado-catingueiro (Mazama gouazoubira). Os resultados obtidos com o presente estudo indicam a exposição dos animais selvagens de vida livre a T. gondii na região noroeste do estado de São Paulo e, portanto, que provavelmente apresentam papel na transmissão e manutenção de T. gondii no meio ambiente em que vivem. Assim, a identificação da infecção em várias espécies de animais na região indica a contaminação ambiental da área. Estudos dessa natureza podem ajudar no entendimento sobre a prevenção e o controle dessa importante doença no Brasil.(AU)

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.

Serum IgG Anti-Toxoplasma gondii Antibody Concentrations Do Not Correlate Nested PCR Results in Blood Donors.

Background:Toxoplasma gondii infects millions of individuals worldwide. This protozoan is food and water-borne transmitted but blood transfusion and organ transplantation constitute alternative forms for transmission. However, the influence of IgG anti-T. gondii antibodies in molecular analysis carried out in peripheral blood still remain unclear. This study aimed to investigate the serum IgG anti-T. gondii antibody concentrations correlate Nested PCR results in blood donors. Methods: 750 blood donors were enrolled. IgM and IgG anti-T. gondii antibodies were assessed by ELISA (DiaSorin, Italy). Nested PCR was performed with primers JW62/JW63 (288 bp) and B22/B23 (115 bp) of the T. gondii B1 gene. The mean values of IgG concentration were compared for PCR positive and PCR Negative blood donors using the t-test or Mann-Whitney according to the normal distribution (p-value ≤ 0.05). Results: 361 (48.1%) blood donors presented positive serology as follow: IgM+/IgG-: 5 (0.6%); IgM+/IgG+: 21 (2.8%); IgM-/IgG+: 335 (44.7%) and 389 (51.9%), negative serology. From 353 blood donors with positive serology tested, the Nested PCR was positive in 38 (10.8%) and negative in 315 (89.2%). There were no differences statistically significant between the mean values of serum IgG anti-T. gondii antibody concentrations and the Nested PCR results. Conclusions: In conclusion, our data show that variations in the serum IgG anti-T. gondii antibody concentrations do not correlate T. gondii parasitemia detected by Nested PCR in chronically infected healthy blood donors.

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.

Evaluation of serological and molecular tests used to identify Toxoplasma gondii infection in pregnant women attended in a public health service in São Paulo state, Brazil.

Toxoplasmosis during pregnancy can have severe consequences. The use of sensitive and specific serological and molecular methods is extremely important for the correct diagnosis of the disease. We compared the ELISA and ELFA serological methods, conventional PCR (cPCR), Nested PCR and quantitative PCR (qPCR) in the diagnosis of Toxoplasma gondii infection in pregnant women without clinical suspicion of toxoplasmosis (G1=94) and with clinical suspicion of toxoplasmosis (G2=53). The results were compared using the Kappa index, and the sensitivity, specificity, positive predictive value and negative predictive value were calculated. The results of the serological methods showed concordance between the ELISA and ELFA methods even though ELFA identified more positive cases than ELISA. Molecular methods were discrepant with cPCR using B22/23 primers having greater sensitivity and lower specificity compared to the other molecular methods.

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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Frequency of anti- Toxoplasma gondii IgA, IgM, and IgG antibodies in high-risk pregnancies, in Brazil.

INTRODUCTION: Toxoplasmosis during pregnancy can be severe; thus, it is essential to diagnose the disease via serological tests. METHODS: An enzyme-linked immunosorbent assay (ELISA) was used to investigate anti-Toxoplasma gondii immunoglobulin A (IgA), M (IgM) and G (IgG) antibodies in 62 high-risk pregnant women. RESULTS: Forty-three (69.4%) women were positive for IgA, 31 (50%) for IgG, and 57 (91.9%) for IgM; 4 (6,5%) were positive for IgA but negative for IgM; 10 (16.1%) were negative for IgA and IgM but positive for IgG. CONCLUSIONS: Testing for these antibodies can help diagnose infection in pregnant women, thereby contributing to clinical management.

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...

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.

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.

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.