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.

Human extracellular vesicles and correlation with two clinical forms of toxoplasmosis.

AIM: This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS: Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS: Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION: These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.

Gastrointestinal, skin and blood parasites in Didelphis spp. from urban and sylvatic areas in São Paulo state, Brazil.

Didelphis (Marsupialia, Didelphimorphia) are synanthropic mammals, whose omnivorous diet predisposes them to infections caused by endoparasites. Their higher frequency in urban areas makes them potential carriers of zoonotic protozoans and helminths, enhancing potential transmission to humans. Our purpose was to study two common species, Didelphis albiventris (54 individuals) and D. aurita (2 individuals), which were screened for blood, skin and intestinal parasites in animals captured in urban areas and in riparian forest regions associated with the Capivari River Basin, in Monte Mor's municipality, São Paulo state (SP), Brazil. Blood and tissue samples were collected for DNA extraction and PCR. Fecal samples were collected and submitted to two sedimentation and two flotation methods. 77.6% of fecal samples were positive for nematode eggs, 34.5% for trematode eggs and 32.7% for protozoans. Two D. aurita specimens were naturally infected by Trypanosoma cruzi. Molecular analysis in a D. albiventris captured on a forested rural area was positive for Leishmania sp. DNA. Several parasites were found infecting Didelphis sp., demonstrating that this group of animals can harbor important zoonotic parasites, potentially playing a role as sylvatic reservoirs for human and domestic animal pathogens.

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.

Gene expression profile of cytokines produced in biopsies from patients with American cutaneous leishmaniasis.

American cutaneous leishmaniasis (ACL) causes a local inflammatory process, inducing expression of several cytokine genes. Particularly, IFN-γ can predict to disease susceptibility. Based in these data, this study was aimed to investigate the gene expression profile of IFN-γ, IL-10, IL-27, TNF-γ, TGF-ß and IL-6 produced in biopsies from ACL patients; and whether the gene expression profile of IFN-γ could determine the disease evolution. Gene expression of 6 cytokines was investigated in 40 formalin-fixed paraffin embedded (FFPE) biopsies from patients with cutaneous leishmaniosis (CL); and 10 FFPE biopsies from patients with mucosal leishmaniasis (ML) (control). All 50 patients were infected with Leishmania (Viannia) braziliensis. Gene expression was determined by qPCR; and a normal control group was used for calculations (5 normal biopsies). Values were expressed as Relative Quantification (RQ). The 40 CL patients were classified into 2 groups. CLlowIFN-γ, 35 patients with RQ for IFN-γ below 100; and CLhighIFN-γ, 5 (12.5%) patients with RQ above 100. Significant increase of mRNA levels of IFN-γ, IL-10 and IL-27 was shown in CLhighIFN-γ group when compared with CLlowIFN-γ and ML groups. TNF-α levels in CLlowIFN-γ group were higher than CLhighIFN-γ and ML groups. TGF-ß and IL-6 were similar in 3 groups. Comparison of cytokine expression/group showed that CLlowIFN-γ group had an equilibrium between the cytokines analyzed. In ML group, IFN-γ was over-expressed; but in CLhighIFN-γ group, besides IFN-γ, IL-27 was also over-expressed. The immune response to Leishmania induces to identification of some markers, which can be determined by analysis by gene expression of cytokines produced in biopsies.

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.

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

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.

Molecular detection of Trypanosoma cruzi in acai pulp and sugarcane juice.

Chagas disease, caused by Trypanosoma cruzi affects about 6-8 million people worldwide. Although transmission by triatomine insects has been controlled, other means of transmission maintain the infection. These forms of transmission are responsible for introducing Chagas disease in other non-endemic countries of the world. Thus, Chagas disease, nowadays is a worldwide health problem. In Brazil, acai pulp and sugarcane juice have been associated with Chagas disease outbreaks. The difficulties in isolation of the parasite from foods are hampering source tracking which could allow the confirmation of an implicated food commodity in these outbreak investigations. To address this scientific gap, we evaluated the performance of real-time PCR (qPCR) for detecting T. cruzi in acai pulp and sugarcane juice. All experiments were performed with acai pulp and sugarcane juice samples contaminated with different concentrations of T. cruzi. In assays with qPCR, the results showed that the ideal procedure for T. cruzi identification in acai pulp and sugarcane juice consisted of: i. centrifugation; ii. DNA extraction with a commercial kit for stool matrix; and iii. qPCR using a specific molecular marker for T. cruzi. The seeding in LIT medium of experimentally contaminated foods was effective in detecting the parasitic load by qPCR. The efficacy of qPCR was also verified testing food samples crushed with infected Triatomines. In conclusion, this methodology can be used to perform rapid diagnosis in outbreaks, facilitating measures in disease control.

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.

Molecular detection of Trypanosoma cruzi in acai pulp and sugarcane juice

Chagas disease, caused by Trypanosoma cruzi affects about 6-8 million people worldwide. Although transmission by triatomine insects has been controlled, other means of transmission maintain the infection. These forms of transmission are responsible for introducing Chagas disease in other non-endemic countries of the world. Thus, Chagas disease, nowadays is a worldwide health problem. In Brazil, acai pulp and sugarcane juice have been associated with Chagas disease outbreaks. The difficulties in isolation of the parasite from foods are hampering source tracking which could allow the confirmation of an implicated food commodity in these outbreak investigations. To address this scientific gap, we evaluated the performance of real-time PCR (qPCR) for detecting T. cruzi in acai pulp and sugarcane juice. All experiments were performed with acai pulp and sugarcane juice samples contaminated with different concentrations of T. cruzi. In assays with qPCR, the results showed that the ideal procedure for T. cruzi identification in acai pulp and sugarcane juice consisted of: i. centrifugation; ii. DNA extraction with a commercial kit for stool matrix; and iii. qPCR using a specific molecular marker for T. cruzi. The seeding in LIT medium of experimentally contaminated foods was effective in detecting the parasitic load by qPCR. The efficacy of qPCR was also verified testing food samples crushed with infected Triatomines. In conclusion, this methodology can be used to perform rapid diagnosis in outbreaks, facilitating measures in disease control.