International Journal for Parasitology, Mar 1, 2021
The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EV... more The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.
Ocular toxoplasmosis (OT) is one of the most common manifestations of Toxoplasma gondii infection... more Ocular toxoplasmosis (OT) is one of the most common manifestations of Toxoplasma gondii infection and can be related with congenital or acquired infections. OT cause posterior uveitis that cause serious sequelae as complete loss of vision. microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. This study evaluated gene expression of miR-155-5p, miR-146a-5p, miR-21-5p, miR-29c-3p and miR-125b-5p in plasma of 51 patients with ocular toxoplasmosis (OT Group), 26 individuals with asymptomatic toxoplasmosis (AT Group), and 25 healthy individuals seronegative for toxoplasmosis (NC Group). Peripherical blood samples were collected in tube with EDTA for plasma isolation, laboratorial diagnosis for toxoplasmosis and RNA extraction. miRNA expression of each sample was performed by qPCR and values were expressed in Relative Quantification (RQ). Results showed that miR-155-5p and miR-29c-3p were up-expressed in OT patients than AT individuals. On the other hand, miR-21-5p and miR-125b-5p were down-expressed in OT patients. Differences were statistically significant. miR-146a-5p expression was similar in OT patients and AT individuals, without significant difference. In addition, comparative analysis for miRNA levels between AT and OT groups confirms these results. So far, this is the first study to evaluate circulating miRNA levels in ocular toxoplasmosis. These findings may contribute to further studies evaluating the exact role of these miRNAs in the course of infection, which may help in understanding the complex parasite-host interaction and future use in diagnosis, prognosis and therapeutic control in ocular toxoplasmosis.
This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excr... more This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra‐centrifuged to recover EVs (Can‐EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate‐poli‐acrylammide gel eletroforesis (SDS‐PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum‐EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can‐EVs from dogs with CanVL (7.78 × 1010 Can‐EVs/mL) were higher (p < .0001) than the non‐infected dogs (mean: 1.47 × 1010 Can‐EVs/mL). These results suggested that concentrations of Can‐EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR‐21‐5p, miR‐146a‐5p, miR‐125b‐5p, miR‐144‐3p, miR‐194‐5p, miR‐346, miR‐29c‐3p, miR‐155‐5p, miR‐24‐3p, miR‐181a‐5p, and miR‐9‐5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up‐expressed miR‐21‐5p and miR‐146a‐5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can‐EVs, as well as, the up‐expression of miR‐21‐5p and miR‐146a‐5p in infected dogs.
ABSTRACT Purpose We analyzed the frequency, viability, and genetic characteristics of T. gondii i... more ABSTRACT Purpose We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples Methods Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis. Results Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed. Conclusion Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.
Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and... more Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and a diagnostic challenge. Conventional methods have low accuracy, hardly discriminating colonization from infection, while some new high-cost or broncho-alveolar lavage-based methods have limited usefulness in developing countries. Quantitative PCR (qPCR) tests may overcome these limitations due to their high accuracy, possibility of automation, and decreasing cost. We evaluated an in-house qPCR targeting the fungus mtSSU gene using induced sputum. Sensitivity of the assay (ten target gene copies/assay) was determined using recombinant plasmids. We prospectively studied 86 AIDS patients with subacute respiratory symptoms in whom PcP was suspected. qPCR results were determined as quantification cycles (Cq) and compared with a qualitative PCR performed in the same IS, serum 1,3-β-D-Glucan assay, and a clinical/laboratory/radiology index for PcP. The qPCR clustered the patients in three grou...
The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases, Jan 29, 2017
Symptomatic forms of toxoplasmosis are a serious public health problem and occur in around 10-20%... more 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 convent...
Background Toxoplasmosis was recently included as a neglected disease by the Center for Disease C... more 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...
Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic t... more Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic tools are restricted. We carried-out a 1-year prospective cohort study at a referral hospital in São Paulo, Brazil. Participants had > or =18 years old, were hospitalized due to any indication and had CD4+ < 200 cells/µl. A urine commercial monoclonal Histoplasma galactomannan enzyme-linked immunosorbent assay (IMMY, Norman, OK, USA) and ‘in house’ Histoplasma blood nested PCR were performed in all cases. Probable/proven DH cases were defined according to international guidelines. Conventional mycological methods were available in routine conditions to investigate suspected DH cases. Treatment of participants followed the institutional routine. One-hundred six participants were included. Median age (interquartile range [IQR]) was 39.5 years (30.0–47.3) and 80 individuals (75.5%) were males. Median (IQR) CD4 cell count was 26.5 (9.4–89.3) cells/mm3. DH was diagnosed in 8/106 patients...
International Journal for Parasitology, Mar 1, 2021
The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EV... more The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.
Ocular toxoplasmosis (OT) is one of the most common manifestations of Toxoplasma gondii infection... more Ocular toxoplasmosis (OT) is one of the most common manifestations of Toxoplasma gondii infection and can be related with congenital or acquired infections. OT cause posterior uveitis that cause serious sequelae as complete loss of vision. microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. This study evaluated gene expression of miR-155-5p, miR-146a-5p, miR-21-5p, miR-29c-3p and miR-125b-5p in plasma of 51 patients with ocular toxoplasmosis (OT Group), 26 individuals with asymptomatic toxoplasmosis (AT Group), and 25 healthy individuals seronegative for toxoplasmosis (NC Group). Peripherical blood samples were collected in tube with EDTA for plasma isolation, laboratorial diagnosis for toxoplasmosis and RNA extraction. miRNA expression of each sample was performed by qPCR and values were expressed in Relative Quantification (RQ). Results showed that miR-155-5p and miR-29c-3p were up-expressed in OT patients than AT individuals. On the other hand, miR-21-5p and miR-125b-5p were down-expressed in OT patients. Differences were statistically significant. miR-146a-5p expression was similar in OT patients and AT individuals, without significant difference. In addition, comparative analysis for miRNA levels between AT and OT groups confirms these results. So far, this is the first study to evaluate circulating miRNA levels in ocular toxoplasmosis. These findings may contribute to further studies evaluating the exact role of these miRNAs in the course of infection, which may help in understanding the complex parasite-host interaction and future use in diagnosis, prognosis and therapeutic control in ocular toxoplasmosis.
This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excr... more This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra‐centrifuged to recover EVs (Can‐EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate‐poli‐acrylammide gel eletroforesis (SDS‐PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum‐EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can‐EVs from dogs with CanVL (7.78 × 1010 Can‐EVs/mL) were higher (p &lt; .0001) than the non‐infected dogs (mean: 1.47 × 1010 Can‐EVs/mL). These results suggested that concentrations of Can‐EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR‐21‐5p, miR‐146a‐5p, miR‐125b‐5p, miR‐144‐3p, miR‐194‐5p, miR‐346, miR‐29c‐3p, miR‐155‐5p, miR‐24‐3p, miR‐181a‐5p, and miR‐9‐5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up‐expressed miR‐21‐5p and miR‐146a‐5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can‐EVs, as well as, the up‐expression of miR‐21‐5p and miR‐146a‐5p in infected dogs.
ABSTRACT Purpose We analyzed the frequency, viability, and genetic characteristics of T. gondii i... more ABSTRACT Purpose We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples Methods Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis. Results Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed. Conclusion Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.
Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and... more Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and a diagnostic challenge. Conventional methods have low accuracy, hardly discriminating colonization from infection, while some new high-cost or broncho-alveolar lavage-based methods have limited usefulness in developing countries. Quantitative PCR (qPCR) tests may overcome these limitations due to their high accuracy, possibility of automation, and decreasing cost. We evaluated an in-house qPCR targeting the fungus mtSSU gene using induced sputum. Sensitivity of the assay (ten target gene copies/assay) was determined using recombinant plasmids. We prospectively studied 86 AIDS patients with subacute respiratory symptoms in whom PcP was suspected. qPCR results were determined as quantification cycles (Cq) and compared with a qualitative PCR performed in the same IS, serum 1,3-β-D-Glucan assay, and a clinical/laboratory/radiology index for PcP. The qPCR clustered the patients in three grou...
The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases, Jan 29, 2017
Symptomatic forms of toxoplasmosis are a serious public health problem and occur in around 10-20%... more 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 convent...
Background Toxoplasmosis was recently included as a neglected disease by the Center for Disease C... more 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...
Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic t... more Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic tools are restricted. We carried-out a 1-year prospective cohort study at a referral hospital in São Paulo, Brazil. Participants had > or =18 years old, were hospitalized due to any indication and had CD4+ < 200 cells/µl. A urine commercial monoclonal Histoplasma galactomannan enzyme-linked immunosorbent assay (IMMY, Norman, OK, USA) and ‘in house’ Histoplasma blood nested PCR were performed in all cases. Probable/proven DH cases were defined according to international guidelines. Conventional mycological methods were available in routine conditions to investigate suspected DH cases. Treatment of participants followed the institutional routine. One-hundred six participants were included. Median age (interquartile range [IQR]) was 39.5 years (30.0–47.3) and 80 individuals (75.5%) were males. Median (IQR) CD4 cell count was 26.5 (9.4–89.3) cells/mm3. DH was diagnosed in 8/106 patients...
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Papers by Ricardo Gava