VERA LUCIA TEIXEIRA DE FREITAS
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
6 resultados
Resultados de Busca
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- A Real Time PCR strategy for the detection and quantification of Candida albicans in human blood(2020) BUSSER, Felipe Delatorre; COELHO, Vivian Caso; FONSECA, Claudia de Abreu; NEGRO, Gilda Maria Barbaro Del; SHIKANAI-YASUDA, Maria Aparecida; LOPES, Marta Heloisa; MAGRI, Marcello Mihailenko Chaves; FREITAS, Vera Lucia Teixeira deCandidemia is a significant cause of bloodstream infections (BSI) in nosocomial settings. The identification of species can potentially improve the quality of care and decrease human mortality. Quantitative PCR (qPCR) was evaluated for Candida albicans detection using culture suspensions containing C. albicans, spiked human blood. the cloned qPCR target fragment (ITS2 region) and the results of these assays were compared. The assays showed a good detection limit: C. albicans DNA extracted from yeast (sensitivity 0.2 CFU/mu L), spiked human blood (sensitivity 10 CFU/mL), and cloned fragment of ITS2 region (sensitivity 20 target copies/mu L). The efficiency of ITS2 fragment-qPCR ranged from 89.67 to 97.07, and the linearity (R-2) of the standard curve ranged from 0.992 to 0.999. The results showed that this ITS2-qPCR has a great potential as a molecular prototype model for the development of a test to be applied in clinical practice, greatly reducing the time of candidemia diagnosis, which is extremely important in this clinical setting.
- Usefulness of PCR for Trypanosoma cruzi DNA in blood and endomyocardial biopsies for detection of Chagas disease reactivation after heart transplantation: A comparative study(2021) BENVENUTI, L. A.; FREITAS, V. L. T.; ROGGéRIO, A.; NISHIYA, A. S.; MANGINI, S.; STRABELLI, T. M. V.Background: Chagas disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease with proliferation and dissemination of Trypanosoma cruzi parasites. Serial blood PCR testing is consensually recommended for CDR monitoring, but there is uncertainty about the incremental value in performing the molecular tests in endomyocardial biopsies (EMB). Methods: We compared qualitative and quantitative results of PCR for T cruzi DNA in 62 pairs of blood and EMB collected with a maximum time interval of 7 days, from 34 heart-transplanted, chagasic patients. Results: Blood PCR resulted positive in 39/62 (62.9%) samples, with PL ranging from 0.14 to 1610.73 (median: 3.31). PCR resulted positive in 8/60 (13.3%) EMB, with PL ranging from 2.82 to 1670.55 (median: 65.63). All blood samples which tested negative presented a paired EMB which also tested negative. However, 31/39 (79.5%) blood samples which tested positive presented a paired EMB which tested negative. There was poor agreement between blood and EMB PCR (kappa = 0.153). CDR affecting the myocardium (myo-CDR) was diagnosed in three occasions. PCR resulted positive in both blood and EMB at the time of myo-CDR, with PL ranging from 0.61 to 1610.73 in blood and 13.8 to 1670.55 in EMB. Conclusions: Negative PCR for T cruzi in blood rules out myo-CDR, with no value of testing EMB. Positive PCR in blood with high PL is diagnostic for myo-CDR. If PCR in blood results positive with low PL, testing EMB is useful: negative PCR turns unlikely, and positive PCR reinforces greatly the possibility of myo-CDR. © 2021 Wiley Periodicals LLC
- Quantitative PCR as a marker for preemptive therapy and its role in therapeutic control in Trypanosoma cruzi/HIV coinfection(2024) FREITAS, Vera Lucia Teixeira de; NOVAES, Christina Terra Gallafrio; SARTORI, Ana Marli Christovam; CARVALHO, Noemia Barbosa; SILVA, Sheila Cristina Vicente da; NAKANISHI, erika Shimoda; SALVADOR, Fernando; CASTRO, Cleudson Nery de; BEZERRA, Rita Cristina; WESTPHALEN, Elizabeth Visone Nunes; OLIVEIRA, Caroline Medeji Ramos de; BUSSER, Felipe Delatorre; HO, Yeh-Li; BUCCHERI, Renata; BONILLA, Carolina; SHIKANAI-YASUDA, Maria AparecidaBackground Trypanosoma cruzi and HIV coinfection can evolve with depression of cellular immunity and increased parasitemia. We applied quantitative PCR (qPCR) as a marker for preemptive antiparasitic treatment to avoid fatal Chagas disease reactivation and analyzed the outcome of treated cases. Methodology This mixed cross-sectional and longitudinal study included 171 Chagas disease patients, 60 coinfected with HIV. Of these 60 patients, ten showed Chagas disease reactivation, confirmed by parasites identified in the blood, cerebrospinal fluid, or tissues, 12 exhibited high parasitemia without reactivation, and 38 had low parasitemia and no reactivation. Results We showed, for the first time, the success of the timely introduction of benznidazole in the non-reactivated group with high levels of parasitemia detected by qPCR and the absence of parasites in reactivated cases with at least 58 days of benznidazole. All HIV+ patients with or without reactivation had a 4.0-5.1 higher chance of having parasitemia than HIV seronegative cases. A positive correlation was found between parasites and viral loads. Remarkably, treated T. cruzi/HIV-coinfected patients had 77.3% conversion from positive to negative parasitemia compared to 19.1% of untreated patients. Additionally, untreated patients showed similar to 13.6 times higher Odds Ratio of having positive parasitemia in the follow-up period compared with treated patients. Treated and untreated patients showed no differences regarding the evolution of Chagas disease. The main factors associated with all-cause mortality were higher parasitemia, lower CD4 counts/mu L, higher viral load, and absence of antiretroviral therapy. Conclusion We recommend qPCR prospective monitoring of T. cruzi parasitemia in HIV+ coinfected patients and point out the value of pre-emptive therapy for those with high parasitemia. In parallel, early antiretroviral therapy introduction is advisable, aiming at viral load control, immune response restoration, and increasing survival. We also suggest an early antiparasitic treatment for all coinfected patients, followed by effectiveness analysis alongside antiretroviral therapy.
- Performance of a Real-Time PCR Assay for the Detection of Five Candida Species in Blood Samples from ICU Patients at Risk of Candidemia(2023) FELIX, Gabriel N.; FREITAS, Vera L. T. de; SILVA JUNIOR, Afonso R. da; MAGRI, Marcello M. C.; ROSSI, Flavia; SEJAS, Odeli N. E.; ABDALA, Edson; MALBOUISSON, Luiz M. S.; GUIMARAES, Thais; BENARD, Gil; NEGRO, Gilda M. B. DelThe gold standard for diagnosing invasive candidiasis still relies on blood cultures, which are inefficient and time-consuming to analyze. We developed an in-house qPCR assay to identify the 5 major Candida species in 78 peripheral blood (PB) samples from ICU patients at risk of candidemia. Blood cultures and (1,3)-beta-D-glucan (BDG) testing were performed concurrently to evaluate the performance of the qPCR. The qPCR was positive for DNA samples from all 20 patients with proven candidemia (positive PB cultures), showing complete concordance with Candida species identification in blood cultures, except for detection of dual candidemia in 4 patients, which was missed by blood cultures. Additionally, the qPCR detected Candida species in six DNA samples from patients with positive central venous catheters blood (CB) but negative PB cultures. BDG values were similarly high in these six samples and the ones with proven candidemia, strongly suggesting the diagnosis of a true candidemia episode despite the negative PB cultures. Samples from patients neither infected nor colonized yielded negative results in both the qPCR and BDG testing. Our qPCR assay was at least as sensitive as blood cultures, but with a shorter turnaround time. Furthermore, negative results from the qPCR provided strong evidence for the absence of candidemia caused by the five major Candida species.
- Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level(2011) FREITAS, Vera Lucia Teixeira de; SILVA, Sheila Cristina Vicente da; SARTORI, Ana Marli; BEZERRA, Rita Cristina; WESTPHALEN, Elizabeth Visone Nunes; MOLINA, Tatiane Decaris; TEIXEIRA, Antonio R. L.; IBRAHIM, Karim Yaqub; SHIKANAI-YASUDA, Maria AparecidaBackground: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.
- A Specific IL6 Polymorphic Genotype Modulates the Risk of Trypanosoma cruzi Parasitemia While IL18, IL17A, and IL1B Variant Profiles and HIV Infection Protect Against Cardiomyopathy in Chagas Disease(2020) SANTOS, Alexandra Gomes dos; WATANABE, Elieser Hitoshi; FERREIRA, Daiane Tomomi; OLIVEIRA, Jamille; NAKANISHI, Erika Shimoda; OLIVEIRA, Claudia Silva; BOCCHI, Edimar; NOVAES, Cristina Terra Gallafrio; CRUZ, Fatima; CARVALHO, Noemia Barbosa; SATO, Paula Keiko; YAMASHIRO-KANASHIRO, Edite Hatsumi; PONTILLO, Alessandra; FREITAS, Vera Lucia Teixeira de; ONUCHIC, Luiz Fernando; SHIKANAI-YASUDA, Maria AparecidaBackground Chagas disease caused by Trypanosoma cruzi (T. cruzi) affects approximately six million individuals worldwide. Clinical manifestations are expected to occur due to the parasite persistence and host immune response. Herein we investigated potential associations between IL1B, IL6, IL17A, or IL18 polymorphism profiles and cardiomyopathy or T. cruzi parasitemia, as well as the impact of HIV infection on cardiopathy. Methods Two hundred twenty-six patients and 90 control individuals were analyzed. IL1B rs1143627 T>C, IL6 rs1800795 C>G, IL17A rs2275913 G>A, IL18 rs187238 C>G, and IL18 rs1946518 C>A SNVs were analyzed by real-time PCR and T. cruzi parasitemia by PCR. Results Our data revealed association between a cytokine gene polymorphism and parasitemia never previously reported. The IL6 rs1800795 CG genotype lowered the risk of positive parasitemia (OR = 0.45, 95% CI 0.24-0.86, P = 0.015). Original findings included associations between IL17A rs2275913 AA and IL18 s1946518 AA genotypes with decreased risk of developing cardiomyopathy (OR = 0.27, 95% CI 0.07-0.97, P = 0.044; and OR = 0.35, 95% CI 0.14-0.87, P = 0.023, respectively). IL18 rs1946518 AA and IL1B rs1143627 TC were associated with reduced risk for cardiomyopathy severity, including NYHA (New York Heart Association) class >= 2 (OR = 0.21, 95% CI 0.06-0.68, P = 0.009; and OR = 0.48, 95% CI 0.24-0.95, P = 0.036, respectively) and LVEF (left ventricular ejection fraction) <45% for IL18 rs1946518 AA (OR = 0.22, 95% CI 0.05-0.89, P = 0.034). A novel, unexpected protective effect of HIV infection against development/progression of cardiomyopathy was identified, based on a lower risk of developing cardiopathy (OR = 0.48, 95% CI 0.23-0.96, P = 0.039), NYHA class >= 2 (OR = 0.15, 95% CI 0.06-0.39, P < 0.001), and LVEF < 45% (OR = 0.03, 95% CI 0.00-0.25, P = 0.001). Digestive involvement was negatively associated with NYHA >= 2 and LVEF < 45% (OR = 0.20, 95% CI 0.09-0.47, P < 0.001; and OR = 0.24, 95% CI 0.09-0.62, P = 0.004, respectively). Conclusions Our data support a protective role of IL17A AA, IL18 AA, and IL1B TC genotypes against development/progression of cardiomyopathy and a modulatory effect of the IL6 CG genotype on the risk of parasitemia in Chagas disease. Notably, HIV infection was shown to protect against development/progression of cardiopathy, potentially associated with a synergistic effect of HIV and highly active antiretroviral therapy (HAART), attenuating a Th1-mediated response in the myocardium. This proposed hypothesis requires confirmation, however, in larger and more comprehensive future studies.