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
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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.
- Suspected vertical transmission of Chagas disease caused by DTU TcIV in an infection probably transmitted orally, during anoutbreak in the Brazilian Amazon(2021) FREITAS, Vera Lucia Teixeira de; ESPER, Helena Rangel; NAKANISHI, Erika Shimoda; PIOTTO, Mariana Ramos; ASSY, Joao Guilherme Pontes Lima; BERRETA, Olivia Campos Pinheiro; SAID, Renato do Carmo; SEGURADO, Aluisio Augusto Cotrim; CARVALHO, Noemia Barbosa; FRANCA, Francisco Oscar de Siqueira; LOPES, Marta HeloisaThis study describes difficulties in the monitoring of a child born during an oral outbreak of Chagas disease, in which there are several indications that the transmission occurred through the congenital route: 1. the mother was in the third trimester of pregnancy when she was infected; 2. She presented high parasitemia at the time of delivery; 3. In both, the mother and her daughter, T cruzi was classified as DTU TcIV. The parasites were not found in the blood at birth and the infection was detected only three months later in an asymptomatic infant. As the mother and her child live in a highly endemic area, vector transmission could not be excluded during this period.
- Detection of Trypanosoma cruzi DTUs TcI and TcIV in two outbreaks of orally-transmitted Chagas disease in the Northern region of Brazil(2023) FREITAS, Vera Lucia Teixeira de; PIOTTO, Mariana Ramos; ESPER, Helena Rangel; NAKANISHI, Erika Yoshie Shimoda; FONSECA, Claudia de Abreu; ASSY, Joao Guilherme Pontes Lima; BERRETA, Olivia Campos Pinheiro; FRANCA, Francisco Oscar de Siqueira; LOPES, Marta HeloisaThis study describes the laboratory investigation of two acute Chagas disease outbreaks that occurred in the riverside communities of Marimarituba and Cachoeira do Arua, in the Santarem municipality, Para State, located in the Northern region of Brazil, and occurred in March 2016 and August 2017, respectively. The generation of data regarding the diversity of Trypanosoma cruzi parasites circulating in the Amazon region is key for understanding the emergence and expansion of Chagas disease. This study aimed to identify T. cruzi Discrete Typing Units (DTUs) involved in two outbreaks of acute Chagas disease (ACD) directly from the patient's biological sample. Nested and multiplex PCR targeting the symbolscript (rRNA) and mini-exon genes, respectively, were used to identify T. cruzi DTU in blood samples from patients diagnosed with ACD. The samples with positive cPCR were submitted for analysis for T. cruzi DTUs, which included 13 samples from the patients with ACD by oral transmission and two samples collected from two newborns of two women with ACD, from Marimarituba and Cachoeira do Arua. The samples were classified as T. cruzi TcIV, from Marimarituba's outbreak, and T. cruzi TcI, from Cachoeira do Arua's outbreak. The molecular identification of T. cruzi may increase understanding of the role of this parasite in Chagas disease's emergence within the Amazon region, contributing to the improvement of the management of this important, but also neglected, disease.
- Interdisciplinary approach at the primary healthcare level for Bolivian immigrants with Chagas disease in the city of Sao Paulo(2017) YASUDA, Maria Aparecida Shikanai; SATOLO, Camila Goncalves; CARVALHO, Noemia Barbosa; ATALA, Magda Maya; FERRUFINO, Rosarlo Quiroga; LEITE, Ruth Moreira; FURUCHO, Celia Regina; LUNA, Expedito; SILVA, Rubens Antonio; HAGE, Marcia; OLIVEIRA, Caroline Medeji Ramos de; BUSSER, Felipe Delatorre; FREITAS, Vera Lucia Teixeira de; WANDERLEY, Dalva Marli Valerio; MARTINELLI, Luzia; ALMEIDA, Sonia Regina; VINAS, Pedro Albajar; CARNEIRO JR., NivaldoBackground/Methods In a pioneering cross-sectional study among Bolivian immigrants in the city of Sao Paulo, Brazil, the epidemiological profile, clinical manifestations and morbidity of Chagas disease were described. The feasibility of the management of Chagas disease at primary healthcare clinics using a biomedical and psychosocial interdisciplinary approach was also tested. Previously, a Trypanosoma cruzi (T. cruzi) infection rate of 4.4% among 633 immigrants was reported. The samples were screened using two commercial enzyme-linked immunoassay (ELISA) tests generated with epimastigote antigens, and those with discrepant or seropositive results were analyzed by confirmatory tests: indirect immunofluorescence (IFI), TESA-blot and a commercial recombinant ELISA. PCR and blood cultures were performed in seropositive patients. Results The majority of the 28 seropositive patients were women, of whom 88.89% were of childbearing age. The predominant clinical forms of Chagas disease were the indeterminate and atypical cardiac forms. Less than 50% received the recommended antiparasitic treatment of benznidazole. An interdisciplinary team was centered on primary healthcare physicians who applied guidelines for the management of patients. Infectologists, cardiologists, pediatricians and other specialists acted as reference professionals. Confirmatory serology and molecular biology tests, as well as echocardiography, Holter and other tests, were performed for the assessment of affected organs in secondary healthcare centers. The published high performance of two commercial ELISA tests was not confirmed. Conclusion An interdisciplinary approach including antiparasitic treatment is feasible at the primary healthcare level for the management of Chagas disease in Bolivian immigrants. The itinerant feature of immigration was associated with a lack of adherence to antiparasitic treatment and was considered a main challenge for the clinical management of this population. This approach is recommended for management of the infected population in endemic and nonendemic areas, although different strategies are needed depending on the severity of the disease and the structure of the healthcare system.
- 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.
- Multiple myeloma and Chagas disease: qPCR as a marker forpreemptive antiparasitic therapy: a case reports series and review(2024) CARVALHO, Noemia Barbosa; FREITAS, Vera Lucia Teixeira de; SEGURO, Fernanda Salles; BEZERRA, Rita Cristina; FATOBENE, Giancarlo; NAKANISHI, erika Yoshie Shimoda; VISNADI, Helena; MARTINEZ, Gracia; BATISTA, Marjorie Vieira; ROCHA, Vanderson; DULLEY, Frederico Luis; COSTA, Silvia Figueiredo; SHIKANAI-YASUDA, Maria AparecidaMultiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56 +/- 32.10 months (mean +/- SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR. KEYWORDS Multiple myeloma; Chagas disease; T. cruzi parasitemia; Conventional PCR; Quantitative PCR
- Aplastic Anemia and Chagas Disease: T. cruzi Parasitemia Monitoring by Quantitative PCR and Preemptive Antiparasitic Therapy(2022) CARVALHO, Noemia Barbosa; FREITAS, Vera Teixeira de; BEZERRA, Rita Cristina; NAKANISHI, Erika Shimoda; VELLOSO, Elvira Pereira; HIGASHINO, Hermes Ryoiti; BATISTA, Marjorie Vieira; FONSECA, Guilherme Henrique; ROCHA, Vanderson; COSTA, Silvia Figueiredo; SHIKANAI-YASUDA, Maria AparecidaBackground: Aplastic anemia is a rare and life-threatening condition, seldomly witnessed concomitantly with Chagas disease. We aim to discuss the management of these patients under risk of chronic Chagas disease reactivation (CDR), a severe condition with a high morbimortality that occurs in chronic Chagas disease patients under immunosuppression. Case reports: Trypanosoma cruzi (T. cruzi) parasitemia was monitored in three patients for 4-58 months by conventional PCR (cPCR), quantitative PCR (qPCR), microhematocrit/buffy coat, blood culture, and/or xenodiagnosis. One patient received antiparasitic treatment (benznidazole) and the other received allopurinol. Although parasitemia was controlled during and after benznidazole treatment at 300 mg/d for 51 days, in one patient, hematologic parameters worsened continuously before, during, and after treatment. Allopurinol led only to the temporary suppression of T. cruzi parasitemia in the second patient, but after danazol and hematological improvement, parasitemia became undetectable until the end of monitoring. Discussion and Conclusion: Unexpected undetectable or low parasitemia by cPCR/qPCR was reported. We show that the monitoring of parasitemia by qPCR and the use of preemptive therapy when the parasitemia was positive proved to be beneficial to our patients. As a result of the toxicity of more effective antiparasitics, shorter regimens of benznidazole or less toxic drugs in preemptive therapy are options that deserve future studies.
- 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.
- Comparative analysis of diagnostic methods for the detection of Cryptococcus neoformans meningitis(2023) DANTAS, Katia Cristina; FREITAS-XAVIER, Roseli Santos de; LOMBARDI, Suzete Cleusa Ferreira Spina; MENDRONI JUNIOR, Alfredo; SILVA, Marcos Vinicius da; CRIADO, Paulo Ricardo; FREITAS, Vera Lucia Teixeira de; ALMEIDA, Terezinha Morato Bastos deAuthor summaryCryptococcal meningitis is an infectious disease of global importance with high morbidity and mortality, especially among individuals with HIV/AIDS. While there have been improvements in the last two decades in the diagnosis of Cryptococcus neoformans, the methods presently employed are problematic for public hospitals in Brazil and other locations due to their extreme cost. In this study, we present a low-cost option for detection and identification of C. neoformans in noninvasive serum sample in immunosuppressed individuals, including those with HIV/AIDS. A nested PCR (5.8SrDNA-ITS) associated with the latex agglutination test has high precision in detection of suspected Cryptococcus spp. BackgroundCryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples. MethodsThe 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains. Principal findingsThe 5.8S DNA-ITS PCR was more sensitive (89-100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96-100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF. ConclusionUse of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future.
- Assessment of biomarkers and clinical parameters as predictors of survival in patients with chagasic heart failure(2023) BOCCHI, Edimar Alcides; GUIMARAES, Guilherme Veiga; ROMERO, Cristhian Espinoza; SATO, Paula Keiko; FREITAS, Vera Lucia Teixeira de; KANASHIRO, Edite Hatsumi Yamashiro; FURUCHO, Celia Regina; CRUZ, Fatima Das Dores; NAKANISHI, Erika Shimoda; BUSSER, Felipe Delatorre; BEZERRA, Rita Cristina; WESTPHALEN, Elizabeth Visone Nunes; ROCHA, Mussya Cisotto; YASUDA, Maria Aparecida ShikanaiBackground Chagas disease, endemic in Latin America and spreading globally due to emigration, has a significant health burden, particularly in relation to chagasic heart failure (HF). Chagasic cardiomyopathy (CCM) is characterized by chronic inflammatory myocardial disease. This study aimed to identify inflammatory parameters and biomarkers that could aid in the management of patients with chagasic HF. Methods and findings A cohort study was conducted at a tertiary cardiology single-center over a mean follow-up period of 2.4 years. The study included patients with HF secondary to CCM enrolled between October 2013 and July 2017. Various clinical parameters, echocardiography findings, parasitemia status, brain natriuretic peptide (BNP) and troponin T (TnT) levels, and inflammatory biomarkers (IL-6, IL-10, IL-12p70, IL-17A, adiponectin, and IFN-gamma) were assessed. The study encompassed a cohort of 103 patients, with a median age of 53 years and 70% being male. The left ventricular ejection fraction (LVEF) was 28%, with 40% of patients classified as NYHA II functional class. The median BNP level was 291 pg/ml. The observed mortality rate during the study period was 38.8%. Predictors of lower survival were identified as elevated levels of BNP, TnT, reduced LVEF, and increased adiponectin (thresholds: BNP > 309 pg/ml, TnT > 27.5 ng/ml, LVEF < 25.5%, adiponectin > 38 mu g/mL). Notably, there was no evidence indicating a relationship between parasitemia and the inflammatory parameters with lower survival in these patients, including INF-gamma, IL-6, IL-10, IL12-(p70), and IL17a. Conclusion Despite the presence of a chronic inflammatory process, the evaluated inflammatory biomarkers in this cohort were not predictive of survival in patients with chagasic HF with reduced ejection fraction (HFrEF). However, reduced LVEF, elevated BNP, adiponectin levels, and troponin T were identified as predictors of lower survival in these patients.