MARIA APARECIDA SHIKANAI YASUDA
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina - Docente
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
15 resultados
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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.
conferenceObject CHAGAS DISEASE CARDIOMYOPATHY: ASSOCIATION WITH IL-17 AND IL-18 GENETIC POLYMORPHISMS(2018) SANTOS, Alexandra dos; FERREIRA, Daiane; OLIVEIRA, Jamile; OLIVEIRA, Claudia; BOCCHI, Edimar; NOVAES, Cristina; CRUZ, Fatima; CARVALHO, Noemia; SATO, Paula; FREITAS, Vera; SHIKANAI-YASUDA, Maria- Multilocus Sequence Typing of Candida tropicalis Shows the Presence of Different Clonal Clusters and Fluconazole Susceptibility Profiles in Sequential Isolates from Candidemia Patients in Sao Paulo, Brazil(2013) MAGRI, Marcello Mihailenko Chaves; GOMES-GOUVEA, Michele Soares; FREITAS, Vera Lucia Teixeira de; MOTTA, Adriana Lopes; MORETTI, Maria Luiza; SHIKANAI-YASUDA, Maria AparecidaThe profiles of 61 Candida tropicalis isolates from 43 patients (28 adults and 15 children) diagnosed with candidemia at two teaching hospitals in Sao Paulo, Brazil, were characterized by multilocus sequence typing (MLST). For the 14 patients who had bloodstream infections, 32 isolates were serially collected from their blood and/or catheters. Thirty-nine diploid sequence types (DSTs) were differentiated. According to the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/), 36 DSTs and 23 genotypes identified from the 61 isolates had not previously been described. This report represents the first study to characterize sequential isolates of C. tropicalis from candidemia cases in South America. Microvariation in a single gene was found in the sequential isolates from 7 patients. The main polymorphisms occurred in the alleles of the XYR1 gene, specifically at nucleotide positions 215, 242, and 344. Macrovariation in six gene fragments was detected in the isolates from 3 patients. eBURST analysis added two new groups to this study (groups 6 and 18). Additionally, susceptibility tests indicate that 3 isolates were resistant to fluconazole. No correlation was found between the DSTs and susceptibility to fluconazole and/or selective antifungal pressure. Two patients were sequentially infected with resistant and susceptible strains. MLST is an important tool for studying the genetic diversity of multiple/sequential isolates of patients with candidemia, allowing the comparison of our data with those from other regions of the world, as well as allowing an analysis of the genetic relationship among several clones in sequential isolates from the same or different candidemia patient sites (blood or catheter).
- Polymorphisms on IFNG, IL12B and IL12RB1 genes and paracoccidioidomycosis in the Brazilian population(2016) CARVALHO, F. M. C.; BUSSER, F. D.; FREITAS, V. L. T.; FURUCHO, C. R.; SADAHIRO, A.; KONO, A. S. G.; CRIADO, P. R.; MORETTI, M. L.; SATO, P. K.; SHIKANAI-YASUDA, M. A.Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, especially Brazil, and is the eighth leading cause of death among chronic and recurrent infectious diseases. PCM infection is characterized by the presence of Th1 immune response; the acute form, by a mixed Th2/Th9, while the chronic form is characterized by Th17/Th22 profiles. The occurrence and severity of human PCM may also be associated with genetic factors such as single nucleotide polymorphisms (SNP) on cytokines encoding genes. We investigated the association between these polymorphisms and the different clinical forms of PCM. We included 156 patients with PCM(40 with the acute form, 99 with the chronic multifocal and 17 with the chronic unifocal form) and assayed their DNA samples for IFNG + 874 T/A SNP by PCR-ARMS (Amplification Refractory Mutational System), IL12B + 1188 A/C SNP on 3' UTR and IL12RB1 641 A/G SNP on exon 7 by PCR-RFLP (Restriction Fragment Length Polymorphism). We found similar genotypic and allelic frequencies of the investigated SNPs among the clinical forms of PCM. Considering male patients, the IL12RB1 641 AA genotype was more frequent in the chronic multifocal form while heterozygosis was in the chronic unifocal form of PCM (p = 0.048). Although our data suggest that the AA genotype (IL12RB1) may be associated with the more disseminated chronic disease, more patients of the chronic unifocal PCM group need to be analyzed as well as the secretion patterns of IFN-gamma combined with the IL-12R beta 1 expression for a better comprehension of this association.
conferenceObject Interferon c and interleukin-12p40 genetic polymorphisms in brazilian patients with paracoccidioidomycosis(2012) HOLANDA, F. M. C.; SATO, P. K.; FREITAS, V. L. T.; YASUDA, M. A. ShikanaiParacoccidioidomycosis, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis, is a chronic systemic granulomatous disease, endemic in South America, mainly in Brazil, being the eighth leading cause of death among infectious and parasitic diseases. Paracoccidioidomycosis infection is characterized by a Th1 response with secretion of IFN-c and basal levels of IL-10 and IL-4. In the acute form or juvenile a Th2 profile is predominant, with production of IL-10 and IL-4 and low levels of IFN-c. In chronic or adult, a mixed Th1 and Th2 profile is observed. The IFN-c can be induced by another cytokine, IL-12, and both are important in cellular immune response. In experimental models, IFN-c confers resistance to infection caused by Paracoccidioides brasiliensis and deficiency of IL-12p40 can cause susceptibility to this mycosis. The differential production of these cytokines may be associated with genetic polymorphisms. Objective: To investigate the association between the single nucleotide polymorphisms (SNP) IL-12B 3¢ UTR +1188 A/C and IFNG +874 T/A and the clinical forms of paracoccidioidomycosis. Materials and methods: DNA was obtained from peripheral blood samples from patients with paracoccidioidomycosis and healthy subjects. SNP IL-12B 3¢ UTR +1188 A/C was investigated with PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and SNP IFNG +874 T/A was investigated with PCR-ARMS (Polymerase Chain Reaction - Amplification Refractory Mutational System). Amplified products were visualized with electrophoresis on agarose gel. Results: In the SNP IFNG +874 T/A, the AA genotype was more frequent in the chronic multifocal and TA genotype in the unifocal. Considering the SNP IL-12B 3¢ UTR +1188 A/C there was a tendency for predominance of AC genotype in patients with chronic form, while the AA genotype was more frequent in acute. Despite this apparent difference among the groups, no statistically significant difference was observed in genotype distribution and alleles A, T and C. Conclusions: In this study there was no association between genetic polymorphisms of IL-12B 3¢ UTR +1188 A/C or IFNG +874 T/A and paracoccidioidomycosis, as well as with different clinical forms.- 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
- Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients(2017) TOZETTO-MENDOZA, Tania Regina; VASCONCELOS, Dewton de Moraes; IBRAHIM, Karim Yaqub; SARTORI, Ana Marli Christovam; BEZERRA, Rita C.; FREITAS, Vera Lucia Teixeira de; SHIKANAI-YASUDA, Maria AparecidaOBJECTIVES: The impact of Chagas disease (CD) in HIV-infected patients is relevant throughout the world. In fact, the characterization of the adaptive immune response in the context of co-infection is important for predicting the need for interventions in areas in which HIV and Chagas disease co-exist. METHODS: We described and compared the frequency of cytokine-producing T cells stimulated with soluble antigen of Trypanosoma cruzi (T. cruzi) using a cytometric assay for the following groups: individuals with chronic Chagas disease (CHR, n=10), those with Chagas disease and HIV infection (CO, n=11), those with only HIV (HIV, n=14) and healthy individuals (C, n=15). RESULTS: We found 1) a constitutively lower frequency of IL-2+ and IFN-gamma+T cells in the CHR group compared with the HIV, CO and healthy groups; 2) a suppressive activity of soluble T. cruzi antigen, which down-regulated IL-2+CD4+ and IFN-gamma+ CD4+ phenotypes, notably in the healthy group; 3) a down-regulation of inflammatory cytokines on CD8+ T cells in the indeterminate form of Chagas disease; and 4) a significant increase in IL-10 + CD8+ cells distinguishing the indeterminate form from the cardiac/digestive form of Chagas disease, even in the presence of HIV infection. CONCLUSIONS: Taken together, our data suggest the presence of an immunoregulatory response in chronic Chagas disease, which seems to be driven by T. cruzi antigens. Our findings provide new insights into immunotherapeutic strategies for people living with HIV/AIDS and Chagas disease.
- 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.