THELMA SUELY OKAY
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Pediatria, Faculdade de Medicina - Docente
LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina
LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina
18 resultados
Resultados de Busca
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- Single-round multiplex PCR with species-specific mitochondrial primers of P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum: Comparison with standard techniques(2022) DOMINGUES, Wilson; SANTOS, Emilly Henrique dos; YAMAMOTO, Lidia; SANTI, Silvia Maria Di; KANUNFRE, Kelly Aparecida; OKAY, Thelma SuelyA single-round multiplex PCR (mPCR) with species-specific primers (SSP) of three mitochondrial genes of Plasmodium, namely COX I, COX III and CYT B, was compared to microscopy and 18S rRNA semi-nested PCR, nested-PCR and Real Time PCRs (*PCRs). Each parasite has between 20 and 150 mitochondria and each mitochondria has one copy of each target gene, while 18S rRNA gene is repeated 4 to 8 times. The specificity of mPCR was assessed by testing Plasmodium from rodents and birds, parasites responsible for other endemic diseases in the country such as schistosomiasis, Chagas disease and leishmaniasis in addition to microorganisms that, like Plasmodium, can cause anemia (Bartonella henselae, Babesia vogeli, Rickettsia vini). No cross-reactions were detected. From a total of 149 specimens from suspected cases of malaria were tested, 97 were positive by microscopy (49 P. falciparum, 38 P. vivax, 6 P. malariae, 4 P. falciparum/P. vivax- mixed infections) and 52 were negative; 148 samples were positive by *PCRs (49 P. falciparum, 53 P. vivax, 7 P. malariae and 39 mixed infections) and one was negative; 146 were positive by mPCR (49 P. falciparum, 56 P. vivax, 9 P. malariae and 32 mixed infections) and three were negative. The comparison of groups found statistically significant differences between microscopy vs.*PCRs or vs. mPCR (p-values <0.0001), but no difference was found between mPCR vs. *PCRs (p=0.946). The agreement in the identification of Plasmodium species was only regular, with Kappa indices of 0.407 (microscopy vs. *PCRs), 0.433 (microscopy vs. mPCR) and 0.558 (*PCRs vs. mPCR). In conclusion, the diagnostic performance of mPCR was comparable to those of *PCRs, and superior to microscopy, although the identification of Plasmodium species showed many disagreements. In conclusion, a sensitive and specific one-round SSP multiplex PCR, capable of simultaneously detecting and identifying P. falciparum, P. vivax/P. simium and P. malariae/P. brasilianum may be useful in resource-constrained countries where quantitative amplifications are not yet fully accessible.
- The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance(2013) CARVALHO, Vagner Oliveira; OKAY, Thelma S.; MELHEM, Marcia S. C.; SZESZS, Maria Walderez; NEGRO, Gilda M. B. delBackground: For many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species. Aims: In this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole. Methods: Screening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing. Results: We identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p. Conclusion: The L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.
- New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil(2023) SANTOS, Emilly Henrique dos; BARREIRA, Gabriel Acca; YAMAMOTO, Lidia; ROCHA, Mussya Cisotto; RODRIGUES, Karen Alessandra; CRUZ, Maria Carolina Pires; KANUNFRE, Kelly Aparecida; OKAY, Thelma SuelyThis study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.
- Myocarditis in children and detection of viruses in myocardial tissue: Implications for immunosuppressive therapy(2011) CAMARGO, Paulo Roberto; OKAY, Thelma Suely; YAMAMOTO, Lidia; NEGRO, Gilda Maria Barbaro Del; LOPES, Antonio AugustoBackground: There is scarce information on the potential benefits of immunosuppression in children with myocarditis and viral genomes in myocardium. We investigated the occurrence of myocarditis in children with a preliminary diagnosis of dilated cardiomyopathy, the frequency of cardiotropic viruses in the myocardium, and the response to immunosuppression. Methods: Thirty patients (nine months to 12 years) with left ventricular ejection fraction of 22.8 +/- 4.1% were subjected to right cardiac catheterization and endomyocardial biopsy. Specimens were analyzed for the presence of inflammatory elements (Dallas criteria) and viral genome (polymerase chain reaction). Patients with active myocarditis received immunosuppressants (azatioprine and prednisone) and were recatheterized nine months later. A historical control group of nine patients with myocarditis who did not receive immunosuppressants was included. Results: Active myocarditis was diagnosed in ten patients (five with viral genomes detected). Immunosuppression resulted in a significant increase in left ventricular ejection fraction from 25.2 +/- 2.8% to 45.7 +/- 8.6% (versus 20.0 +/- 4.0% to 22.0 +/- 9.0% in historical controls, p < 0.01) and cardiac index from 3.28 +/- 0.51 L/min/m(2) to 4.40 +/- 0.49 L/min/m(2) (versus 3.50 +/- 0.40 L/min/m(2) to 3.70 +/- 0.50 L/min/m(2) in controls, p < 0.01), regardless of the presence of viral genomes (p - 0.98 and p - 0.22, respectively for the two variables). No relevant clinical events were observed. Non-inflammatory cardiomyopathy was diagnosed in 20 patients (seven with viral genomes). While on conventional therapy, there were four deaths and three assignments to transplantation, and no improvement of left ventricular ejection fraction in the remaining ones (22.5 +/- 3.6% to 27.5 +/- 10.6%). Conclusion: Children with chronic myocarditis seem to benefit from immunosuppressive therapy, regardless of the presence of viral genome in the myocardium.
- HLA-DQA1/B1 alleles as putative susceptibility markers in congenital toxoplasmosis(2016) SHIMOKAWA, Paulo Tadashi; TARGA, Lilia Spaleta; YAMAMOTO, Lidia; RODRIGUES, Jonatas Cristian; KANUNFRE, Kelly Aparecida; OKAY, Thelma SuelyHost and parasite genotypes are among the factors associated with congenital toxoplasmosis pathogenesis. As HLA class II molecules play a key role in the immune system regulation, the aim of this study was to investigate whether HLA-DQA1/B1 alleles are associated with susceptibility or protection to congenital toxoplasmosis. One hundred and twenty-two fetuses with and 103 without toxoplasmosis were studied. The two study groups were comparable according to a number of socio-demographic and genetic variables. HLA alleles were typed by PCR-SSP. In the HLA-DQA1 region, the allele frequencies showed that *01:03 and *03:02 alleles could confer susceptibility ( OR= 3.06, p = 0.0002 and OR=9.60, p= 0.0001, respectively) as they were more frequent among infected fetuses. Regarding the HLA-DQB1 region, the *05: 04 allele could confer susceptibility ( OR = 6.95, p < 0.0001). Of the 122 infected fetuses, 10 presented susceptibility haplotypes contrasting with only one in the non-infected group. This difference was not statistically significant after correction for multiple comparison ( OR = 9.37, p=0.011). In the casuistic, there were two severely damaged fetuses with high parasite loads determined in amniotic fluid samples and HLA-DQA1 susceptibility alleles. In the present study, a discriminatory potential of HLA-DQA1/B1 alleles to identify susceptibility to congenital toxoplasmosis and the most severe cases has been shown.
- Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections(2020) YAMAMOTO, Lidia; AMORIM FILHO, Antonio G.; QUEIROZ, Joelma A.; CARVALHO, Mario H. B. de; RODRIGUES, Jonatas C.; KANUNFRE, Kelly A.; V, Rossana P. Francisco; OKAY, Thelma SuelyContext.-Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. Objective.-To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Design.-Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Results.-Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. Conclusions.-The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.
- Gastrointestinal manifestations are associated with severe pediatric COVID-19: A study in tertiary hospital(2021) PAULA, Camila Sanson Yoshino de; PALANDRI, Giovanna Gavros; FONSECA, Taiane Siraisi; VENDRAMINI, Thais Cristina Annibale; FARHAT, Sylvia Costa Lima; PEREIRA, Maria Fernanda Badue; LITVINOV, Nadia; TOMA, Ricardo Katsuya; SA, Fernanda Viveiros Moreira de; RODRIGUES, Katharina Reichmann; SCHVARTSMAN, Claudio; FORSAIT, Silvana; SAKITA, Neusa Keico; KANUNFRE, Kelly Aparecida; ROCHA, Mussya Cisotto; SANTOS, Emilly Henrique dos; OKAY, Thelma Suely; PINHO, Joao Renato Rebello; CARVALHO, Werther Brunow de; CARNEIRO-SAMPAIO, Magda; SILVA, Clovis Artur Almeida; MARQUES, Heloisa Helena de Sousa; EISENCRAFT, Adriana Pasmanik; ROSSI JUNIOR, Alfio; DELGADO, Artur Figueiredo; LEAL, Gabriela Nunes; FRAMIL, Juliana Valeria de Souza; GIBELLI, Maria Augusta Bento Cicaroni; JORGE, Patricia Palmeira Daenekas
- Differences in children and adolescents with SARS-CoV-2 infection: a cohort study in a Brazilian tertiary referral hospital(2021) MARQUES, Heloisa Helena de Sousa; PEREIRA, Maria Fernanda Badue; SANTOS, Angelica Carreira dos; FINK, Thais Toledo; PAULA, Camila Sanson Yoshino de; LITVINOV, Nadia; SCHVARTSMAN, Claudio; DELGADO, Artur Figueiredo; GIBELLI, Maria Augusta Bento Cicaroni; CARVALHOL, Werther Brunow de; ODONE FILHO, Vicente; TANNURI, Uenis; CARNEIRO-SAMPAIO, Magda; GRISI, Sandra; DUARTE, Alberto Jose da Silva; ANTONANGELO, Leila; FRANCISCO, Rossana Pucineli Vieira; OKAY, Thelma Suely; BATISTTELLA, Linamara Rizzo; CARVALHO, Carlos Roberto Ribeiro de; BRENTANI, Alexandra Valeria Maria; SILVA, Clovis ArturOBJECTIVES: To compare demographic/clinical/laboratory/treatments and outcomes among children and adolescents with laboratory-confirmed coronavirus disease 2019 (COVID-19). METHODS: This was a cross-sectional study that included patients diagnosed with pediatric COVID-19 (aged <18 years) between April 11, 2020 and April 22, 2021. During this period, 102/5,951 (1.7%) of all admissions occurred in neonates, children, and adolescents. Furthermore, 3,962 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection samples were processed in patients aged <18 years, and laboratory-confirmed COVID19 occurred in 155 (4%) inpatients and outpatients. Six/155 pediatric patients were excluded from the study. Therefore, the final group included 149 children and adolescents (n=97 inpatients and 52 outpatients) with positive SARS-CoV-2 results. RESULTS: The frequencies of sore throat, anosmia, dysgeusia, headache, myalgia, nausea, lymphopenia, pre-existing chronic conditions, immunosuppressive conditions, and autoimmune diseases were significantly reduced in children and adolescents (p < 0.05). Likewise, the frequencies of enoxaparin use (p=0.037), current immunosuppressant use (p=0.008), vasoactive agents (p=0.045), arterial hypotension (p <0.001), and shock (p=0.024) were significantly lower in children than in adolescents. Logistic regression analysis showed that adolescents with laboratory-confirmed COVID-19 had increased odds ratios (ORs) for sore throat (OR 13.054; 95% confidence interval [CI] 2.750-61.977; p=0.001), nausea (OR 8.875; 95% CI 1.660-47.446; p=0.011), and lymphopenia (OR 3.575; 95% CI 1.355-9.430; p=0.010), but also had less hospitalizations (OR 0.355; 95% CI 0.138-0.916; p=0.032). The additional logistic regression analysis on patients with preexisting chronic conditions (n=108) showed that death as an outcome was significantly associated with pediatric severe acute respiratory syndrome (SARS) (OR 22.300; 95% CI 2.341-212.421; p=0.007) and multisystem inflammatory syndrome in children (MIS-C) (OR 11.261; 95% CI 1.189-106. 581; p=0.035). CONCLUSIONS: Half of the laboratory-confirmed COVID-19 cases occurred in adolescents. Individuals belonging to this age group had an acute systemic involvement of SARS-CoV-2 infection. Pediatric SARS and MIS-C were the most important factors associated with the mortality rate in pediatric chronic conditions with COVID-19.
- Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?(2021) TOZETTO-MENDOZA, Tania Regina; KANUNFRE, Kelly Aparecida; VILAS-BOAS, Lucy Santos; ESPINOZA, Evelyn Patricia Sanchez; PAIAO, Heuder Gustavo Oliveira; ROCHA, Mussya Cisotto; PAULA, Anderson Vicente de; OLIVEIRA, Maura Salaroli de; ZAMPELLI, Daniella Bosco; JR, Jose Mauro Vieira; BUSS, Lewis; COSTA, Silvia Figueiredo; SABINO, Ester Cerdeira; WITKIN, Steven S.; OKAY, Thelma Suely; MENDES-CORREA, Maria CassiaObjectives: We evaluated the performance of a nucleoprotein-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to SARS-CoV-2. Methods: The ELISA was based on serum IgG reactivity to a 46-kDa protein derived from the recombinant SARSCoV2 nucleoprotein. Assay sensitivity was assessed using serum samples from 134 COVID-19 confirmed cases obtained > 15 days after symptom onset. Specificity was determined by testing sera from 94 healthy controls. Cross-reactivity was evaluated with sera from 96 individuals with previous dengue or zika virus-confirmed infections, with 44 sera from individuals with confirmed infections to other respiratory viruses or with bacterial and fungal infections that cause pneumonia and with 40 sera negative for SARS-CoV-2 nucleoprotein by commercial ELISA kits. Results: The majority of subjects were male and >= 60 years old. Assay sensitivity was 90.3 % (95 % confidence interval 84.1 %-94.2 %) and specificity was 97.9 % (92.6 %-99.4 %). There was no cross-reactivity with sera from individuals diagnosed with dengue, zika virus, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus, seasonal coronavirus, Mycobacterium tuberculosis, Staphylococcus (S. aureus and coagulase-negative), Streptococcus pneumoniae, Klebsiella pneumoniae and the fungus Aspergillus fumigatus. The level of concordance of our test with results from commercial ELISA kits was 100 %. Conclusion: The nucleoprotein-based ELISA was specific for detection of IgG anti-nucleoprotein antibodies to SARS-CoV-2. It utilizes a frequently employed low expense assay protocol and is easier to perform than other currently available commercial SARS-CoV2 antibody detection tests.
- A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene(2020) SANTOS, Emilly Henrique dos; YAMAMOTO, Lidia; DOMINGUES, Wilson; SANTI, Silvia Maria di; KANUNFRE, Kelly Aparecida; OKAY, Thelma SuelyPlasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/mu L). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.