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
39 resultados
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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.
- Accuracy of the QuantiFERON-TB Gold in Tube for diagnosing tuberculosis in a young pediatric population previously vaccinated with Bacille Calmette-Guerin(2014) VALLADA, Marcelo Genofre; OKAY, Thelma Suely; DEL NEGRO, Gilda Maria B.; ANTONIO, Claudio Amaral; YAMAMOTO, Lidia; RAMOS, Sonia Regina T. S.Objective: To evaluate the accuracy of an interferongamma release assay (QuantiFERON-TB Gold in Tube) for diagnosing Mycobacterium tuberculosis infection in a young pediatric population. Methods: 195 children previously vaccinated with BCG were evaluated, being 184 healthy individuals with no clinical or epidemiological evidence of mycobacterial infection, and 11 with Mycobacterium tuberculosis infection, according to clinical, radiological, and laboratory parameters. A blood sample was obtained from each child and processed according to the manufacturer's instructions. The assay performance was evaluated by a Receiver Operating Characteristic (ROC) curve. Results: In the group of 184 non-infected children, 130 (70.6%) were under the age of four years (mean age of 35 months). In this group, 177 children (96.2%) had negative test results, six (3.2%) had indeterminate results, and one (0.5%) had a positive result. In the group of 11 infected children, the mean age was 58.5 months, and two of them (18%) had negative results. The ROC curve had an area under the curve of 0.88 (95%CI 0.82-0.92; p<0.001), disclosing a predictive positive value of 81.8% for the test (95%CI 46.3-97.4). The assay sensitivity was 81.8% (95%CI 48.2-97.2) and the specificity was 98.8% (95%CI 96-99.8). Conclusions: In the present study, the QuantiFERON-TB Gold in Tube performance for diagnosing M. tuberculosis infection was appropriate in a young pediatric population.
- PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10-3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS(2016) DOMINGUES, Wilson; KANUNFRE, Kelly Aparecida; RODRIGUES, Jonatas Cristian; TEIXEIRA, Leandro Emidio; YAMAMOTO, Lidia; OKAY, Thelma SuelyOnly a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGF beta 1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (-3575), TGF beta 1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (chi(2) = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (chi(2) = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.
- The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples(2013) TEIXEIRA, Leandro Emidio; KANUNFRE, Kelly Aparecida; SHIMOKAWA, Paulo Tadashi; TARGA, Lilia Spaleta; RODRIGUES, Jonatas Cristian; DOMINGUES, Wilson; YAMAMOTO, Lidia; OKAY, Thelma SuelyIntroduction: Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods: Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results: Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions: The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (co), NLR (0.017), and Ef (99%).
- 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.
- A PCR and RFLP-based molecular diagnostic algorithm for visceral leishmaniasis(2020) GODOY, Natalia Souza de; LIMA-JUNIOR, Manoel Sebastiao da Costa; LINDOSO, Jose Angelo Lauletta; PEREIRA-CHIOCCOLA, Vera Lucia; OKAY, Thelma Suely; BRAZ, Lucia Maria AlmeidaObjective: To determine an algorithm for molecular diagnosis of visceral leishmaniasis (VL) by kinetoplast DNA (kDNA) (RV1/ RV2) and internal transcriber spacer (ITS1) (LITSR/L5.8S) polymerase chain reaction (PCR), complemented by ITS1 PCR restriction fragment length polymorphism (RFLP), using peripheral blood or bone marrow aspirate from patients with suspected VL. Methods: Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS1 PCR, kDNA PCR, and ITS1 PCR RFLP. The samples were obtained from seven groups: group I, 82 samples from patients with confirmed VL; group H , 16 samples from patients under treatment for VL; groupII, 14 samples from dogs with canine visceral leishmaniasis (CVL); group II, a pool of six experimentally infected sandflies (Lutzomya longipalpis); group IV, 18 samples from patients with confirmed tegumentary leishmaniasis (TL) and groups ? and VI were from control groups without VII. Results: The following gold standard and molecular examination results were obtained for each of the seven groups: group I : parasitologic and immunochromatographic tests showed a sensitivity of 76.3% (61 of 80) and 68.8% (55 of 80), respectively, and a sensitivity of 97.6% (80 of 82) and 92.7% (76 of 82) by ITS1 and kDNA PCR, respectively. After ITS1 PCR RFLP (Hae III) analysis of the 80 positive samples, 52.5% (42 of 80) generated three fragments of 180, 70, and 50 bp, corresponding to the pattern of Leishmania infantum infantum; group Pi : negative for the parasitologic methods and positive for IrK39 (100%, 16 of 16), presented 12.5% (2 of 16) of positivity by ITS1 PCR and 25.0% (4 of 16) by kDNA PCR; group III: positive in the parasitologic and serologic tests (100%, 14 of 14), presented 85.7%(12 of 14) of positivity by ITS1 PCR and kDNA PCR. ITS1 PCR RFLP showed that 83.3% (10 of 12) of the canine samples contained parasites with profiles similar to L. infantum; groupIVpresented amplifications by ITS1 PCR and kDNA PCR. ITS1 PCR products were analyzed by RFLP, generating a profile similar to that of L. infantum; group V: positive in the parasitologic examination (100%, 18 of 18), presented 72.2% (13 of 18) of the samples by ITS1 PCR positive. A total of 69.2% (9 of 13) showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP; and group ? and group W were negative by ITS1 and kDNA molecular tests. Comparing the molecular results with the parasitologic and serologic diagnosis from group I, almost perfect agreement was found ( kappa both>0.80, P<0.001). ITS1 and RV1/RV2 PCR detected 90.2% (74 of 82) of the samples. Two samples positive by RV1/RV2 were negative by LITSR/L5.8S, and six samples positive by LITSR/L5.8S were negative by RV1/RV2. Therefore, these two systems complemented each other; they diagnosed 100% of the samples as belonging to the Leishmania genus. Conclusions: We suggest an algorithm for the molecular diagnosis of VL, which must consider previous parasitologic and serologic (immunochromatographic) diagnoses, and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L. infantum species.
- 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.