LUCIA MARIA ALMEIDA BRAZ
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Medicina Preventiva, Faculdade de Medicina
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
6 resultados
Resultados de Busca
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- 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.
- USEFULNESS OF kDNA PCR IN THE DIAGNOSIS OF VISCERAL LEISHMANIASIS REACTIVATION IN CO-INFECTED PATIENTS(2013) NICODEMO, Antonio Carlos; AMATO, Valdir Sabbaga; TUON, Felipe Francisco; SOUZA, Regina Maia de; OKAY, Thelma Suely; ALMEIDA, Lucia MariaIt is important to develop new methods for diagnosing relapses in the co-infection of visceral leishmaniasis (VL) and HIV to enable earlier detection using less invasive methods. We report a case of a co-infected patient who had relapses after VL treatment, where the qualitative kDNA PCR showed a good performance. The kDNA PCR seems to be a useful tool for diagnosing VL and may be a good marker for predicting VL relapses after treatment of co-infected patients with clinical symptoms of the disease.
- Visceral leishmaniasis reactivation diagnosed by molecular technique in blood sample(2012) BRAZ, L.; NICODEMO, A.; SOUZA, R.; SANTOS, N.; GODOY, N.; OKAY, T.; AMATO, V.Background: It is possible to perform the serological diagnosis of visceral leishmaniasis through k39 immunochromatographic test. However, usually in the co-infection visceral leishmaniasis with HIV k39 strip results are not conclusive. Then, investigation by microscopic examination of smear, culture medium NNN/BHI and PCR can be performed in bone marrow aspirates (gold standard) or blood sample. In this case report PCR in blood sample proved to be useful for monitoring reactivation of visceral leishmaniasis in a patient with HIV. Methods: In four different periods of a patient hospitalization with visceral leishmaniasis, samples of bone marrow aspirates and peripheral blood were examined. They were evaluated by microscopic examination of smear stained by Panóptico, culture medium NNN/BHI and PCR targeting the kDNA. Results: In four different periods of patient hospitalization the samples from bone marrow aspirates and blood sample presented amastigotes after smear microscopic examination. By PCR (kDNA) the samples from bone marrow aspirates and blood sample showed a 120-bp band on the electrophoresis gel. And promastigotes were found in the culture medium NNN/BHI from aspirated bone marrow only in the last hospitalization. Conclusion: Aspiration of bone marrow, the gold-standard diagnostic laboratory of visceral leishmaniasis, is known to be invasive and painful. However, it is possible to diagnose the disease through k39 immunochromatographic test. But usually in patients coinfected with HIV the k39 results are not conclusive. And the microscopic examination of smear is subjective and extremely time consuming. Our results of positive PCR (120 bp) in both bone marrow aspirates and in the blood sample demonstrated the importance of PCR in detection of visceral leishmaniasis reactivation. PCR results in blood samples suggest the possibility of replacing the PCR and even the smear examination in bone marrow aspirates to detect reactivation.
- A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area(2021) FERREIRA-FILHO, Julio Cesar Rente; BRAZ, Lucia Maria Almeida; ANDRINO, Marcos Luiz Alves; YAMAMOTO, Lidia; KANASHIRO, Edite Hatsumi Yamashiro; SILVA, Ana Maria Goncalves da; KANUNFRE, Kelly Aparecida; OKAY, Thelma SuelyQuantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A100 parasites/50 ng of DNA; group B-10 parasites; group C - around 1 parasite and group D less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.
article 4 Citação(ões) na Scopus Unusual Clinical Manifestations of Leishmania (L.) infantum chagasi in an HIV-coinfected Patient and the Relevance of ITS1-PCR-RFLP: A Case Report(2018) DE, De Godoy Natalia Souza; DEMARCHI, Aiello Vera; MAIA, De Souza Regina; THELMA, Okay; ALMEIDA, Braz Lucia MariaPatients coinfected with Leishmania/HIV can develop atypical forms of visceral leishmaniasis (VL), making it indispensable to identify the etiological agent. We are presenting a postmortemspecie definition by ITS1-PCR-RFLP in a larynx tissue of a patient presented coinfection Leishmania/HIV. This patient was from a leishmaniasis endemic region in Sao Paulo(SP), Brazil, and was diagnosed clinically with mucocutaneous leishmaniasis. Before a rK39 immunochromatographic test positive, a tiny stored paraffin-embedded larynx tissue wasobtained post-mortem and submitted to 3 conventional PCR assays: kDNA (K20/K22 and RV1/RV2), and ITS1 (LITSR/L5.8S). The last one was followed by RFLP (HaeIII) and analyzed by 4% Metaphor agarose gel electrophoresis. Leishmania genus and Leishmania (Leishmania) subgenus were defined by kDNA-PCR, with K20/K22 (120 bp) and RV1/RV2 (145 bp), respectively. ITS1-PCR-RFLP identified L. (L.) infantum chagasi species visualized by the restriction patterns of 180, 70 and 50 bp. This case draws attention to the necessity for a clear identification of the etiological agent causing infection, especially in endemicregions of cutaneous and visceral leishmaniasis, and particularly in patients with comorbidities who often present atypical forms of the disease. L. (L.) infantum chagasi, which is usually responsible for VL, had changed its clinical spectrum for mucocutaneous. Unequivocal identification was carried out by ITS-PCR-RFLP, therefore confirming rK39 result. These techniques, which complemented each other, have a convenient cost-benefit ratio that makes them suitable to be applied in developing countries.- Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load(2019) FERREIRA FILHO, Julio Cesar Rente; BRAZ, Lucia Maria Almeida; ANDRINO, Marcos Luiz Alves; YAMAMOTOA, Lidia; KANUNFREA, Kelly Aparecida; OKAYA, Thelma SuelyThe choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 degrees C +/- 1 degrees C for satellite-DNA and 78.1 degrees C +/- 1 degrees C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 x 10(-3) parasite or 240 target copies, and for kDNA, 2 x 10(-4) parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (+/-0.002) and 0.991 (+/-0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.