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
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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.
- Nitro-Heterocyclic compounds induce apoptosis-like effects in Leishmania (L). amazonensis promastigotes(2019) MENDONCA, Daiane Barros Dias; SILVA, Renata Ellen Costa; PALACE-BERL, Fanny; TAKAKURA, Cleusa F. H.; SOARES, Sandra Regina C.; BRAZ, Lucia Maria Almeida; TAVARES, Leoberto Costa; LINDOSO, Jose Angelo LaulettaBackground: Three drugs - pentavalent antimonials, amphotericin B and pentamidine - are currently used for leishmaniasis treatment. They are administered for long periods, only parenterally, and have high cardiac, renal and hepatic toxicities. Therefore, the investigation of new compounds is required. Nitro-heterocyclic derivatives have been used as possible drug candidates to treat diseases caused by trypanosomatids. Methods: Leishmania (L.) amazonensis promastigotes (MHO/BR/73/M2269), maintained in the Laboratorio de Soroepidemiologia - Instituto de Medicina Tropical-USP, were exposed to five nitroheterocyclic derivatives, with differences at phenyl-ring position 4: BSF-C4H9, BSF-H, BSF-NO2, BSF-CH3 and BSF-Cl, for 48 hours. After analyzing viability (MTT assay), we evaluated cellular-morphology activity of compounds by transmission electron microscopy (TEM) and measurement of apoptosis (phosphatidylserine expression) by flow cytometry. Results: EC50 of amphotericin B and BSF-CH3 were 0.50 mu M and 0.39 mu M respective. Other nitro-heterocyclic compounds presented EC50 higher than amphotericin B. All compounds showed greater AV - and PI-positive expression than amphotericin B at 100 mu M, except BSF-NO2. TEM showed complete nuclear disfigurement with 100 mu M of BSF-NO2, 25 and 6.25 mu M of BSF-H, and 6.25 mu M BSF-Cl; presence of vesicles within the flagellar pocket with 25 mu M BSF-H; alteration of the kinetoplast with 25 mu M BSF-C4H9, 25 mu M of BSF-H, 6.25 mu M BSF-CH3 and 6.25 mu M of BSF-Cl. Conclusions: Nitro-heterocyclic compounds have shown activity against promastigotes of L. amazonensis, at lower concentrations. However, improvement of compound scaffolds are needed to assist the elucidation of the mechanism of action and to achieve greater activity.
- 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.
- 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.- Genetic variability of Leishmania (Leishmania) infantum causing human visceral leishmaniasis in the Southeastern Brazil(2023) LIMA, Vinicius Alves; SILVA, Renata Elen Costa; CAMARGO, Luiz Henrique Moraes Caetano; HIRAMOTO, Roberto Mitsuyoshi; LEAL, Elcio de Souza; BRAZ, Lucia Maria Almeida; LINDOSO, Jose Angelo LaulettaLeishmania infantum is a protozoan that causes visceral leishmaniasis (VL) in the Americas and some regions of Europe. The disease is mainly characterized by hepatosplenomegaly and fever, and can be fatal. Factors related to the host and parasite can contribute to the transmission of Leishmania and the clinical outcome. The intraspecific genetic variability of L. infantum strains may be one of these factors. In this study, we evaluated the genetic variability of L. infantum obtained from bone marrow smear slides from patients in the Sao Paulo State, Brazil. For this, the minicircle of the kDNA hypervariable region was used as target by Sanger sequencing. By analyzing the similarity of the nucleotides and the maximum likelihood tree (Fasttree), we observed a high similarity (98%) among samples. Moreover, we identified four different profiles of L. infantum. In conclusion, L. infantum strains from Sao Paulo State, Brazil, showed low diversity measured by minicircle of the kDNA hypervariable region.
- Reactivation of cutaneous and mucocutaneous tegumentary leishmaniasis in rheumatoid arthritis patients: an emerging problem?(2017) SOUZA, Regina Maia de; ANDRADE JUNIOR, Heitor Franco de; DUARTE, Maria Irma Seixas; BRAZ, Lucia Maria Almeida; SCHUBACH, Armando de Oliveira; SILVA, Fatima Conceicao; AMATO, Valdir SabbagaRheumatoid arthritis (RA) is a chronic condition that is frequent in patients living in tropical areas exposed to leishmaniasis. RA therapy involves immunosuppressant drugs such as methotrexate (MTX), monoclonal antibodies (mAbs) and prednisone. We report an unusual presentation of cutaneous (CL) or mucocutaneous leishmaniasis (ML) in RA patients from an endemic area of leishmaniasis. A 51-year-old woman noted a cutaneous ulcer on her left ankle during MTX and prednisone RA therapy. Initially diagnosed as a venous stasis ulcer, the aspirate of the injury revealed the presence of Leishmania DNA. A 73-year-old woman presenting non-ulcerated, infiltrated and painful erythematous nodules inside her nostrils while receiving MTX, anti-TNF mAb, and prednisone for RA, had also the aspirate of injuries showing the presence of Leishmania DNA. Both patients healed after the therapy with liposomal amphotericin. The RA therapy has changed to low-dose prednisone, without further reactivation episodes. Both cases suggest that CL or ML can reactivate after administration of an immunosuppressant for RA treatment. Therefore, immunosuppressive treatments for RA should be carefully prescribed in patients from endemic areas or with a history of CL and ML.
- Unusual manifestation of genital cutaneous leishmaniasis in an immunocompetent patient from Sao Paulo, Brazil: A case report(2021) REIS, Luiza Campos; LINDOSO, Jose Angelo Lauletta; CELESTE, Beatriz Julieta; BRAZ, Lucia Maria Almeida; RAMOS-SANCHEZ, Eduardo Milton; YAMASHIRO-KANASHIRO, Edite Hatsumi; GOTO, Hiro; OYAFUSO, Luiza Keiko MatsukaA 31-year-old male patient developed an ulcer on the glans penis that evolved for three months without healing. We diagnosed it as leishmaniasis using polymerase chain reaction. No immunosuppression or associated diseases were observed. The patient was treated with meglumine antimoniate that cured the lesion in a month post-treatment. Here, we report this case of cutaneous leishmaniasis lesion at the unusual location of glans penis in an immunocompetent individual. The lesion likely developed due to the bite of a vector, highlighting the need for considering cutaneous leishmaniasis among differential diagnosis of sexually transmitted diseases in areas endemic for leishmaniasis.