Quantificação do kDNA de Leishmania Viannia por qPCR em diferentes profundidades de lesões cutâneas de Leishmaniose Tegumentar Americana

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Ciências Médicas, 2018.A Leishmaniose Tegumentar Americana (LTA) é um grande problema de saúde pública, e no Brasil ocorreu por todo o território nacional. Na época atual, ainda representa um grande desafio para o clínico, pela complexidade das formas de manifestação da doença. Os exames parasitológicos (esfregaço e cultura) e histopatológico apresentam uma boa especificidade diagnóstica, mas uma sensibilidade insatisfatória, em relação ao exame de imunofluorescência indireta (IFI) é caracterizado pela alta sensibilidade, mas uma baixa especificidade diagnóstica. A toxicidade dos tratamentos disponíveis fundamenta a busca frequente por técnicas mais precisas e menos invasivas no reconhecimento da LTA. Nesse estudo, utilizou-se a Reação em Cadeia da Polimerase em tempo real (qPCR) para quantificar o cinetoplasto do DNA (kDNA) de Leishmania spp. em lesões de LTA nas diferentes camadas da pele. O estudo recrutou pacientes com suspeita clínica de LTA, no Hospital Universitário de Brasília – HUB. Exames de IFI, esfregaço, cultura do aspirado da lesão, histopatológico e Reação em Cadeia da Polimerase (PCR) convencional foram utilizados para alocar os pacientes em dois grupos: LTA e controle. A separação da epiderme da derme superior foi feita pela técnica de salt split skin, e as demais camadas foram separadas por bisturi estéril. A qPCR foi realizada de forma qualitativa e quantitativa utilizando os primers PPf-5’- GGC CCA CTA TAT TAC ACC AAC CCC-3’ e PPr-5’-GGG GTA GGG GCG TTC TGC GAA-3’, resultando em um amplificado de 120pb, para encontrar e quantificar o kDNA de Leishmania spp.. Foram incluídos 59 pacientes na pesquisa, sendo que 40 foram designados para o grupo LTA e 19 para o grupo controle. Dentre todos os exames utilizados para a alocação dos grupos, o histopatológico em relação à compatibilidade apresentou a maior acurácia diagnóstica (82,35% IC 95% = 69,75 - 90,43). A epiderme (159,1 x 106) e a derme superior (75,4 x 106) respectivamente, mostraram um maior número de parasitos do que na derme inferior (54,6 x 106) e hipoderme (16,8 x 106), porém, essa diferença não foi significantiva (p > 0.05). A acurácia obtida na derme superior (77,9% IC 95% = 65,9 - 86,7), foi significativamente maior que a acurácia das amostras da epiderme e hipoderme (p = 0,039 em ambas as comparações). No grupo LTA, a maior sensibilidade alcançada foi nas amostras da derme superior (67,5% IC 95% = 50,9 - 81,43), e no grupo controle, a epiderme, derme superior e hipoderme alcançaram 100% (IC 95% 82,3 - 100 / 82,5 - 100 / 81,5 - 100) de especificidade. Futuros estudos sobre a eliminação transepidérmica como mecanismo de defesa do parasito, devem levar em consideração o kDNA de Leishmania spp. encontrado na epiderme no presente estudo. Concluiu-se que técnicas de coletas mais superficiais e menos invasivas são suficientes para encontrar um grande número de parasitos de Leishmania spp..Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).American Tegumentary Leishmaniasis (ATL) is a major public health problem, and in Brazil occurred throughout the national territory. At the present time, it still represents a great challenge for the clinician, due to the complexity form of the manifestation of the disease. Parasitological (smear and culture) and histopathological exams have good diagnostic specificity, but an unsatisfactory sensitivity, in relation to indirect immunofluorescence (IFI) is characterized by high sensitivity, but low diagnostic specificity. The toxicity of the available treatments fundamental the frequent search for more accurate and less invasive techniques in the recognition of ATL. In this study, was used the real-time Polymerase Chain Reaction (qPCR) to quantify the DNA kinetoplast (kDNA) of Leishmania spp. in ATL lesions on different layers of the skin. The study recruited patients with clinical suspicion of ATL at the University Hospital of Brasília - HUB. IFI exams, smear, culture of the aspirate of the lesion, histopathological and conventional Polymerase Chain Reaction (PCR) were used to allocate the patients in two groups: ATL and control. The separation of the epidermis from the upper dermis was done by the salt split skin technique, and the other layers were separated by sterile scalpel. The qPCR was performed of form qualitative and quantitative using primers PPf-5'-GGC CCA CTA TAT TAC ACC AAC CCC-3 'and PPr-5'-GGG GTA GGG GCG TTC TGC GAA-3', resulting in an amplification of 120pb, to find and quantify the kDNA of Leishmania spp.. Were included 59 patients in the study, of whom 40 were assigned to the ATL group and 19 were assigned to the control group. Among all the tests used for the allocation of the groups, the histopathological aspects in relation to compatibility showed the highest diagnostic accuracy (82.35% CI 95% = 69.75 - 90.43). The epidermis (159.1 x 106) and the upper dermis (75.4 x 106), respectively, showed a higher number of parasites than in the lower dermis (54.6 x 106) and hypodermis (16.8 x 106) however, this difference was not significant (p> 0.05). Accuracy achieved in the upper dermis (77.9% CI 95% = 65.9-86.7) was significantly higher than the accuracy of the epidermis and hypodermis samples (p = 0.039 in both comparisons). In the ATL group, the highest sensitivity was found in the samples of the upper dermis (67.5% CI 95% = 50.9 - 81.43), and in the control group, the epidermis, upper dermis and hypodermis reached 100% % 82.3 - 100 / 82.5 - 100 / 81.5 - 100) of specificity. Future studies on transepidermal elimination as a defense mechanism of the parasite should take into consideration the kDNA of Leishmania spp. found in the epidermis in the present study. Was concluded that more superficial and less invasive collection techniques are sufficient to find large numbers of parasites of Leishmania spp .

PD-L1 may mediate T-cell exhaustion in a case of early diffuse Leishmaniasis caused by Leishmania (L.) amazonensis

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient

PD-L1 May Mediate T-Cell Exhaustion in a Case of Early Diffuse Leishmaniasis Caused by Leishmania (L.) amazonensis

IntroductionDiffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry.Case presentationA 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation.ConclusionThe dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient