ABSTRACT
Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.
Subject(s)
Leishmania infantum/genetics , Leishmania mexicana/genetics , Real-Time Polymerase Chain Reaction/methods , Animals , Brazil , Dogs , HumansABSTRACT
This article contains the data regarding Leishmania species identification in human and canine clinical samples from a Brazilian region endemic for Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis, using a previously developed approach involving two qPCR assays (qPCR-ML and qPCR-ama). The data are related to the article "Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: application on Brazilian clinical samples" [1], and include also details of clinical evaluation/diagnosis of human patients and primer sequences used in the qPCR assays. The Leishmania species has been determined in 27 canine samples and 11 human samples, exploiting HRM analysis of qPCR-ML and Cq values of qPCR-ML and qPCR-ama, as reported previously [2]. The qPCR data were in agreement with the species characterization obtained with other methods such as conventional species-specific PCR, ITS1 PCR-RFLP or DNA sequencing. Despite the limited number of clinical samples, these data are encouraging for a potential application in regions where L. (Viannia) spp., L. (L.) infantum and L. (L.) amazonensis are co-endemic.
ABSTRACT
Paciente do sexo feminino, queixando-se de astenia e dor em hipogastro, foi admitida no pronto-socorro do Hospital Universitário do Oeste do Paraná (HUOP). Durante a anamnese relatou tratamento de infecção crônica pelo vírus da hepatite C (VHC) com inteferon peguilado e ribavirina. Dentre os exames laboratoriais solicitados, a pesquisa de autoanticorpos contra antígenos celulares (PAAC-HEp-2), conhecido tradicionalmente como fator antinúcleo (FAN), apresentou fluorescência em forma de bastões e/ou anéis no citoplasma das células. Esse padrão é caracterizado por bastões de 3-10 µm e anéis com 2-5 µm de diâmetro espalhados através do citoplasma da célula. Portanto, esse novo padrão tem sido designado como "bastões e anéis" (traduzido do inglês: Rods and Rings, RR). O alvo antigênico dessa reação foi identificado como inosina-5'-monofosfato desidrogenase tipo 2 (IMPDH2) que é uma enzima chave na síntese de nucleotídeos púricos. A enzima IMPDH2 agregada ou modificada em forma de RR nos pacientes tratados com ribavirina pode tornar-se antigênica e induzir uma resposta autoimune. É possível que o interferon alfa estimule a ocorrência de reatividade anti-RR aparentemente induzida pela ribavirina. Até o momento não se sabe por que o padrão RR em células HEp-2 ocorrem apenas em uma fração de pacientes portadores do VHC. Os dados apresentados em trabalhos anteriores possibilitam afirmar que esses anticorpos associados ao padrão RR estão fortemente relacionados com o tratamento da hepatite C. Além disso, pode-se afirmar que a ocorrência de reatividade anti-RR é promovida pela terapia combinada com interferon alfa e ribavirina.
Female patient, complaining of weakness and pain in hypogastric, was admitted to the emergency department of the University Hospital of the West of Paraná (HUOP). During the interview reported treatment of chronic infection with hepatitis C virus (HCV) with peginterferon and ribavirin. Among the laboratory tests ordered, the search for self-antibodies against cellular antigens, traditionally known as antinuclear factor, showed fluorescence shaped like rods and/or rings in the cytoplasm of cells. This study attempts to clarify the relationship between this pattern not yet completely understood and the clinical picture of the patient. This pattern is characterized by 3–10 µm rods or rings with 2–5 µm in diameter scattered throughout the cytoplasm of the cell. Therefore, this new standard has been designated as "rods and rings" (RR). The antigenic target of this reaction was identified as inosine-5'-monophosphate dehydrogenase type 2 (IMPDH2) which is a key enzyme in the synthesis of purine nucleotides. The IMPDH2 enzyme aggregated or modified shaped RR in those patients treated with ribavirin may become antigenic and induce an autoimmune response. It is possible that interferon alpha stimulates the occurrence of anti-RR reactivity apparently induced by ribavirin. So far it is not known why the standard RR in HEp2 cells occurs only in a fraction of patients with HCV. Previous studies presented in this paper allow affirming that these antibodies associated with the standard RR are strongly related to hepatitis C. Moreover, it can be stated that the occurrence of anti-RR reactivity is promoted by combination therapy with interferon and ribavirin.
Subject(s)
Humans , Female , Antiviral Agents/therapeutic use , Cytoplasm/ultrastructure , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/pathologyABSTRACT
Female patient, complaining of weakness and pain in hypogastric, was admitted to the emergency department of the University Hospital of the West of Paraná (HUOP). During the interview reported treatment of chronic infection with hepatitis C virus (HCV) with peginterferon and ribavirin. Among the laboratory tests ordered, the search for self-antibodies against cellular antigens, traditionally known as antinuclear factor, showed fluorescence shaped like rods and/or rings in the cytoplasm of cells. This study attempts to clarify the relationship between this pattern not yet completely understood and the clinical picture of the patient. This pattern is characterized by 3-10 µm rods or rings with 2-5 µm in diameter scattered throughout the cytoplasm of the cell. Therefore, this new standard has been designated as "rods and rings" (RR). The antigenic target of this reaction was identified as inosine-5'-monophosphate dehydrogenase type 2 (IMPDH2) which is a key enzyme in the synthesis of purine nucleotides. The IMPDH2 enzyme aggregated or modified shaped RR in those patients treated with ribavirin may become antigenic and induce an autoimmune response. It is possible that interferon alpha stimulates the occurrence of anti-RR reactivity apparently induced by ribavirin. So far it is not known why the standard RR in HEp2 cells occurs only in a fraction of patients with HCV. Previous studies presented in this paper allow affirming that these antibodies associated with the standard RR are strongly related to hepatitis C. Moreover, it can be stated that the occurrence of anti-RR reactivity is promoted by combination therapy with interferon and ribavirin.
