Ledice Inácia de Araújo Pereira

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B

Resistance of Leishmania parasites to specific chemotherapy has become a well‐documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT‐test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC50, EC90 and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNgamma KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Graces insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNgamma KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNgamma KO mice to improve the possibility to isolate New World Leishmania species.

Leishmania braziliensis amastigotes stimulate production of IL-1β, IL-6, IL-10 and TGF-β by peripheral blood mononuclear cells from nonendemic area healthy residents.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL‐1β, IL‐6, TGF‐β and IL‐23 production, whereas IL‐10 and TGF‐β are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon‐γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL‐10 production in PBMCs; however, only amastigotes induced IL‐1β, IL‐6 and TGF‐β. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10

BackgroundThe interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis.MethodsIL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP.ResultsWe showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals.ConclusionsThese data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

In Vitro Metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Clinical Field Isolates, as Evaluated by Morphology, Complement Resistance, and Infectivity to Human Macrophages

This study was designed to assess in vitro metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis clinical field isolates obtained from patient lesions (L. braziliensis IMG3 and PPS6m; L. amazonensis MAB6). Metacyclogenesis was evaluated by different criteria, namely, promastigote size (morphometric analysis and flow cytometry), surface modifications (loss of lectin or monoclonal antibody (mAb) binding, complement resistance), and infectivity to human macrophages. Growth curves were similar for all parasites evaluated. The various features analyzed were expressed in a high percentage of promastigotes at 6th and 10th days of culture and a low percentage at the 2nd day. However, in most isolates, these features, considered as markers of metacyclogenesis, seemed to develop with different time courses, since the percentages of metacyclic forms detected with each technique were usually different. Parasites from 6th or 10th day and those negatively selected with lectin or mAb similarly infected human macrophages. From all isolates analyzed, L. amazonensis PH8 and MAB6 showed the highest and the lowest levels of susceptibility, respectively, to leishmanicidal activity of IFN-γ/LPS-activated macrophages. Our results showed that by using different techniques to evaluate different aspects of metacyclogenesis (morphological and biochemical modifications) different percentages of metacyclic promastigotes can be detected in each isolate culture.

Improvements in obtaining New World Leishmania sp from mucosal lesions: Notes on isolating and stocking parasites

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.

Leishmania (Viannia) braziliensis amastigotes from patients with mucosal leishmaniasis have increased ability to disseminate and are controlled by nitric oxide at the early stage of murine infection.

Mucosal leishmaniasis (ML) caused by Leishmania (Vianna) braziliensis usually appears after the healing of the primary lesion when amastigotes disseminate from the infection site to the mucosal area. Here, we investigated murine infection with amastigotes obtained from patients with ML or localized cutaneous leishmaniasis (LCL). Amastigotes were used to infect wild type, IFN-γ KO and inducible nitric oxide synthase (iNOS) KO mice. Amastigotes from patients with LCL induced lesions that appeared earlier in IFN-γ KO than parasites from ML. The lesion after infection with ML appeared early in iNOS KO than in IFN-γ KO mice and in iNOS KO mice parasites from ML and LCL cause similar lesions at the initial phase of infection, while parasites from ML induced greater lesions than the ones from LCL at the late phase. A greater number of parasites were observed in spleen of IFN-γ KO and iNOS KO mice infected with amastigotes from patients with ML than those with LCL. Parasites from ML infect a lower percentage of macrophages and are killed independent on IFN-γ and dependent on NO. The data suggest that amastigotes responsible for mucosal lesion in humans develop slowly on the initial phase of infection due to high susceptibility to NO and they have an increased ability to disseminate.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Susceptibility to Miltefosine in Brazilian Clinical Isolates of Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is the main causative species of tegumentary leishmaniasis in Brazil. In this study, we evaluated the susceptibility of 16 clinical isolates of L. (V.) braziliensis from different regions of Brazil to miltefosine in vitro. Half-maximal inhibitory concentrations of miltefosine varied from 22.9 to 144.2 μM against promastigotes and from 0.3 to 4.2 μM against intracellular amastigotes. No significant differences were found between isolates of different geographical origins. A clear correlation between the EC50 against promastigotes and amastigotes within each isolate was found. These findings contribute to the evaluation of miltefosines potential and limitations for the treatment of tegumentary leishmaniasis in Brazil.

Identification and Biological Characterization of Leishmania (Viannia) guyanensis Isolated from a Patient with Tegumentary Leishmaniasis in Goias, a Nonendemic Area for This Species in Brazil

The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil.