Memorias do Instituto Oswaldo Cruz最新文献

Background: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening.

Objectives: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability.

Methods: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry.

Findings: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed.

Main conclusions: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.

Background: Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis.

Objectives: In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil.

Methods and findings: We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens.

Main conclusions: This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.

Background: Leprosy is a highly neglected disease that is considered a serious public health problem in many countries. This illness is characterised by a variety of clinical and histopathological manifestations that are related to the patient immune response.

Objectives: This work aimed evaluate the profile of circulating immune mediators in the plasma from patients classified clinically as paucibacillary (PB), multibacillary (MB), households contacts (HHC), type1 leprosy reaction (T1R), type2 leprosy reaction (T2R) and control individuals without medical history of leprosy (CTL).

Methods: To assessment of the plasma immune mediators was used multiplex microbeads immunoassay "Luminex".

Findings: The results showed that patients (PB) had a regulatory-biased profile, while MB revealed a pro-inflammatory trend of highly expressed biomarkers. HHC display conspicuously increased levels in the plasma of the chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8), pro-inflammatory cytokines (IFN-γ,TNF and IL-1β), modulating cytokines (IL-9 and IL-1Ra) and growth factors (PDGF, G-CSF and IL-2). Interestingly, HHC displayed superior production of IFN-γ as compared to other leprosy groups, indicating a putative protective role for this cytokine during chronic Mycobacterium leprae exposure.

Main conclusion: Further investigations are currently underway to elucidate the potential of these mediators as biomarkers applicable to the diagnosis/prognosis of leprosy and also T1R and T2R leprosy reactions.

Twenty-five years have passed since the initial observation of endemic zoonotic sporotrichosis in Rio de Janeiro, Brazil. Since then, this disease has spread throughout South America. Accompanying the emergence of this mycosis, some progress has been made, including the expansion of a research network in this field and higher visibility of sporotrichosis within government authorities and funding agencies. However, there are still some challenges to curbing the expansion of this disease in the coming years. These include the development of rapid and accurate diagnostic tests, new antifungal drugs, particularly for the treatment of extracutaneous manifestations of sporotrichosis, and more comprehensive care for cats with sporotrichosis. Including these actions in the sporotrichosis research agenda is required so as to change the development of this disease in the years to come.

Background: American tegumentary leishmaniasis (ATL) is an endemic neglected tropical disease (NTD), its conventional treatment is toxic, slow, and invasive. Rapid diagnosis is crucial for the clinical management of suspected patients, so the development and use of low-cost, miniaturised and portable devices could be the key.

Objectives: This work aimed to develop a simple paper-based electrochemical platform for the serological detection of ATL.

Methods: Platform was fabricated in Whatman N°1 paper, contains a hydrophobic zone generated by wax printing, two pencil graphite electrodes, and uses specific crude extracts (CA) antigens for ATL immuno-determination. The platform performance was analysed by measuring the relative impedance change for different antigen-antibody combinations. Then, 10 serum human samples previously diagnosed by the gold standard (five positive ATL cases and five non-ATL cases) were evaluated.

Findings: The platform presented a linear response for the charge transfer resistance (ΔRct) and the interface reactance (ΔXc). Also, optimal working conditions were established (1/60 serum dilution and 180 µg/mL CA concentration). Then, the platform permits to distinguish between ATL and non-ATL (p < 0.05) human serum samples.

Main conclusions: Our platform could allow the diagnosis, management, and monitoring of leishmaniasis while being an extremely simple and environmentally friendly technology.

Background: In Brazil, transmission of visceral and cutaneous leishmaniasis has expanded geographically over the last decades, with both clinical forms occurring simultaneously in the same area.

Objectives: This study characterised the clinical, spatial, and temporal distribution, and performed entomological surveillance and natural infection analysis of a leishmaniasis-endemic area.

Methods: In order to characterise the risk of leishmaniasis transmission in Altos, Piauí, we described the clinical and socio-demographic variables and the spatial and temporal distribution of cases of American visceral leishmaniasis (AVL) and American cutaneous leishmaniasis (ACL) cases and identified potential phlebotomine vectors.

Findings: The urban area concentrated almost 54% of ACL and 86.8% of AVL cases. The temporal and spatial distribution of AVL and ACL cases in Altos show a reduction in the number of risk areas, but the presence of permanent disease transmission foci is observed especially in the urban area. 3,808 phlebotomine specimens were captured, with Lutzomyia longipalpis as the most frequent species (98.45%). Of the 35 females assessed for natural infection, one specimen of Lu. longipalpis tested positive for the presence of Leishmania infantum and Leishmania braziliensis DNA.

Main conclusion: Our results indicate the presence of risk areas for ACL and AVL in the municipality of Altos and highlight the importance of entomological surveillance to further understand a possible role of Lu. longipalpis in ACL transmission.

Background: Eosinophils are granulocytes that rapidly increase frequency in the bloodstream during helminthic infections and allergic responses. They are found in tissue infected by Leishmania during early disease, but their role during infection is not entirely understood.

Objectives: We aim to compare the disease due to Leishmania amazonensis in BALB/c and Δdbl-GATA1 mice, which lack eosinophils.

Methods: BALB/c and Δdbl-GATA1 mice infected with L. amazonensis were observed for several weeks. The parasite load and dissemination pattern were assessed.

Findings: The Δdbl-GATA1 mice developed an anticipated dissemination of L. amazonensis and a worsening disease. No differences were found in the lesion development or the parasite load in the footpad among Δdbl-GATA1 mice and BALB/c eight weeks after infection. However, nine weeks after infection, massive growth of metastatic lesions appeared in several parts of the skin in Δdbl-GATA1 mice, weeks earlier than BALB/c. We observed increased parasites in the bloodstream, probably an essential dissemination route. Thirteen weeks after infection, metastatic lesions were found in all Δdbl-GATA1 mice.

Main conclusion: These results suggest a protective role of eosinophils in delaying the disease caused by L. amazonensis, although several limitations of this mice strain must be considered.

Background: Tuberculosis (TB) is a major public health problem, which has been aggravated by the alarming growth of drug-resistant tuberculosis. Therefore, the development of a safer and more effective treatment is needed.

Objectives: The aim of this work was repositioning and evaluate histone deacetylases (HDAC) inhibitors- based drugs with potential antimycobacterial activity.

Methods: Using an in silico pharmacological repositioning strategy, three molecules that bind to the catalytic site of histone deacetylase were selected. Pneumocytes type II and macrophages were infected with Mycobacterium tuberculosis and treated with pre-selected HDAC inhibitors (HDACi). Subsequently, the ability of each of these molecules to directly promote the elimination of M. tuberculosis was evaluated by colony-forming unit (CFU)/mL. We assessed the expression of antimicrobial peptides and respiratory burst using reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Findings: Aminoacetanilide (ACE), N-Boc-1,2-phenylenediamine (N-BOC), 1,3-Diphenylurea (DFU), reduce bacillary loads in macrophages and increase the production of β-defensin-2, LL-37, superoxide dismutase (SOD) 3 and inducible nitric oxide synthase (iNOS). While only the use of ACE in type II pneumocytes decreases the bacterial load through increasing LL-37 expression. Furthermore, the use of ACE and rifampicin inhibited the survival of intracellular multi-drug resistance M. tuberculosis.

Main conclusions: Our data support the usefulness of in silico approaches for drug repositioning to provide a potential adjunctive therapy for TB.

Background: A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD).

Objectives: To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD.

Methods: This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant.

Findings: Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR.

Main conclusions: Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

Background: Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs.

Objectives: We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis.

Methods: Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays.

Findings: Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism.

Main conclusions: γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.