Scielo RSS <![CDATA[Memórias do Instituto Oswaldo Cruz]]> vol. 113 num. 4 lang. en <![CDATA[SciELO Logo]]> <![CDATA[RNA viruses in trypanosomatid parasites: a historical overview]]> Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates’ biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts. <![CDATA[Introns in <em>Cryptococcus</em>]]> In Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi. <![CDATA[Advances in <em>Cryptococcus</em> genomics: insights into the evolution of pathogenesis]]> Cryptococcus species are the causative agents of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals. Initial work on the molecular epidemiology of this fungal pathogen utilized genotyping approaches to describe the genetic diversity and biogeography of two species, Cryptococcus neoformans and Cryptococcus gattii. Whole genome sequencing of representatives of both species resulted in reference assemblies enabling a wide array of downstream studies and genomic resources. With the increasing availability of whole genome sequencing, both species have now had hundreds of individual isolates sequenced, providing fine-scale insight into the evolution and diversification of Cryptococcus and allowing for the first genome-wide association studies to identify genetic variants associated with human virulence. Sequencing has also begun to examine the microevolution of isolates during prolonged infection and to identify variants specific to outbreak lineages, highlighting the potential role of hyper-mutation in evolving within short time scales. We can anticipate that further advances in sequencing technology and sequencing microbial genomes at scale, including metagenomics approaches, will continue to refine our view of how the evolution of Cryptococcus drives its success as a pathogen. <![CDATA[The war on cryptococcosis: A Review of the antifungal arsenal]]> Cryptococcal meningitis is the most common central nervous system infection in the world today. It occurs primarily, but not exclusively, in immunocompromised individuals and despite substantial improvement in management of clinical events like AIDS, the numbers of cases of cryptococcosis remain very high. Unfortunately, despite several antifungal agents available for treatment, morbidity and mortality rates remain high with this fungal infection. In this Review, we will describe the treatments and strategies for success, identify the failures, and provide insights into the future developments / improvements for management. This sugar-coated yeast can play havoc within the human brain. Our goals must be to either prevent or diagnose disease early and treat aggressively with all our clinical tools when disease is detected. <![CDATA[<em>In vitro</em> evaluation of the anti-leishmanial activity and toxicity of PK11195]]> BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent. <![CDATA[The influence of a light and dark cycle on the egg laying activity of <em>Aedes aegypti</em> (Linnaeus, 1762) (Diptera: Culicidae)]]> BACKGROUND The epidemiological importance of the mosquito Aedes aegypti as a vector of multiple human pathogens has generated a growing number of studies on the physiology and behaviour of its blood-feeding females. The activity of oviposition is one of the critical elements contributing to the expansion of Ae. aegypti's populations. Although there is a vast literature about oviposition behaviour, significant specific knowledge about egg viability and female fertility under light and dark conditions is still lacking. OBJECTIVES We studied, in controlled laboratory conditions, the effect that light and dark cycles have on the efficiency of oviposition by Ae. aegypti females. METHODS Physiological assays were performed using synchronised eggs obtained from forced egg laying. The number and viability of eggs was analysed under three different light/dark regimes: LD12:12 (12 h of light and 12 h of dark), DD (constant darkness) and LL (constant light). FINDINGS and CONCLUSIONS Our results show that females prefer to lay their eggs in dark conditions, but maximising the number and viability of eggs requires the occurrence of a light/dark cycle. Ongoing research on this theme has the potential of contributing to the proposition of new strategies for control based on the failure of egg laying and hatching. <![CDATA[Chagas disease and systemic autoimmune diseases among Bolivian patients in Switzerland]]> BACKGROUND Chronic cardiomyopathy occurs in 20-40% of the patients with Chagas disease. Autoimmune mechanisms may contribute to its pathogenesis. We diagnosed several cases of systemic autoimmune diseases among Bolivian migrants in Geneva with a high prevalence of Chagas disease. OBJECTIVES We tested the hypothesis of a clinical association between systemic autoimmune diseases and Chagas disease, particularly with the development of cardiomyopathy. METHODS We retrospectively searched the medical records of all Bolivian patients visiting Geneva University Hospitals between 2012 and 2015 for diagnosis of Chagas disease or systemic autoimmune diseases. FINDINGS Of the 2,189 eligible patients, 28 [1.3%; 95% confidence interval (CI) = 0.9-1.9%] presented with systemic autoimmune disease. The Chagas status was known in 903 (41.3%) patient, of whom 244 (27.0%; 95% CI = 24.2-30.0%) were positive. Eight (28.6%; 95% CI = 15.3-47.1%) of the 28 cases of systemic autoimmune disease had Chagas disease. We found no association between both entities (p = 1.000) or with Chagasic cardiomyopathy (p = 0.729). Moreover, there was no evidence of a temporal relationship between antiparasitic chemotherapy and the development of systemic autoimmune diseases. CONCLUSIONS Our results do not support a clinical association between chronic Chagas disease and systemic autoimmune diseases. However, prospective studies in areas endemic for Chagas disease should better assess the prevalence of systemic autoimmune diseases and thus a possible relationship with this infection. <![CDATA[Characterisation of an ABC transporter of a resistant <em>Candida glabrata</em> clinical isolate]]> BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion. <![CDATA[Type-2 diabetes alters the basal phenotype of human macrophages and diminishes their capacity to respond, internalise, and control <em>Mycobacterium tuberculosis</em>]]> BACKGROUND Type 2 diabetes (T2D) is a risk factor for the development of tuberculosis (TB), although the associated mechanisms are not known. OBJECTIVES To study the association between T2D and the basal phenotype of macrophages, and their immune response to Mycobacterium tuberculosis (Mtb) infection. METHODS We evaluated the influence of T2D on the response of monocyte-derived macrophages (MDM) to Mtb in patients with T2D (n = 10) compared to healthy subjects (n = 9), before and after infection with Mtb clinical isolates bearing different degrees of virulence. The levels of cell surface markers for activation secreted cytokines and chemokines, bacterial association, and intracellular bacterial growth were evaluated. FINDINGS The expression levels of HLA-DR, CD80, and CD86 were low while those of of PD-L1 were high in uninfected MDMs derived from patients with diabetes; as a result of Mtb infection, changes were only observed in the expression levels of PD-L1. The levels of cytokines (e.g., IL-6, IL-1β, IL-10, and IL-12) and chemokines (e.g., MCP-1, MIG, and RANTES) are perturbed in MDMs derived from patients with diabetes, both before infection and in response to Mtb infection. In response to the more virulent Mtb strains, the levels of association and bacterial clearance were diminished in MDMs derived from patients with diabetes. CONCLUSIONS T2D affects the basal activation state of the macrophages and its capacity to respond and control Mtb infection. <![CDATA[Correlation of transforming growth factor-β1 and tumour necrosis factor levels with left ventricular function in Chagas disease]]> BACKGROUND Transforming growth factor β1 (TGF-β1) and tumour necrosis factor (TNF) have been implicated in Chagas disease pathophysiology and may correlate with left ventricular (LV) function. OBJECTIVES We determined whether TGF-β1 and TNF serum levels correlate with LV systolic and diastolic functions and brain natriuretic peptide (BNP) serum levels in chronic Chagas disease. METHODS This cross-sectional study included 152 patients with Chagas disease (43% men; 57 ± 12 years old), classified as 53 patients with indeterminate form and 99 patients with cardiac form (stage A: 24, stage B: 25, stage C: 44, stage D: 6). TGF-β1, TNF, and BNP were determined by enzyme-linked immunosorbent assay ELISA. Echocardiogram was used to determine left atrial and LV diameters, as well as LV ejection fraction and diastolic function. FINDINGS TGF-b1 serum levels were lower in stages B, C, and D, while TNF serum levels were higher in stages C and D of the cardiac form. TGF-β1 presented a weak correlation with LV diastolic function and LV ejection fraction. TNF presented a weak correlation with left atrial and LV diameters and LV ejection fraction. CONCLUSIONS TNF is increased, while TGF-β1 is decreased in the cardiac form of chronic Chagas disease. TNF and TGF-β1 serum levels present a weak correlation with LV systolic and diastolic function in Chagas disease patients. <![CDATA[Lower galactosylation levels of the Lipophosphoglycan from <em>Leishmania (Leishmania) major</em>-like strains affect interaction with <em>Phlebotomus papatasi</em> and <em>Lutzomyia longipalpis</em>]]> BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host. <![CDATA[Complement system contributes to modulate the infectivity of susceptible TcI strains of <em>Trypanosoma cruzi</em>]]> BACKGROUND Trypanosoma cruzi is a protozoan parasite and an etiological agent of Chagas disease. There is a wide variability in the clinical outcome of its infection, ranging from asymptomatic individuals to those with chronic fatal mega syndromes. Both parasite and host factors, as well as their interplay, are thought to be involved in the process. OBJECTIVES To evaluate the resistance to complement-mediated killing in two T. cruzi TcI strains with differential virulence and the subsequent effect on their infectivity in mammalian cells. METHODS Tissue-culture derived trypomastigotes of both strains were incubated in guinea pig serum and subjected to flow cytometry in order to determine their viability and complement activations. Trypomastigotes were also incubated on host cells monolayers in the presence of serum, and infectivity was evaluated under different conditions of complement pathway inhibition. Relative expression of the main parasite-specific complement receptors between the two strains was assessed by quantitative real-time polymerase chain reaction. FINDINGS In this work, we showed that two TcI strains, one with lower virulence (Ninoa) compared to the other (Qro), differ in their resistance to the lytic activity of complement system, hence causing a compromised ability of Ninoa strain to invade mammalian cells. These results correlate with the three-fold lower messenger RNA (mRNA) levels of complement regulatory protein (CRP), trypomastigote-decay acceleration factor (T-DAF), and complement C2 receptor inhibitor trispanning (CRIT) in Ninoa compared to those in Qro. On the other hand, calreticulin (CRT) mRNA and surface protein levels were higher in Ninoa strain and promoted its infectivity when the lectin pathway of the complement system was inhibited. MAIN CONCLUSIONS This work suggests the complex interplay of CRP, T-DAF, CRIT, and CRT, and the diagnostic value of mRNA levels in the assessment of virulence potential of T. cruzi strains, particularly when dealing with isolates with similar genetic background. <![CDATA[First detection of dengue virus in the saliva of immunocompetent murine model]]> The lack of an experimental animal model for the study of dengue pathogenesis is a limiting factor for the development of vaccines and drugs. In previous studies, our group demonstrated the susceptibility of BALB/c mice to infection by dengue virus (DENV) 1 and 2, and the virus was successfully isolated in several organs. In this study, BALB/c mice were experimentally infected intravenously with DENV-4, and samples of their saliva were collected. Viral RNA extracted from the saliva samples was subjected to qRT-PCR, with a detection limit of 0.002 PFU/mL. The presence of DENV-4 viral RNA was detected in the saliva of two mice, presenting viral titers of 109 RNA/mL. The detection of DENV RNA via saliva sampling is not a common practice in dengue diagnosis, due to the lower detection rates in human patients. However, the results observed in this study seem to indicate that, as in humans, detection rates of DENV RNA in mouse saliva are also low, correlating the infection in both cases. This study reports the first DENV detection in the saliva of BALB/c immunocompetent mice experimentally infected with non-neuroadapted DENV-4. <![CDATA[<em>Leishmania (Viannia) braziliensis</em> isolated from the saliva of patients in a cutaneous leishmaniasis-endemic area of northeastern Brazil]]> Several studies have described the use of non-invasive collection methods, mostly based on the detection of parasite DNA, for diagnosis. However, no Leishmania specimens have been isolated from saliva. Here, we report the first isolation of Leishmania braziliensis from the saliva of humans with cutaneous leishmaniasis but without lesions on their mucosa. The isolates were obtained from salivary fluid inoculated in hamsters and were tested by multilocus enzyme electrophoresis. Seven samples from 43 patients suspected of having the disease were identified for in vivo culture. These findings suggest that saliva is a clinical sample that allows the isolation of Leishmania sp. <![CDATA[Erratum: Trichuris trichiura in a post-Colonial Brazilian mummy]]> Several studies have described the use of non-invasive collection methods, mostly based on the detection of parasite DNA, for diagnosis. However, no Leishmania specimens have been isolated from saliva. Here, we report the first isolation of Leishmania braziliensis from the saliva of humans with cutaneous leishmaniasis but without lesions on their mucosa. The isolates were obtained from salivary fluid inoculated in hamsters and were tested by multilocus enzyme electrophoresis. Seven samples from 43 patients suspected of having the disease were identified for in vivo culture. These findings suggest that saliva is a clinical sample that allows the isolation of Leishmania sp. <![CDATA[Erratum]]> Several studies have described the use of non-invasive collection methods, mostly based on the detection of parasite DNA, for diagnosis. However, no Leishmania specimens have been isolated from saliva. Here, we report the first isolation of Leishmania braziliensis from the saliva of humans with cutaneous leishmaniasis but without lesions on their mucosa. The isolates were obtained from salivary fluid inoculated in hamsters and were tested by multilocus enzyme electrophoresis. Seven samples from 43 patients suspected of having the disease were identified for in vivo culture. These findings suggest that saliva is a clinical sample that allows the isolation of Leishmania sp.