Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Genotyping of the MTL loci and susceptibility to two antifungal agents of Candida glabrata clinical isolates

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Candida albicans isolated from human fungaemia induces apoptosis in an experimental endocarditis model

Candida albicans is the most common fungal pathogen known to cause endovascular infections, such as vascular catheter sepsis, infections of vascular prostheses and infective endocarditis. A C. albicans isolate was used to determine the apoptotic potential of the fungus in a rat endocarditis model. This study confirms the ability of C. albicans to induce apoptosis in myocardial tissue.

Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model

We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

Molecular and antigenic characterisation of ribosomal phosphoprotein P0 from Babesia bovis

Babesia bovis is a tick-borne pathogen that remains an important constraint for the development of cattle industries in tropical and subtropical regions of the world. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these phenotypes have a number of drawbacks, which justifies the search for new, more efficient immunogens based mainly on recombinant protein technology. In the present paper, ribosomal phosphoprotein P0 from a Brazilian isolate of B. bovis was produced and evaluated with regard to conservation and antigenicity. The protein sequence displayed high conservation between different Brazilian isolates of B. bovis and several Apicomplexa parasites such as Theileria, Neospora and Toxoplasma. IgG from cattle experimentally and naturally infected with B. bovisas well as IgG1 and IgG2 from naturally infected cattle reacted with the recombinant protein. IgG from cattle experimentally infected with Babesia bigemina cross-reacted with B. bovis recombinant P0. These characteristics suggest that P0 is a potential antigen for recombinant vaccine preparations against bovine babesiosis.

Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate "what must be understood" to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

Transmission blocking vaccines to control insect-borne diseases: a review

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Helicobacter pylori detection in gastric biopsies, saliva and dental plaque of Brazilian dyspeptic patients

Helicobacter pylori is an important human pathogen that causes chronic gastritis and is associated with the development of peptic ulcer disease and gastric malignancies. The oral cavity has been implicated as a potential H. pylori reservoir and may therefore be involved in the reinfection of the stomach, which can sometimes occur following treatment of an H. pylori infection. The objectives of this paper were (i) to determine the presence of H. pylori in the oral cavity and (ii) to examine the relationship between oral H. pylori and subsequent gastritis. Gastric biopsies, saliva samples and dental plaques were obtained from 78 dyspeptic adults. DNA was extracted and evaluated for the presence of H. pylori using polymerase chain reaction and Southern blotting methods. Persons with gastritis were frequently positive for H. pylori in their stomachs (p < 0.0001) and there was a statistically significant correlation between the presence of H. pylori in gastric biopsies and the oral cavity (p < 0.0001). Our results suggest a relationship between gastric infection and the presence of this bacterium in the oral cavity. Despite this, H. pylori were present in the oral cavity with variable distribution between saliva and dental plaques, suggesting the existence of a reservoir for the species and a potential association with gastric reinfection.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9%) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9%), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3%), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Specificity of immunoblotting analyses in eosinophilic meningitis

Angiostrongylus cantonensis and Gnathostoma spinigerum are the two most common causative parasites of eosinophilic meningitis (EOM). Serological tests are helpful tools for confirming the identity of the pathogen. Recent reports determined the specificity of such tests by using normal healthy controls. There have been limited studies done to rule out the cross-reactivity between these two causative parasites of EOM. This study aims to assess the specificity of the serological test in EOM by using each condition as a control for the other. Thirty-three patients with a diagnosis of EOM were enrolled. Sera from 22 patients with a positive 29-kDa antigenic diagnostic band of A. cantonensis were tested for the 21 and 24-kDa antigenic bands of G. spinigerum. Similarly, sera of 11 gnathostomiasis patients were tested for the 29-kDa diagnostic band for A. cantonensis. Only one patient in the angiostrongyliasis group had a positive result for the 21 and 24-kDa antigenic bands of G. spinigerum, while no gnathostomiasis patients showed a positive result for the 29-kDa antigenic band of A. cantonensis. The specificity of the 21 and 24-kDa antigenic bands for gnathostomiasis and the 29-kDa antigenic band for A. cantonensis was 95.5% and 100%, respectively. The antigenic bands for the diagnosis of gnathostomiasis and angiostrongyliasis in EOM were highly specific.

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.

Genotyping of two Neisseria gonorrhoeae fluroquinolone-resistant strains in the Brazilian Amazon Region

We report two ciprofloxacin and ofloxacin-resistant Neisseria gonorrhoeae strains that were isolated from the urethral discharge of male patients at the sexually transmitted diseases outpatient clinic of the Alfredo da Matta Foundation (Manaus, state of Amazonas, Brazil). The gonococci displayed minimal inhibitory concentrations (> 32.00 µg/mL) and three mutations in the quinolone resistance-determining region (S91F and D95G in GyrA and S87R in ParC). Both isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing and the analysis showed that the ST225 which represented an emerging widespread multi-resistant clone that has also been associated with reduced susceptibility to ceftriaxone. We recommend continued surveillance of this pathogen to assess the efficacy of anti-gonococcal antibiotics in Brazil.

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.