A cross-sectional study on the clinical and immunological spectrum of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

The objectives of this study were to identify individuals with symptomatic and/or asymptomatic infection due to Leishmania (L.) infantum chagasi; to study the two types of infection, both clinically and immunologically, and to determine the prevalence rate of infection at the beginning of the study. This was a cross-sectional study with a cohort of 946 individuals, of both genders, from the age of 1 year, living in the municipality of Barcarena, PA, Brazil, an area endemic for American visceral leishmaniasis (AVL). The leishmanin skin test (LST) and the indirect fluorescent test (IFAT), were used for the diagnosis of infection. One hundred and twenty cases of infection were diagnosed, with a prevalence rate of 12.6%; eight cases showed high seroreactivity (1280-10240, IgG) in IFAT and no LST reaction; four of these cases were typical AVL and four had subclinical oligosymptomatic infection. Using two immunological methods with a clinical examination of the infected individuals enabled the identification of five clinical-immunological profiles which may promote a better understanding of the interaction between L. (L.) i. chagasi and the human immune response: asymptomatic infection (AI) 73.4%; subclinical resistant infection (SRI) 15%; subclinicat oligosymptomatic infection (SOI) 3%; symptomatic infection (AVL) 3% and indeterminate initial infection (III) 5%. (C) 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Detection of dengue viral RNA in Aedes aegypti (Diptera : Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 mul of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degreesC. Following 7,10,14, and 28 d application, 10 mosquitoes each were removed from the lure pooled and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21 and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.

GLUT4 - At the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this 'translocation hypothesis' (Cushman SW. Wardzala LJ. J Biol Chem 1980;255: 4758-4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542-2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process) Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.

Effect of neutralization and cross-linking on the thermal degradation of chitosan electrospun membranes

Thermal degradation of as electrospun chitosan membranes and samples subsequently treated with ethanol and cross-linked with glutaraldehyde (GA) have been studied by thermogravimetry (TG) coupled with an infrared spectrometer (FTIR). The influence of the electrospinning process and cross-linking in the electrospun chitosan thermal stability was evaluated. Up to three degradation steps were observed in the TG data, corresponding to water dehydration reaction at temperatures below 100 ºC, loss of side groups formed between the amine groups of chitosan and trifluoroacetic acid between 150 – 270 ºC and chitosan thermal degradation that starts around 250 ºC and goes up to 400 ºC. The Kissinger model was employed to evaluate the activation energies of the electrospun membranes during isothermal experiments and revealed that thermal degradation activation energy increases for the samples processed by electrospinning and subsequent neutralization and cross-linking treatments with respect to the neat chitosan powder.

Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area, Brazil

A parasitological, clinical, serological and molecular cross-sectional study carried out in a highly endemic malaria area of Rio Negro in the Amazon State, Brazil, revealed a high prevalence of asymptomatic Plasmodium vivax infection. A total of 109 persons from 25 families were studied in five villages. Ninety-nine inhabitants (90.8%) had at least one previous episode of malaria. Serology showed 85.7% and 46.9% of positivity when P. falciparum antigens and P. vivax MSP-1, respectively, were used. Twenty blood samples were PCR positive for P. vivax (20.4%) and no P. falciparum infection was evidenced by this technique. No individual presenting positive PCR reaction had clinical malaria during the survey neither in the six months before nor after, confirming that they were cases of asymptomatic infection. Only one 12 year old girl presented a positive thick blood smear for P. vivax. This is the first description of asymptomatic Plasmodium infection in this area studied.

PREVALENCE OF PARACOCCIDIOIDOMYCOSIS INFECTION BY INTRADERMAL REACTION IN RURAL AREAS IN ALFENAS, MINAS GERAIS, BRAZIL

This study aimed to estimate the prevalence of paracoccidioidal infection by intradermal reaction (Delayed-Type Hypersensitivity, DTH) to Paracoccidioides brasiliensis in rural areas in Alfenas, Southern Minas Gerais (MG) State, Brazil, and to assess risk factors (gender, occupation, age, alcohol intake and smoking) associated with infection. We conducted a population-based cross-sectional study using intradermal tests with gp 43 paracoccidioidin in 542 participants, who were previously contacted by local health agents and so spontaneously attended the test. Participants underwent an interview by filling out a registration form with epidemiological data and were tested with an intradermal administration of 0.1 mL of paracoccidioidin in the left forearm. The test was read 48 hours after injection and was considered positive if induration was greater than or equal to 5 mm. Out of 542 participants, 46.67% were positive to the skin test. Prevalence increased in accordance with an increase of age. There was statistical significance only for males. Occupation, alcohol intake and smoking habits were not significantly associated with the risk of paracoccidioidomycosis infection. There is relevance of paracoccidioidomycosis infection in such rural areas, which suggests that further epidemiological and clinical studies on this mycosis should be done in the southern part of Minas Gerais State.

Evidence of a higher prevalence of HPV infection in HTLV-1-infected women: a cross-sectional study

INTRODUCTION:HTLV-1 infection increases susceptibility to other infections. Few studies have addressed the co-infection between HPV and HTLV-1 and the immune response involved in this interaction. The aim of this study was to determine the prevalence of cervical HPV infection in HTLV-1-infected women and to establish the risk factors involved in this co-infection. METHODS: A cross-sectional study was carried out in Salvador, Brazil, between September 2005 and December 2008, involving 50 HTLV-1-infected women from the HTLV Reference Center and 40 uninfected patients from gynecological clinic, both at the Bahiana School of Medicine. HPV infection was assessed using hybrid capture. HTLV-1 proviral load was quantified using real-time polymerase chain reaction (PCR). RESULTS: The mean age of HTLV-1-infected women (38 ± 10 years) was similar to that of the control group (36 ± 13 years). The prevalence of HPV infection was 44% in the HTLV-1-infected group and 22.5% in uninfected women (p = 0.03). HTLV-1-infected women had lower mean age at onset of sexual life (17 ± 3 years versus 19 ± 3 years; p = 0.03) and greater number of lifetime partners compared with the control group (4 ± 3 versus 2 ± 1; p < 0.01). In the group of HTLV-1-infected patients, there was neither difference in HTLV-1 proviral load between HPV-infected women and the uninfected. CONCLUSIONS: The prevalence of HPV infection was higher in HTLV-1-infected women. Further studies should be performed to evaluate the progression of this co-infection.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. Conclusions HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

Search for H→γγ produced in association with top quarks and constraints on the Yukawa coupling between the top quark and the Higgs boson using data taken at 7 TeV and 8 TeV with the ATLAS detector

A search is performed for Higgs bosons produced in association with top quarks using the diphoton decay mode of the Higgs boson. Selection requirements are optimized separately for leptonic and fully hadronic final states from the top quark decays. The dataset used corresponds to an integrated luminosity of 4.5 fb−1 of proton--proton collisions at a center-of-mass energy of 7 TeV and 20.3 fb−1 at 8 TeV recorded by the ATLAS detector at the CERN Large Hadron Collider. No significant excess over the background prediction is observed and upper limits are set on the tt¯H production cross section. The observed exclusion upper limit at 95% confidence level is 6.7 times the predicted Standard Model cross section value. In addition, limits are set on the strength of the Yukawa coupling between the top quark and the Higgs boson, taking into account the dependence of the tt¯H and tH cross sections as well as the H→γγ branching fraction on the Yukawa coupling. Lower and upper limits at 95% confidence level are set at −1.3 and +8.0 times the Yukawa coupling strength in the Standard Model.

Measurement of the WW+WZ cross section and limits on anomalous triple gauge couplings using final states with one lepton, missing transverse momentum, and two jets with the ATLAS detector at s√=7 TeV

The production of a W boson decaying to eν or μν in association with a W or Z boson decaying to two jets is studied using 4.6 fb−1 of proton--proton collision data at s√=7 TeV recorded with the ATLAS detector at the LHC. The combined WW+WZ cross section is measured with a significance of 3.4σ and is found to be 68±7 (stat.)±19 (syst.) pb, in agreement with the Standard Model expectation of 61.1±2.2 pb. The distribution of the transverse momentum of the dijet system is used to set limits on anomalous contributions to the triple gauge coupling vertices and on parameters of an effective-field-theory model.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in São Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.

Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories

BACKGROUND. The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. RESULTS. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. CONCLUSION. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Histological behavior of glutaraldehyde cross-linked bovine collagen injected into the human bladder for the treatment of vesicoureteral reflux

Endoscopic subureteral collagen injection has become an accepted means for the treatment of vesicoureteral reflux in children. The aim of this study was to evaluate the histological behavior of glutaraldehyde cross-linked bovine collagen implants. The specimens were harvested from 29 patients who underwent reimplant surgery 2 to 30 months (mean 9.5) after unsuccessful subureteral injection therapy. In addition to routine hematoxylin and eosin staining, a new staining method (solophenyl red 3BL) able to demonstrate selectively neoformation of types I and III human collagen, was applied. Invasion of host fibroblasts into the bovine implant and the formation of endogenous types I and III collagen were demonstrated in all 29 cases. Adverse histological reactions were rare and, if present, they were predominantly of an inflammatory nature.