Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 µg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.

Quantitave and qualitative interferences of pentoxifillyne on hepatic Schistosoma mansoni granulomas: effects on extracellular matrix and eosinophil population

Mast cells and eosinophils actively participate in tissue repair and are prominent components of Schistosoma mansoni granulomas. Since pentoxifillyne (PTX) is an immunomodulatory and antifibrotic substance, we aimed to characterize, by morphological techniques, the effect of this drug on fibrosis developed inside murine hepatic schistosomal granulomatous reaction, beyond the quantification of eosinophil and mast cell populations. The drug (1 mg/100 g animal weight) was administrated from 35 to 90 days post-infection, when the animals were killed. The intragranulomatous interstitial collagen network was analyzed by confocal laser scanning microscopy, the number of eosinophils and mast cells was quantified and the results were validated by t-student test. Treatment did not interfere on the granuloma evolution but caused a significant decrease in the total and involutive number of hepatic granulomas (p = 0.01 and 0.001, respectivelly), and in the intragranulomatous accumulation of eosinophils (p = 0.0001). Otherwise, the number of mast cells was not significantly altered (p = 0.9); however, it was positively correlated with the number of granulomatous structures (r = 0.955). In conclusion, PTX does not affect development and collagen deposition in S. mansoni murine granuloma, but decreases the intragranulomatous eosinophil accumulation possibly due to its immunomodulatory capability, interfering in cellular recruitment and/or differentiation.

Derris (Lonchocarpus) urucu (Leguminosae) Extract Modifies the Peritrophic Matrix Structure of Aedes aegypti (Diptera:Culicidae)

Aqueous suspension of ethanol extracts of Derris (Lonchocarpus) urucu (Leguminosae), collected in the state of Amazonas, Brazil, were tested for larvicidal activity against the mosquito Aedes aegypti (Diptera:Culicidae). The aim of this study was to observe the alterations of peritrophic matrix in Ae. aegypti larvae treated with an aqueous suspension of D. urucu extract. Different concentrations of D. urucu root extract were tested against fourth instar larvae. One hundred percent mortality was observed at 150 µg/ml (LC50 17.6 µg/ml) 24 h following treatment. In response to D. urucu feeding, larvae excreted a large amount of amorphous feces, while control larvae did not produce feces during the assay period. Ultrastructural studies showed that larvae fed with 150 µg/ml of D. urucu extract for 4 h have an imperfect peritrophic matrix and extensive damage of the midgut epithelium. Data indicate a protective role for the peritrophic matrix. The structural modification of the peritrophic matrix is intrinsically associated with larval mortality.

Fibrogenesis and collagen resorption in the heart and skeletal muscle of Calomys callosus experimentally infected with Trypanosoma cruzi: immunohistochemical identification of extracellular matrix components

Intense inflammatory lesions and early development of interstitial fibrosis of the myocardium and skeletal muscle with spontaneous regression, have been described in Calomys callosus infected with Trypanosoma cruzi. The genetic types of collagen present in this model were investigated through immunohistochemistry using specific antibodies, combined with histopathology and Picro-Sirius staining of collagen. Thirty-five calomys were infected with the Colombian strain of T. cruzi and sacrificed at 24, 30, 40, 60 and 90 days post-infection. Inflammatory lesions and fibrogenesis were prominent at the early phase of infection and significantly decreased during late infection. Immunoisotyping of the matrix components was performed by indirect immunofluorescence on 5 µm thick cryostat sections using specific antibodies against laminin, fibronectin and isotypes I, III and IV of collagen. In the early phase, positive deposits of all the matrix components were present, with predominance of fibronectin, laminin and collagens types I and III in the myocardium and of types III and IV in the skeletal muscles. From the 40th day, type IV collagen predominates in the heart. At the late phase of infection (60th to 90th day), a clear fragmentation and decrease of all the matrix components were detected. Findings of the present study indicate that a modulation of the inflammatory process occurs in the model of C. callosus, leading to spontaneous regression of fibrosis independent of the genetic types of collagen involved in this process.

Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.

The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Comparative randomised trial of high and conventional doses of praziquantel in the treatment of schistosomiasis mansoni

The efficacy of oral praziquantel in the treatment of schistosomiasis has been considered low by most public health institutions. In this paper, we compared the efficacy of two dosages of praziquantel (80 mg/kg vs. 50 mg/kg) in patients with chronic schistosomiasis mansoni. Two hundred eighty-eight patients with schistosomiasis from a community in Brazil were randomly divided into two groups: 145 patients (Group 1) received 80 mg/kg body weight of oral praziquantel divided in two equal doses with 1 h interval and 143 patients (Group 2) received 50 mg/kg body weight of oral praziquantel. To keep the study masked, patients in Group 2 received placebo 1 h after the first dose. All patients were subjected to clinical and ultrasonographic examination. Cure assessment was performed by repeating two stool examinations, by a quantitative method, at 30, 90 and 180 days after treatment. The morbidity of schistosomiasis was low, with a few cases of light periportal thickening and 16 cases of mild splenomegaly. The cure rates were 89.7% for Group 1 and 83.9% for Group 2. There was no difference in the efficacy of both therapeutic dosages of praziquantel assayed. The adverse reactions were more frequent with higher dosage.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.

Serial QuantiFERON-TB Gold In-Tube assay and tuberculin skin test to diagnose latent tuberculosis in household Mexican contacts: conversion and reversion rates and associated factors using conventional and borderline zone definitions

A cohort of 123 adult contacts was followed for 18‐24 months (86 completed the follow-up) to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT) and tuberculin skin test (TST) (PPD from TUBERSOL, Aventis Pasteur, Canada) for diagnosing latent tuberculosis (TB) in household contacts of TB patients using conventional (C) and borderline zone (BZ) definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL) conversion was defined as <0.35 to ≥0.35 (C) or <0.35 to >0.70 (BZ) and reversion was defined as ≥0.35 to <0.35 (C) or ≥0.35 to <0.20 (BZ); TST (mm) conversion was defined as <5 to ≥5 (C) or <5 to >10 (BZ) and reversion was defined as ≥5 to <5 (C). The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%). The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.

Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.