[Hepatitis virus. Identification and immuno-pathogenic mechanisms].
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Carrier State↗
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Results for “pathogen identification”
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Isolation & identification of Pseudomonas aeruginosa pathogenic to insect larvae.
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Animals↗
Identification of a major pathogenic epitope in the human IRBP molecule recognized by mice of the H-2r haplotype.
PURPOSE: Mice of the H-2b, H-2k, and H-2r haplotypes develop experimental autoimmune uveoretinitis (EAU) after immunization with interphotoreceptor retinoid-binding protein (IRBP) of bovine or monkey origin. The purpose of this study was to identify putative pathogenic epitope(s) of IRBP and to establish their immunodominance within the IRBP molecule. METHODS: Overlapping 20-amino acid peptides, spanning the entire human IRBP molecule, were synthesized and used to immunize C57BL/10 (H-2b), B10.BR (H-2k), and B10.RIII (H-2r) mice. Bovine IRBP was used as a positive control. Experimental autoimmune uveoretinitis was examined by histopathology 21 days after immunization. Immunologic responses were assessed by delayed-type hypersensitivity (DH) and lymphocyte proliferation assays. RESULTS: Peptide 161-180, spanning the sequence SGIPYIISYLHPGNTILHVD, was found to be highly pathogenic for B10.RIII mice but not for the other strains. A dose-response curve showed that peptide 161-180 was maximally pathogenic at 50 micrograms, but incidence and scores were reduced at 10 micrograms. The truncated 13-mer 165-177 was also highly pathogenic (100 to 200 micrograms), suggesting that it contained the pathogenic epitope. Mice immunized with the peptide, or with whole IRBP, had positive DH and lymphocyte responses to the immunizing as well as to the reciprocal antigen. A cell line derived to peptide 161-180 was also pathogenic for B10.RIII mice after adoptive transfer and responded (proliferation) to native IRBP. CONCLUSIONS: High incidence and high severity scores, as well as immunologic cross-recognition of peptide 161-180 and native IRBP in vivo and in vitro, suggest that this peptide contains a major epitope recognized as pathogenic by B10.RIII mice.
Amino Acid Sequence↗
[Isolation and identification of coli enteritis pathogens].
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Bacteriological Techniques↗
[Methodologic problems in the detection of indole formation by anaerobic pathogens].
The demonstration of indole formation is of great value for the identification of pathogenic anaerobes. A reduction of the pH to values below the critical range of 5.1-5.3 in complex media 5.5 in simple testing substrates, respectively, which is caused by the presence of fermentable carbohydrates and will result in false-negative reactions. For this reason, the indol test should be performed with an adequate buffer capacity and an addition of fermentable carbohydrates should be avoided. Strains showing a false-negative reaction in the spot indole test can be reliably accounted for by means of the rapid test in buffered tryptophan solution described here without having to use additional anaerobic culture methods.
Bacteriological Techniques↗
Strategies for the identification of virulence determinants in human pathogenic fungi.
The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.
Fungi↗
H. pylori infection in non-ulcer patients--to treat or not to treat. The case against treatment.
Heretofore regarded as a strict pathogen, recent identification of multiple mutants of H. pylori, varying in pathogenicity, genomic composition, antigenic structure and other characteristics, has led to speculation that not all strains of the organism merit elimination. Affecting half the world's population, H. pylori appears to cause clinically significant disease in <20% of cases. The costs of eradicating harmless infection in over 2 billion people are prohibitive, particularly in countries lacking resources, and are questionable even in advanced countries where infection, gastritis and related diseases are declining as social conditions improve. Major controversies surround empiric eradication of helicobacter infection in patients with asymptomatic gastritis or non-specific dyspepsia. Apart from cost, and feasibility, there are concerns that widespread campaigns to eradicate H. pylori might cause major increases in esophageal reflux disease and esophageal adenocarcinoma, while causing some serious iatrogenic illness and increasing antibiotic resistance, with uncertain consequences to affected populations.
Dyspepsia↗
[Development of molecular tests for rapid detection of enteropathogens].
Amplification of specific DNA sequences by polymerase chain reaction (PCR), enables rapid, sensitive and direct, specific identification of pathogens at very low concentrations in clinical samples. Studies in recent years have reported identification of several enteropathogens directly from stool samples by PCR. The amplification process includes the use of primers complementary to the DNA sequences specific to the pathogen, thus relying on the pathogen's genotype, rather than its phenotype on which identification by the methods of classical microbiology were based. We have developed PCR protocols for the differential identification of enteropathogens resembling the normal flora (enterotoxigenic E. coli (ETEC), E. coli O-157), Shigella spp, and the detection of enteropathogens that can not be grown on classic growth media (Norwalk virus). The amplification process is inhibited by several substrates present in fecal material (phenol, hemoglobin), limiting DNA extraction by phenol. The protocols we have developed for direct detection of Shigella spp and ETEC in stools circumvent inhibition of PCR by the use of a 4-hour pre-enrichment step in brain-heart infusion broth. Rapid and accurate identification of enteropathogens is important for prompt and focused intervention to stop the chain of transmission in outbreaks of gastroenteritis in military and civilian populations.
Dysentery, Bacillary↗
Efficacy of the enteric pathogen screen card in the presumptive identification of Salmonella enteritidis.
The efficacy of the Enteric Pathogen Screen (EPS) card as a rapid method for identification of Salmonella spp. isolated in stool cultures was studied. Of the 120 lactose-negative, sucrose-negative, H2S-producing colonies tested, 74 were identified as Salmonella enteritidis by conventional methods. The reading of the EPS card after 2 h of incubation yielded a sensitivity of 47.2% and a specificity and positive predictive value of 100%, with a negative predictive value of 54.1%. In the final reading of 4 h, a sensitivity of 97.2% and a specificity of 86.9% were obtained, with a positive predictive value of 92.3% and a negative predictive value of 95.2%.
Bacteriological Techniques↗
Development of a microfluidic biosensor module for pathogen detection.
The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.
Bacteria↗
Listeria monocytogenes. A model system for the molecular study of intracellular parasitism.
Listeria monocytogenes is a facultative intracellular bacterium which, in its mammalian host, can infect enterocytes and mononuclear phagocytes. It is responsible for severe infections in humans and animals. Recovery from infection and resistance depends on the development of a T-cell response, antibodies not being protective. Several features of L. monocytogenes make it particularly suitable for the study of genetic and molecular aspects of invasion and intracellular parasitism. First, L. monocytogenes not only multiplies rapidly in bacterial broth but also easily infects macrophages and other cells in culture. Second, since it infects primarily immunocompromised individuals or pregnant women, its manipulation does not require extensive containment. Third, the genus Listeria includes several nonpathogenic species, facilitating the identification of species-specific genes and products required for pathogenicity. This identification is now possible due to the parallel development of powerful genus-specific genetic tools (transposons, plasmids, genetic transformation) and improvement of recombinant DNA techniques. Finally, the in vivo relevance of the putative virulence genes or gene products can be tested in the experimental murine infection, which has already proved invaluable in the study of the induction and expression of T-cell-mediated immune response. This review discusses current knowledge concerning these particular features, with an emphasis on listeriolysin O, a major virulence factor, and the only bacterial gene product known to be absolutely required for intracellular growth.
Animals↗
Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens.
Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n=38) or solely histologically proven (n=24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n=10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.
DNA, Fungal↗
Evaluation of new medium for identification of dermatophytes and primary dimorphic pathogens.
Only 25 of 77 dermatophytic isolates caused dermatophyte identification medium (DIM) to turn purple after incubation at the recommended temperature (37 degrees C); the accuracy of the results was improved at 30 degrees C (71 of 77 isolates yielded positive results). Many dimorphic pathogenic fungi also tested positive at both incubation temperatures. Thus, DIM has limited usefulness for presumptive identification of dermatophytes.
Arthrodermataceae↗
[Identification of genomic differences in avian pathogenic Escherichia coli using suppression subtractive hybridization analysis].
To identify unique DNA fragments associated with avian pathogenic Escherichia coli strains, suppression subtractive hybridization (SSH) was used. The genome of nonpathogenic E. coli K-12 strain MG1655 was subtracted from the genome of avian highly pathogenic strain E037 (serotype O78) resulting in the identification of 17 specific fragments. And the genome of avian low pathogenic E. coli strain E526 (serotype O2) was subtracted from the genome of avian highly pathogenic strain E058 (serotype O2) resulting in the identification of 32 specific fragments. Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function. And 12 specific fragments that were not found in E. coli K-12 were identified from two avian E. coli strains. The results suggested that there were some genetic differences between the highly pathogenic strains and low pathogenic or nonpathogenic strains.
Animals↗
Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.
Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900 bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.
Acanthamoeba↗
Rapid identification of bacteria from positive blood cultures by terminal restriction fragment length polymorphism profile analysis of the 16S rRNA gene.
Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5899 predicted TRF profiles from sequence information representing 2860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within approximately 8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.
Bacteria↗
An immunogenic 30-kDa surface antigen of pathogenic clinical isolates of Entamoeba histolytica.
A 30-kDa surface antigen was identified by Western blots with human immune sera in all 15 isolates of E. histolytica from patients with invasive amebiasis (pathogenic) but not in 15 strains from asymptomatic patients (nonpathogenic). This antigen is highly immunogenic in naturally infected humans and was recognized by sera from 22 patients with invasive disease but not by sera from 13 patients harboring nonpathogenic strains. Its surface location is supported by its differential extraction in the detergent phase of Triton X-114 and by surface immunofluorescence of live trophozoites. Unlike previously described amebic surface antigens, this 30-kDa antigen is undetectable in axenic strains that were originally isolated from patients with invasive disease but have been adapted to grow without bacteria. Affinity-purified antibody to the 30-kDa antigen did not promote lysis of complement-resistant pathogenic strains. This surface antigen may be diagnostically important in the identification of pathogenic clinical isolates.
Amebiasis↗
[Microchips based on three dimensional gel cells: history and perspective].
The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.
Gels↗