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PubMed2004

PCR identification system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger.

A PCR system was developed that allowed recognition of three major pathogenic Aspergillus species, namely A. fumigatus, A. niger and A. flavus, in isolates obtained from clinical specimens. The primer pair for PCR was designed from conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA and its flanking regions. Products 521 bp in size were successfully amplified by PCR from the seven Aspergillus species most frequently encountered as opportunistic pathogens, and the three most commonly significant species were identified by separate PCR reactions or nested PCR based on use of species-specific primers. To our knowledge, this is first report of identification of the second and third most frequent pathogenic Aspergillus species using specific PCR amplification. The PCR based identification system reported here will be a powerful tool to control difficult pulmonary fungal infections and to speed the application of effective treatment.

Aspergillus flavus↗

PubMed1995

Application of fluoroimmunoassay to the identification of low avidity specific IgG against pathogenic human viruses and Toxoplasma gondii.

BACKGROUND: Serological diagnosis of primary viral infections is usually made by detection of specific IgM. In some cases, false positive results (mainly due to crossreactions between closely related viruses) can be obtained. Moreover, some primary infections occur without specific IgM response. Thus, alternative serological approaches are required for diagnosis. Detection of low avidity, specific IgG has been applied as a useful serological marker for diagnosing infections caused by several viruses and Toxoplasma gondii. OBJECTIVE: The standardization and application of specific IgG avidity assays using a semiautomated solid phase immunoassay (fluoroimmunoassay (FIA)) on the basis of the urea elution principle, for the characterization of low avidity specific IgG against rubella virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV) and T. gondii. STUDY DESIGN: The method consists of two simultaneous determinations, one as recommended by the manufacturer and the other including a washing step with 8 M urea after the antigen-antibody reaction. A reduction in titer higher than, or equal to, 50% was considered indicative for presence of low avidity specific IgG. RESULTS: When applied to the diagnosis of infections, this method showed sensitivity ranging from 81% to 100%, and absolute specificity. The detection of low avidity specific IgG allowed the differentiation between primary and recurrent infections caused by VZV. Furthermore, it helped in the identification of CMV as the etiological agent of congenital infection in the absence of specific IgM response, as well as in the elucidation of crossreactivity between antigenically related viruses, i.e., VZV and HSV, and Epstein-Barr virus and CMV. CONCLUSION: FIA can be used for the characterization of the avidity of specific IgG antibody as a diagnostic test in clinical laboratories.

Journal Article↗

PubMed1999

Management of bacterial complications in critically ill patients: surgical wound and catheter-related infections.

The occurrence of surgical wound infections and/or bacteremia associated with central venous catheter use are of growing concern to all physicians who treat critically ill patients. The physician must be aware that some patients have an even greater risk for infection, such as those with multiple risk factors, those who are on central lines, or those patients who undergo multiple invasive diagnostic or therapeutic procedures. The emergence of resistant pathogens, particularly Gram-positive pathogens, is an important factor in the morbidity and mortality of hospitalized patients. In the face of this growing resistance among target organisms, the selection of the correct antimicrobial and nonpharmacologic interventions, based on correct identification and susceptibility test data, has become increasingly challenging. Methicillin-resistant Staphylococcus aureus and, more recently, glycopeptide-resistant enterococci and staphylococci represent a significant danger to the patient. As a consequence, earlier and more precise identification of the pathogens most frequently associated with infection is essential. The role of exacting surgical technique, infection control measures, and the appropriate use of prophylactic and therapeutic antibiotics cannot be overestimated in helping to reduce potential morbidity and mortality associated with severe surgical infection. The development of new antibiotics may help treat the difficult cases attributable to resistant Gram-positive bacteria.

Bacteremia↗

PubMedDate not supplied

[Detection of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. Diagnostic validation].

Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen that attacks rainbow trout (Oncorhynchus mykiss) farming. The only diagnostic method recognised by the European community for VHS is based on the detection of the viral agent in cell culture followed by an immunological identification of the pathogen. Reverse transcription followed by a double amplification of the polymerase chain reaction (RT-PCR) for the gene encoding the viral glycoprotein G is proposed here as an alternative method to the virus assay in cell culture. The RT-PCR was found to have three advantages over the viral assay method. First, the RT-PCR was found to be more rapid than the virus assay method (24 h compared to 72-120 h). Second, this method was found to be more sensitive than the virus assay (2.10(-2) pfu of virus were detected per millilitre of viral suspension compared to 2.10(-1) pfu.mL-1 by inoculation of the cell lines EPC and RTG2). Third, the RT-PCR was shown to be specific towards the VHS virus assay (represented by its four serotypes VHS I, VHS II, VHS 23/75 and VHS IV). Moreover no amplification was obtained with the other rhabdoviruses used: infectious haematopoietic necrosis virus (American strain Amend 72, WRAC, RB, SRVC) and French reference strain 69/87, eel viruses, spring viraemia of carp virus and pike fry virus. The validation of this method was performed on organs removed from experimentally infected rainbow trout and ovarian fluid samples from farmed broodfish from D0 to D150. By using RT-PCR between D30 and D60, 13 samples from nine experimentally infected trout (ten kidney-spleen pools and three brains) tested positive, whereas only nine samples (four kidney-spleen pools and five brains) from six fish were positive at D30. The last positive response was obtained by RT-PCR at D60 for kidney-spleen pools from three fish. At D150, all the results were negative. From the 60 ovarian fluid samples tested, 28 were VHS positive by the RT-PCR versus 15 by the virus assay method. Eleven out of the 60 broodfish had neutralised anti-VHS, six were negative by RT-PCR and by the virus assay, four were positive by RT-PCR and negative by virus assay and one positive by both methods. The specificity, sensitivity and rapidity of the RT-PCR method makes it an attractive alternative to classical virological methods currently recommended by European Fish Health Surveillance Programmes.

Animals↗

PubMed1990

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.

Animals↗

PubMedDate not supplied

[Listeria pathogenicity factors and their role in pathogenesis and laboratory diagnosis of listeriosis].

The mechanisms of relationships between Listeria and eukaryotic cells on the level of the main biomolecules are analyzed. These mechanisms determine the penetration and multiplication of Listeria and their importance for the pathogenesis of listeriosis. The theoretical and practical aspects of the regulation of the expression of pathogenicity factors in Listeria, connected with the activation of the regulatory protein PrfA and autoregulatory mechanisms, are considered. The elimination of the autorepressor from the cultivation medium results in the activation of the regulatory protein and the induction of the pathogenicity factors. The effective identification method for L. monocytogenes, permitting the differentiation of pathogenic and non-pathogenic Listeria, has been developed on the basis of the study of lecithinase expression in the presence of activated charcoal.

Animals↗

PubMed1995

[Molecular study of 6 episodes of nosocomial infections produced by methicillin resistant Staphylococcus aureus].

A critical step in any epidemiologic research concerning nosocomial infections is the precise identification of the responsible pathogen. The present work utilized a molecular approach -plasmids identification, restriction length polymorphism DNA analysis, and random amplified polymorphic DNA- for the characterization of 6 nosocomial outbreaks due to 52 strains of methicillin-resistant Staphylococcus aureus (MRSA). In these episodes, the clinic-epidemiologic and phenotypic analysis (antibiotype) pointed to a nosocomial infection. Through molecular analysis it was possible to establish, in a very precise way, clonality due to MRSA strains in 2 of the studied outbreaks; the same type of analysis allowed to eliminate a MRSA clonal origin in the remainder 4 episodes. The antibiogram was not an useful analytic tool due to its poor discriminatory power. Also, through a PCR procedure, it was possible to identify the presence of the gen mecA in every of the 52 MRSA strains studied.

Base Sequence↗

PubMed2006

Rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization.

We report the identification of immune-related molecules from the fat body, and intestine of Rhodnius prolixus, an important vector of Chagas disease. Insects were challenged by introducing pathogens or Trypanosoma cruzi, the parasite that causes Chagas disease, into the hemocoel. RNA from intestines, or fat body were isolated 24h after stimulation. We used suppressive subtractive hybridization to identify immune-related genes, generated three subtracted libraries, sequenced the clones and assembled the sequences. The functional annotation revealed expressed sequence tags (ESTs) generated in response to various stimuli in all tissues, and included pathogen recognition molecules, regulatory molecules, and effector molecules.

Animals↗

PubMed2004

Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value.

Streptococcus iniae is a well-known pathogen of both fish and humans that is difficult to identify by conventional biochemical tests. A PCR assay based on the lactate oxidase (lctO) gene of S. iniae was developed for the rapid and specific detection and identification of this pathogen from different sources. The PCR assay had a detection limit of 62-31 cells, and 25 pg of DNA per PCR reaction mixture. The PCR was also effective in detecting the bacterium from inoculated tissue homogenates, suggesting its potential use for a rapid and accurate diagnosis of S. iniae infections.

Animals↗

PubMed1990

Comparison of serological and DNA probe analyses for detection of suspected periodontal pathogens in subgingival plaque samples.

Several methods are currently being used to identify specific bacteria in dental plaque, namely direct culture, serological techniques and DNA probes. Culture methods are labour-intensive, dependent on the viability of the cells, and require fastidious growth conditions. Serological and DNA probes allow rapid strain-specific identification of periodontal pathogens with limited effort. The purpose of the present study was to compare results from serological and DNA probes in assessing and quantitating the presence of three suspected periodontal pathogens in subgingival plaque: Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bact. intermedius. Subgingival bacterial samples were obtained from 4 periodontal sites of each of 13 patients with untreated moderate to advanced adult periodontitis. Samples were taken using 2 paper points that were simultaneously inserted for 10 s to the bottom of the periodontal pockets. The plaque from one paper point was analysed by immunofluorescence using monoclonal antibodies. The other sample was analysed by species-specific cloned DNA probes. The number of bacteria per millilitre was calculated for both methods, and used for comparisons of results obtained with the two techniques. Results from indirect immunofluorescence and DNA hybridization analyses were negative for A. actinomycetemcomitans across all samples. Fluorescence did not detect bacteria at levels lower than 10(4), while the DNA probes identified organisms at levels of 10(3). Similar numbers of samples positive for Bact. gingivalis were obtained with either method (p = 0.227), and the results were not independent. A significantly greater proportion of Bact. intermedius-positive samples was detected by immunofluorescence (p = 0.0039), and the results of immunofluorescence and DNA hybridization were independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus↗

PubMed1989

Biotypes and O serogroups of Escherichia coli involved in intestinal infections of weaned rabbits: clues to diagnosis of pathogenic strains.

A total of 575 Escherichia coli strains isolated from weaned rabbits experiencing diarrhea in 119 French commercial farms were tested for O serogroups. The results showed a strong predominance of serogroup O103 strains. A sample of 126 strains were further checked for simplified biotypes by using five carbohydrate fermentation reactions. Of 72 O103 strains, 70 were shown to belong to biotypes characterized by a rhamnose-negative reaction. Four of nine serogroup O68 strains also showed this type of reaction. Thirty-nine strains, representative of the serotypes and biotypes found, were further tested for experimental pathogenicity in weaned rabbits and for antibiotic susceptibility. All the rhamnose-negative strains produced life-threatening watery or hemorrhagic diarrhea, whereas rhamnose-positive strains induced only mild diarrheic syndrome without any mortality or no clinical signs at all. Rhamnose-negative, highly pathogenic strains did not belong to related antibiotypes. We think that O serogrouping together with biotyping, or even rhamnose fermentation testing, may be an important clue in the diagnosis of enteropathogenic strains from rabbits in France, permitting rapid identification of highly pathogenic strains and leading to improved prognosis and treatment.

Animals↗

PubMedDate not supplied

Listeria monocytogenes-induced endogenous endophthalmitis in an otherwise healthy individual: rapid PCR-diagnosis as the basis for effective treatment.

PURPOSE: Listeria monocytogenes is a rare cause of endogenous endophthalmitis. To date 15 cases have been published in the literature. All eyes showed similar clinical features and profound visual loss mainly due to delayed diagnosis. METHODS: An additional case of an otherwise healthy 73 year-old male, who was referred to our hospital because of acute iridocyclitis with secondary glaucoma, is reported. Within a few days the severity of the intraocular infection increased dramatically, resulting in the clinical picture of acute endophthalmitis. RESULTS: In contrast to most published cases, early identification of the causative pathogen in the aqueous humor after anterior chamber puncture using polymerase chain reaction (PCR) and the initiation of a specific, systemic antibiotic medication, resulted in-complete recovery of visual acuity. CONCLUSIONS: PCR is very useful for the identification of the pathogen in intraocular infections.

Acute Disease↗

PubMed2006

[Emergence of new pneumonia: besides severe acute respiratory syndrome].

Important epidemiological modifications have been registered in respiratory infections, both in immunocompetent and immunocompromised hosts. Pathogens with modified antibiotic susceptibility patterns have emerged, which display an increased antibiotic resistance, such as S. pneumoniae, S. aureus, H. influenzae. This trait has a strong impact on the therapeutic choices, particularly when an empiric antibiotic treatment is selected. The prevalence of bacterial species showing non-susceptibility to the most common prescribed antibiotics (betalactams, macrolides etc.) follows a different geographic distribution. Some pathogens have acquired a new epidemiological role in patients affected with immune deficiencies: among them P. carinii and other bacterial, fungal and viral pathogens. The emergence of new, previously unknown, species, has been registered, both bacteria (C. pneumoniae) and viruses (Metapneumovirus, Hantavirus etc.). Such aspects must be considered in the diagnosis of respiratory infections, which should include diagnostic tests for the identification of such pathogens. Among the new respiratory infections severe acute respiratory syndrome (SARS) has quickly become a health care emergency, so that efforts have been made to identify the aetiological agent as well as the main epidemiological and clinical characteristics of the disease. Avian influenza has raised great interest immediately after the first cases of human infection caused by the avian virus, especially after the outbreaks in Asian countries and in the Netherlands. A crucial step in containing infection is the prevention of the disease; efforts are directed toward this endpoint.

Animals↗

PubMed2006

[Isolation and identification of Bacillus anthracis in an accidental case].

During June to July 2005, a few farmers in Chengde county of Hebei province were got ill after eating beef of sick cattle. The cattle could be infected with Bacillus anthracis. One beef sample and one soil sample contaminated with cattle blood were collected and used for pathogen isolation and identification in laboratory. Two bacteria strains were isolated from beef and soil sample, respectively, and showed typical morphology of Bacillus anthracis on blood agar and under microscope with Gram stain. The two bacteria strains were also positive to standard positive serum of Bacillus anthracis by slide agglutination test. Biochemical characteristics of the two bacteria were tested using API CHB/E strip and analyzed by API software (version 3.3), result showed that the two isolated bacteria were Bacillus anthracis. Polymerase chain reaction (PCR) was used to further characterize the two isolated bacteria strains. Three pairs of primer were designed and used for PCR, and these primers exactly matched the protective antigen gene, edema factor gene and capsule gene, respectively. By analyzed on agarose gel, PCR products were 423bp, 494bp and 397bp, respectively, and this result showed that the two isolated bacteria contained two plasmids, pX01 and pX02, which encoded anthrax toxin and capsule, respectively. Anthrax toxin and capsule were very important virulent factors for Bacillus anthracis. PCR products were purified and then cloned to T vector, positive clone was chose and sequenced. By BLAST with GenBank, sequence of the three genes of the two bacteria strains had a similarity of 99% with Bacillus anthracis A2012 strain, Ames Ancestor strain and A16R strain. Based on results of colonial morphology, serum test and biochemistry characterization, the two bacteria strains are Bacillus anthracis. They can encode anthrax toxin and capsule, and are virulent to animal and human.

Animals↗

PubMed1967

Preliminary studies of some physiological properties of Torula jeanselmei.

The morphological characteristics of pathogenic and saprophytic strains of Torula jeanselmei (Fungi Imperfecti) are indistinguishable. Some basic physiological properties of T. jeanselmei were investigated in the hope of separating the human pathogenic strain from common saprophytes in a clinical laboratory. Nitrogen and carbon assimilation tests were not useful for distinguishing the pathogen, since all strains, pathogenic and saprophytic, were able to assimilate nitrogen from ammonium, nitrate, and nitrite sources, and all were able to utilize glucose, fructose, galactose, cellobiose, lactose, glycerol, and starch. It appeared that temperature range for growth might be useful in the identification of the pathogen. All pathogenic strains grew well at 37 C but poorly at 10 C; in contrast, all saprophytes could grow at 10 C but were unable to grow at 37 C (with two exceptions).

Carbohydrate Metabolism↗

PubMed2001

Comparative proteomics of bacterial pathogens.

The monitoring of gene expression via the technologies encompassed under the term 'proteomics' allows proteins of significance to be related to phenotypes associated with strain variability, environmental influences and the effects of genetic manipulation. The characterizations of these molecules are routinely performed utilising two-dimensional (2-D) gel electrophoresis in association with mass spectrometry for the identification of proteins. Pathogenic bacteria are suitable for proteomic comparisons in the aim of elucidating proteins with vaccine and diagnostic applications, as well as determining novel targets for drug design and the effects of these drugs on cellular physiology. Strains exhibiting diverse phenotypes including antibiotic or chemical resistances, altered mode of pathogenicity, or differential capability of growth in similar environments, can be compared via protein differential display to correlate relative protein abundances associated with these conditions. Technically, proteins are 'mapped' on 2-D arrays under 'standard' conditions and visually compared to arrays of proteins from a variety of test conditions. High-throughput technologies allow molecules of significance to be elucidated rapidly from within complex mixtures using a combination of cellular pre fractionation to determine cellular location and pathway predictions to aid in overcoming the limitations of 2-D gel technology for the analysis of whole proteomes.

Bacteria↗

PubMed2004

A method using active-site sequence conservation to find functional shifts in protein families: application to the enzymes of central metabolism, leading to the identification of an anomalous isocitrate dehydrogenase in pathogens.

We have introduced a method to identify functional shifts in protein families. Our method is based on the calculation of an active-site conservation ratio, which we call the "ASC ratio." For a structurally based alignment of a protein family, this ratio is the average sequence similarity of the active-site region compared to the full-length protein. The active-site region is defined as all the residues within a certain radius of the known functionally important groups. Using our method, we have analyzed enzymes of central metabolism from a large number of genomes (35). We found that for most of the enzymes, the active-site region is more highly conserved than the full-length sequence. However, for three tricarboxylic acid (TCA)-cycle enzymes, active-site sequences are considerably more diverged (than full-length ones). In particular, we were able to identify in six pathogens a novel isocitrate dehydrogenase that has very low sequence similarity around the active site. Detailed sequence-structure analysis indicates that while the active-site structure of isocitrate dehydrogenase is most likely similar between pathogens and nonpathogens, the unusual sequence divergence could result from an extra domain added at the N-terminus. This domain has a leucine-rich motif similar one in the Yersinia pestis cytotoxin and may therefore confer additional pathogenic functions.

Amino Acid Motifs↗

PubMedDate not supplied

Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons, LEE1, LEE2, LEE3, LEE4, and tir(LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator, ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE. L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenic L0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally, trans expression of L0044 suppressed the expression of the LEE. Furthermore, mRNA of ler increased as a result of deleting L0044, and disrupting ler in a L0044-deleted background reversed the loss of the repression phenotype. Thus, L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation of ler.

Bacterial Proteins↗