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PubMed1998

16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis.

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.

Adult↗
PubMed2003

Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae).

In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses. The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal. For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes. Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences. Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting. For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex. The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I. ricinus nymphs that had fed on B. microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest. The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest. Few pathogens were detected but the discovery of Borrelia valaisiana and B. burgdorferi s.s. in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

Animals↗
PubMed1986

Preliminary evaluation of a rapid colorimetric method for identification of pathogenic Neisseria.

A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on beta-galactosidase, gamma-glutamylaminopeptidase, and gamma-prolylaminopeptidase is described. All 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories.

Chromogenic Compounds↗
PubMed2003

Risk factors for pediatric ventriculoperitoneal shunt infection and predictors of infectious pathogens.

Identification of risk factors for shunt infection and predictors of infectious pathogens may improve current methods to prevent and treat shunt infections. We reviewed data on 820 consecutive ventriculoperitoneal (VP) shunt placement procedures in 442 pediatric patients at our institution during 1992-1998. Ninety-two shunts (11%) developed infection a median of 19 days (interquartile range, 11-35 days) after insertion. Premature birth (relative risk [RR], 4.81; 95% confidence interval [CI], 2.19-10.87), previous shunt infection (RR, 3.83; 95% CI, 2.40-6.13), and intraoperative use of the neuroendoscope (RR, 1.58; 95% CI, 1.01-2.50) were independent risk factors for shunt infection. The bacterial organisms early after shunt surgery (<14 days) were the same as those late after shunt surgery (>14 days). As determined by an analysis of the 92 infected shunts, hospital stay of >3 days at the time of shunt insertion (odds ratio [OR], 5.27; 95% CI, 1.15-25.3) and prior Staphylococcus aureus shunt infection (OR, 5.91; 95% CI, 1.35-25.9) independently increased the odds that S. aureus was the causal pathogen.

Adolescent↗
PubMed2005

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes.

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

Bacteria↗
PubMed1997

Identification of pathogenic yeasts of the imperfect genus Candida by polymerase chain reaction fingerprinting.

With the increase in the number of immunocompromised hosts, the number of fungal pathogens has increased markedly. Identification and classification, especially of yeast species and strains, is often difficult when based solely on phenotypic characteristics. Since it became clear that different fungal pathogens require specific treatment strategies, there is a need for simple, rapid and reliable methods to identify fungal isolates. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to identify yeast isolates. Microsatellite [(GTG)5; (GACA)4] and minisatellite [(5'GAGGGTGGCGGTTCT 3'), derived from the core-sequence of the phage M13] specific primers were used as single primers in the PCR to amplify hypervariable interrepeat DNA sequences from over 200 European, American and Australian clinical isolates within the genus Candida. Each species, represented by its type strain, could be identified by a specific multilocus pattern, allowing for the assignment of all the isolates to the appropriate species. Intra-species variation in the multilocus profiles was about 20% compared to inter-species variation, which was up to 80%. Anamorph-teleomorph pairs could be identified by highly homologous PCR fingerprint patterns. PCR fingerprinting was more discriminatory when compared with routinely used biochemical tests (Vitek YBC and API ID 32C). PCR fingerprinting has proven to be a powerful tool for the identification of medically important yeasts. It is rapid, sensitive, reliable, highly reproducible, stable in vitro and in vivo, and applicable to large scale experiments. Potential applications include: yeast taxonomy, epidemiology, environmental surveys, and improvement of the diagnosis of mycotic diseases.

Australia↗
PubMed2004

Update on identification of pathogenic crystals in joint fluid.

Crystal identification in joint fluid has been an essential part of diagnosis of joint disease. Recent advances have included progress in crystal identification on stained slides, attention to aspiration techniques, and arthrocenteses of asymptomatic joints. Challenges remain to increase use and optimize techniques.

Biopsy, Fine-Needle↗
PubMed2006

Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene.

BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.

Base Sequence↗
PubMed2005

Rapid detection and identification of pathogens in blood cultures by fluorescence in situ hybridization and flow cytometry.

Septicemia is one of the leading causes of death in hospitalized patients. The timely detection and identification of microorganisms from the patient's blood has great diagnostic, prognostic and economic significance. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been proven to be a fast method for the identification of human pathogenic bacteria and yeasts. Data presented herein reveal that the combination of FISH and flow cytometry (FC-FISH) is a rapid and reliable technique for identification of pathogens (Gram-negative rods, Candida spp.) directly from blood cultures without further cultivation and biotyping. Moreover, detection of growing pathogens (e.g., Stenotrophomonas maltophilia) in blood cultures is achieved more rapidly by FC-FISH compared to standard detection methods. Therefore, FC-FISH allows rapid detection and identification of pathogens in blood cultures.

Bacteria↗
PubMed1994

Identification of veterinary pathogens by use of commercial identification systems and new trends in antimicrobial susceptibility testing of veterinary pathogens.

Veterinary diagnostic microbiology is a unique specialty within microbiology. Although isolation and identification techniques are similar to those used for human pathogens, many veterinary pathogens require unique cultivation or identification procedures. Commercial identification systems provide rapid, accurate identification of human pathogens. However, the accuracy of these systems with veterinary pathogens varies widely depending on the bacterial species and the host animal from which it was isolated. Increased numbers of veterinary strains or species in the data bases of the various systems would improve their accuracy. Current procedures and interpretive criteria used for antimicrobial susceptibility testing of veterinary pathogens are based on guidelines used for human pathogens. The validity of these guidelines for use with veterinary pathogens has not been established. As with fastidious human pathogens, standardized methodologies and quality control isolates are needed for tests of organisms such as Actinobacillus pleuropneumoniae and Haemophilus somnus. Furthermore, interpretive criteria for veterinary antimicrobial agents based on the MIC for veterinary pathogens, the pharmacokinetics of the antimicrobial agent in the host animal, and in vivo efficacy of the antimicrobial agent are needed. This article reviews both the commercial identification systems evaluated with veterinary pathogens and current methods for performing and interpreting antimicrobial susceptibility tests with veterinary pathogens. Recommendations for future improvements in both areas are discussed.

Animals↗
PubMed2024

Universal Identification of Pathogenic Viruses by Liquid Chromatography Coupled with Tandem Mass Spectrometry Proteotyping.

Accurate and rapid identification of viruses is crucial for an effective medical diagnosis when dealing with infections. Conventional methods, including DNA amplification techniques or lateral-flow assays, are constrained to a specific set of targets to search for. In this study, we introduce a novel tandem mass spectrometry proteotyping-based method that offers a universal approach for the identification of pathogenic viruses and other components, eliminating the need for a priori knowledge of the sample composition. Our protocol relies on a time and cost-efficient peptide sample preparation, followed by an analysis with liquid chromatography coupled to high-resolution tandem mass spectrometry. As a proof of concept, we first assessed our method on publicly available shotgun proteomics datasets obtained from virus preparations and fecal samples of infected individuals. Successful virus identification was achieved with 53 public datasets, spanning 23 distinct viral species. Furthermore, we illustrated the method's capability to discriminate closely related viruses within the same sample, using alphaviruses as an example. The clinical applicability of our method was demonstrated by the accurate detection of the vaccinia virus in spiked saliva, a matrix of paramount clinical significance due to its non-invasive and easily obtainable nature. This innovative approach represents a significant advancement in pathogen detection and paves the way for enhanced diagnostic capabilities.

Tandem Mass Spectrometry↗
PubMed1985

Comparative evaluation of five commercial systems for the rapid identification of pathogenic Neisseria species.

Prompt diagnosis and effective treatment of urogenital gonococcal infections require rapid isolation and identification of Neisseria gonorrhoeae from urogenital specimens. We evaluated a new, rapid (30-min) test called Gonochek II (E-Y Laboratories, San Mateo, Calif.) which utilizes chromogenic substrates for the identification of pathogenic Neisseria species. It was compared with the API NeIdent (Analytab Products, Inc., Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and RapID NH (Innovative Diagnostics, Atlanta, Ga.), systems and the Phadebact GC (Pharmacia Diagnostics, Piscataway, N.J.) test for its performance in identifying known strains of N. gonorrhoeae (39 strains), Neisseria meningitidis (22 strains), Neisseria lactamica (12 strains), and Branhamella catarrhalis (17 strains). The Gonochek II system correctly identified 100% of N. gonorrhoeae, N. lactamica, and B. catarrhalis strains and 95.4% of N. meningitidis strains. The percent agreement for correct identification of all strains tested was 98.8%. In contrast, the Minitek, RapID NH, and API NeIdent systems correctly identified 86.6, 80.0, and 73.3% of the strains, respectively. The Phadebact GC test identified 94.9% of the N. gonorrhoeae isolates but also cross-reacted with 41.6% of the N. lactamica strains. The Gonochek II system is rapid, simple to perform, and easy to interpret, requires 1 to 2 min to set up, and more accurately identifies pathogenic Neisseria species when compared with other systems used in this study.

Cost-Benefit Analysis↗
PubMed2005

Rapid Identification of pathogenic rapidly growing mycobacteria by PCR-restriction endonuclease analysis.

INTRODUCTION: The accuracy and practicality of PCR-restriction endonuclease analysis (PRA) for rapid identification of pathogenic rapidly growing mycobacteria (RGM) isolates were evaluated. MATERIALS AND METHOD: PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by conventional methods, for 39 clinically significant RGM isolates. RESULTS: The accuracy of PRA in the identification of RGM isolates was comparable to that of conventional methods. Moreover, PRA was able to identify RGM faster, within 2 to 3 working days compared to conventional methods which require 2 to 4 weeks to perform and complete different tests. CONCLUSION: PRA methodology could be easily incorporated into the clinical laboratory setting. This would be beneficial for the management of patients with infections due to pathogenic RGM.

Bacterial Proteins↗
PubMed1994

Rapid identification of pathogenic neisserias using the Identicult-Neisseria test.

A rapid enzymatic method using chromogenic substrates for the rapid identification of pathogenic neisseria (Identicult-Neisseria, Scott Laboratories Inc., CA, USA) was tested in parallel with the rapid carbohydrate utilization test (RCUT) and the Phadebact Monoclonal GC Test against 198 consecutive clinical isolates of oxidase-positive Gram-negative diplococci (118 Neisseria gonorrhoeae, 76 N. meningitidis and four N. lactamica). On initial testing the Identicult-Neisseria gave a 95% overall concordance (97.5% N. gonorrhoeae, 90.8% N. meningitidis) with the RCUT and Phadebact tests; the corresponding figures after repeat testing were 98% overall concordance (98.3% N. gonorrhoeae, 97.4% N. meningitidis). Two of the three strains of N. gonorrhoeae mis-identified as N. meningitidis on primary testing were also mis-identified on repeat testing. Seven strains of N. meningitidis were mis-identified on initial testing (six as Moraxella catarrhalis and one as N. lactamica) and two on repeat testing (both as Mor. catarrhalis). We conclude that the Identicult-Neisseria is not sufficiently reliable for the culture confirmation of gonococci and meningococci.

Agglutination Tests↗
PubMedDate not supplied

Rapid detection and identification of pathogenic fungi by polymerase chain reaction amplification of large subunit ribosomal DNA.

We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens.

Base Sequence↗
PubMed1996

Appraisal of various random amplified polymorphic DNA-polymerase chain reaction primers for Leishmania identification.

Pathogenic Leishmania can cause a variety of symptoms ranging from asymptomatic or mild infections to severe mucocutaneous disease, partly according to the species of Leishmania involved. Genomic and kinetoplast-derived DNA probes as well as species complex-specific kinetoplast-derived polymerase chain reaction (PCR) primers have been successful for parasite identification in epidemiologic and taxonomic studies. However, the lack of a species DNA probe or PCR primer set, their relatively poor availability, and unknown comparative sensitivity and specificity have precluded their routine and widespread use as identification tools in many laboratories. This study addresses this problem for 28 different random amplified polymorphic DNA (RAPD)-PCR primers that have been assessed regarding their sensitivity, specificity, and reliability for distinguishing each of four closely related New World Leishmania species. The degree of relatedness between species was quantified and estimates were made of the accuracy and precision of the determinations. The results compared well with standard methods for Leishmania classification. The application of RAPD-PCR for screening isolates for possible interspecific hybrids is also demonstrated.

Animals↗
PubMed1992

[Detection and identification of pathogenic bacteria by polymerase chain reaction with primers from DNA sequence of ribosomal RNA].

Applicability of the polymerase chain reaction method for identification of pathogenic bacteria was examined with the primers synthesized from the ribosomal RNA gene sequence containing both homologous and species-specific regions of bacterial species from Mycoplasma to Mycobacteria. Two out of the nine sets of promoters prepared, each covering about 650 nucleotides spanning from 16S RNA to 23S RNA regions, produced the corresponding DNA fragments from all the strains tested, and another set did so from all species but Mycoplasma. This method enabled one to detect and identify E. coli in a sample containing 2 x 10(2) CFU. The restriction enzyme patterns of the PCR products obtained with Hae-III, Hha-I, Mbo-I, Msp-I, Rsa-I and Taq-I were so characteristic as to differentiate one species from another. Ten strains of E. coli showed identical restriction patterns and 10 of S. aureus also showed identical patterns indicating that the restriction pattern is species-specific. The method may be applicable to detection and identification of a certain species bacteria which are suspected to be consealed in water or food samples, or clinical specimens, especially when the consealed bacterial genus or species can not be predicted.

Base Sequence↗