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PubMed2006

Acute musculoskeletal infection: comparison of different methods for intraoperative bacterial identification.

PURPOSE OF THE STUDY: Various techniques are used for detection of pathogens in musculoskeletal infection. These methods differ with respect to reliability and ease of handling. A prospective study was performed to evaluate the efficacy of three intraoperative techniques. MATERIAL AND METHODS: In 20 cases (18 patients) with clinically confirmed acute musculoskeletal infections, intraoperative collected swab samples, tissue samples and fluid samples injected into standard blood culture vials were used for microbiological diagnosis. Identification of bacteria, time necessary for detection and ease of handling during surgery was evaluated. RESULTS: In 19 cases bacterial growth was demonstrated using either intraoperative swabs or blood culture technique (95% sensitivity), whereas 18 tissue biopsies were positive (90% sensitivity. 27 bacterial species were isolated. In 18 instances for the swab technique, 14 instances for the tissue biopsy and 4 operations for the blood culture vials, ease of handling was rated as excellent. DISCUSSION: The study demonstrated differences between the three tested methods with respect to ease of handling. With respect to the number of detected organisms and time for their detection there are no significant differences. These last findings are in contrast to of the results of other authors. The reason for this could be that during operative dissection an accurate and specific collection of specimens from the acute deep infected soft tissues and bones independent from the type of surgical procedure is possible. Therefore, even with the swab method a high amount of microorganisms can be recovered. Especially for intraarticular infections, fluid samples injected into standard blood vials is a practical method for the surgeon. In acute musculoskeletal infections other than joint infections, there is less benefit for the blood culture vials. CONCLUSION: Intraoperative swab technique yields valid results comparable to other techniques and is an accurate technique for detection of pathogens from acute musculoskeletal infections. Key words: implant, infections, bacteriological techniques, comparative study.

Acute Disease↗

PubMed2003

A microfluidic biosensor based on nucleic acid sequence recognition.

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome-target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol microL(-1) (10 pmol L(-1)). Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.

Base Sequence↗

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↗

PubMed2006

Mid-IR biosensor: detection and fingerprinting of pathogens on gold island functionalized chalcogenide films.

Antibody (human IgG, anti-E. coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR fingerprinting. Highly selective detection of bacterial targets was achieved at both the species (E. coli vs. S. enteriditis) and strain level (E. coli O157:H7 vs E. coli K12). A mid-infrared approach could thus be used as a biosensor as well as a molecular fingerprinting tool.

Antibodies, Bacterial↗

PubMed2005

Detection and removal of pathogenic biofilms on medical implant surfaces.

Advances in sensor technology have had a significant impact in medical research and practice in the last decade. However, within the hospital environment problems still exist where the application of sensing technology could provide the solution. The presence of antibiotic resistant bacteria within hospitals and the risk of serious infection that they pose is a cause for concern. This paper describes a research project that has recently started at the University of Ulster investigating the potential of "Sense and Destroy" tactics to reduce the spread of medical device related infections. It is proposed that Electrical Impedance Spectroscopy (EIS) probes implanted within a catheter may be used to detect subclinical biofilm formation. Furthermore, if the presence of a biofilm is detected, activation of a photocatalytic coating on the catheter wall may be used to inactivate the responsible microorganisms.

Biofilms↗

PubMed1988

Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods.

The combination of radiometric methodology (BACTEC 12B) and probe technology for recovery and identification of mycobacteria was studied in two large hospital laboratories. The sediment from vials with positive growth indices was tested with DNA probes specific for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The sensitivity of the radiometric method and the specificity of the probes resulted in a marked reduction in the time to the final report. Biochemical testing could be eliminated on isolates giving a positive reaction with one of the probes. Some 176 isolates of M. tuberculosis, 110 of M. avium, and 5 of M. intracellulare were recovered. Two-thirds of these isolates were detected and identified within 2 weeks of inoculation and the remainder was detected by 4 weeks, a reduction of 5 to 7 weeks to the final report.

DNA, Bacterial↗

PubMed1999

Methodology for detection and typing of foodborne microorganisms.

Over the past decade many improvements have been seen in both conventional and modern methods for the detection of pathogenic bacteria in foods. Modifications and automation of conventional methods in food microbiology include sample preparation, plating techniques, counting and identification test kits. ATP bioluminescence techniques are increasingly used for measuring the efficiency of cleaning surfaces and utensils. Cell counting methods, including flow cytometry and the direct epifluorescent filter technique are suitable techniques for rapid detection of microorganisms, especially in fluids. Automated systems based on impedimetry are able to screen high numbers of samples based on total bacterial counts within 1 day. Immunoassays in a wide range of formats make rapid detection of many pathogens possible. Recently, there have been important developments in the use of nucleic acid-based assays for the detection and subtyping of foodborne pathogens. The sensitivity of these methods has been significantly increased by the use of the polymerase chain reaction and other amplification techniques. Alternative and rapid methods must meet several requirements concerning accuracy, validation, speed, automation, sample matrix, etc. Both conventional and rapid methods are used within hazard analysis critical control point programs. Further improvements especially in immunoassays and genetic methods can be expected, including the use of biosensors and DNA chip technology.

Bacterial Typing Techniques↗

PubMed1999

Development of a rapid response biosensor for detection of Salmonella typhimurium.

An integrated optic interferometer for detecting foodborne pathogens was developed. The interferometer is a planar waveguide with two thin antibody-coated channels of immunochemically selective agents that interact with antigen molecules. One channel is coated with antibody to Salmonella as a sample, and the other is coated with human immunoglobulin G as a reference channel by using reductive amination. Salmonella was introduced onto the sensing channels through the flow cell on the channels. Phase shift (pi) generated by refractive index variation, as determined by interfering the perturbed sample channel with an unperturbed reference channel and observing the fringe shift, was used for detection. Salmonella Typhimurium (heat-treated or boiled) was detected by binding to antibody against Salmonella common structural antigen immobilized on a silane-derived sensor surface at concentrations in the range of 1x10(5) to 1x10(7) CFU/ml. Salmonella (1x10(7) CFU/ml) mixed with Escherichia coli (1x10(7) CFU/ml) were readily detected without any decrease in sensitivity by the direct assay. Application of a sandwich assay with a second antibody or a gold-conjugated antibody increased the detection limit to 1x10(5) CFU/ml within a 10-min reaction time. Various methods for the immobilization of the capture antibody to the biosensor channels were compared. The greatest binding response was observed in a direct reductive amination method with a long reaction period and increased the detection limit of direct binding of Salmonella antigen to 1x10(4) CFU/ml. The biosensor was able to detect Salmonella Typhimurium in chicken carcass wash fluid originally inoculated at a level of 20 CFU/ml after 12 h of nonselective enrichment. The planar optic biosensor shows promise as a fast, sensitive, reliable, and economical means of detecting food pathogens in the future.

Animals↗

PubMed2005

Frequency of detection of picornaviruses and seven other respiratory pathogens in infants.

BACKGROUND: Previous studies in which molecular-based techniques have been used to identify the causative pathogens of respiratory tract infection have investigated hospitalized children only. We report a prospective study designed to determine the frequency and clinical presentation of community-acquired respiratory illness in infancy associated with 8 common respiratory pathogens. METHODS: Eighty-eight infants were monitored through their first winter. With each respiratory illness, infants were examined, and a nasal lavage specimen was collected. Individual reverse transcription-polymerase chain reactions were performed to detect infection with picornaviruses (rhinoviruses and enteroviruses), coronaviruses (serotypes OC43 and 229E), adenoviruses, parainfluenza viruses 1-3, influenza viruses (types A and B), respiratory syncytial virus (RSV), Chlamydia pneumoniae and Mycoplasma pneumoniae. RESULTS: Picornaviruses were the most frequently detected pathogen identified in 46% (56 of 123) of episodes, followed by RSV (27%), parainfluenza viruses (13%) and coronaviruses (9%). Dual pathogen infections were identified in 20% of episodes, predominantly caused by picornaviruses together with either RSV or parainfluenza viruses. RSV infection was significantly associated with a diagnosis of bronchiolitis. No other associations were found between pathogen and clinical diagnosis. Dual infection did not predispose infants to a more severe clinical course. CONCLUSIONS: Picornaviruses are the predominant cause of community-acquired respiratory tract infection in the first year of life. Large prospective community-based studies will be needed to fully evaluate the contribution of picornaviruses, both in isolation and in combination with other respiratory pathogens, to the various clinical syndromes of respiratory infection observed during infancy.

Age Distribution↗

PubMed1997

Detection and identification of fungal pathogens in blood by using molecular probes.

A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.

Amphotericin B↗

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↗

PubMed2007

Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction.

BACKGROUND: Saliva has been studied for the presence of subgingival pathogens in periodontitis patients. With the anaerobic culture technique, the discrepancy between salivary recovery and subgingival presence has been significant, which makes this approach not suitable for practical use in the microbial diagnosis of periodontitis patients. The real-time polymerase chain reaction (PCR) technique represents a very sensitive technique to detect and quantify bacterial pathogens. The aim of the study was to compare the presence and numbers of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, and Micromonas micros in subgingival plaque and mouthwash samples by the anaerobic culture and real-time PCR techniques. METHODS: Pooled subgingival plaque samples and 10-ml mouthwash samples were collected from 21 adult patients with periodontitis and analyzed by quantitative anaerobic culture and real-time PCR for A. actinomycetemcomitans, P. gingivalis, T. forsythensis, P. intermedia, and M. micros. RESULTS: The detection frequency of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis in subgingival plaque was identical by culture and real-time PCR and was higher for P. intermedia and M. micros by real-time PCR. The highest detection frequencies for the target bacteria were found in mouthwash samples by real-time PCR. The additional value of the real-time PCR to detect target bacteria was 38% for P. gingivalis, 73% for T. forsythensis, 77% for P. intermedia, and 71% for M. micros. The sensitivity to detect target species in mouthwash by real-time PCR was 100% for all test species except for P. intermedia (93.8%). CONCLUSIONS: Rapid detection and quantification of periodontal pathogens in mouthwash samples are possible by real-time PCR. The procedure is significantly less time-consuming than subgingival sampling with paper points. This approach to detect major periodontal pathogens in mouthwash samples may simplify microbial diagnosis in periodontitis patients and may be used to monitor periodontal treatment.

Adult↗

PubMed2026

Visual Detection and Stratification of Pathogenic mtDNA SNV Heteroplasmy by Balancing FnCas12a Signal Output and Allelic Discrimination.

Assessment of pathogenic mitochondrial DNA (mtDNA) single-nucleotide variant (SNV) heteroplasmy is important for molecular diagnostics, yet rapid visual profiling remains analytically challenging because an assay must combine single-nucleotide allelic discrimination, mutant-fraction-associated readout, and suitable target access. Herein, we report VISTA (visual identification and stratification of targeted mtDNA alleles), a broad-PAM FnCas12a assay that rebalances trans-cleavage signal output and mutant-wild-type discrimination for visual mtDNA SNV heteroplasmy analysis. VISTA uses unmodified FnCas12a with relaxed TTN PAM recognition and integrates crRNA spacer-length engineering with PEG8000/acBSA reaction tuning to improve the practical signal-discrimination balance without nuclease engineering. At the m.3243A>G model locus, spacer truncation enhanced mutant-wild-type discrimination, while molecular-dynamics simulations identified spacer-dependent differences between matched and mismatched complexes at the crRNA-DNA interface. The optimized assay resolved defined synthetic m.3243A>G heteroplasmy gradients by fluorescence imaging and was further adapted to lateral-flow detection. In locus-specific analyses of a deidentified collection of 74 peripheral-blood samples, fluorescence and lateral-flow readouts achieved ROC AUC values above 0.9 for mutant-allele classification after target-region amplification. Fluorescence supported heteroplasmy-associated profiling, whereas lateral flow provided a visual, semiquantitative readout for relative ranking based on the T/C ratio rather than absolute heteroplasmy measurement. VISTA therefore provides an accessible dual-readout analytical strategy for visual detection and heteroplasmy-associated profiling by tuning the FnCas12a signal output and allelic discrimination.

DNA, Mitochondrial↗

PubMed2000

[Diagnosis of Lyme borreliosis. How to corroborate suspected borreliosis].

Lyme borreliosis is a zoonosis transmitted via lxodes ticks. The causal agent--the spirochete Borrelia burgdorferi sensu lato--triggers a multisystem disease that manifests in particular in the skin, heart, nervous system and joints. Lyme borreliosis is the most common tick-borne infectious disease in the Northern hemisphere. In Europe the Lyme disease spirochetes are heterogeneous and comprise three species that infect humans: Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii. The microbiological diagnosis is based primarily on the detection of antibodies, and secondarily on the detection of the pathogen. Suitable material for the detection of the pathogen are various body fluids (cerebrospinal fluid, joint fluid) and biopsy material (in particular skin). Antibodies are usually detected in the serum. Negative serology does not exclude an early manifestation, and a positive finding is no proof of a clinically manifest infection--it may simply reflect an earlier Lyme infection.

Animals↗

PubMed2002

Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens.

Advances in molecular technology show great potential for the rapid detection and identification of fungi for medical, scientific and commercial purposes. Numerous targets within the fungal genome have been evaluated, with much of the current work using sequence areas within the ribosomal DNA (rDNA) gene complex. This section of the genome includes the 18S, 5.8S and 28S genes which code for ribosomal RNA (rRNA) and which have a relatively conserved nucleotide sequence among fungi. It also includes the variable DNA sequence areas of the intervening internal transcribed spacer (ITS) regions called ITS1 and ITS2. Although not translated into proteins, the ITS coding regions have a critical role in the development of functional rRNA, with sequence variations among species showing promise as signature regions for molecular assays. This review of the current literature was conducted to evaluate clinical approaches for using the fungal ITS regions as molecular targets. Multiple applications using the fungal ITS sequences are summarized here including those for culture identification, phylogenetic research, direct detection from clinical specimens or the environment, and molecular typing for epidemiological investigations. The breadth of applications shows that ITS regions have great potential as targets in molecular-based assays for the characterization and identification of fungi. Development of rapid and accurate amplification-based ITS assays to diagnose invasive fungal infections could potentially impact care and improve outcome for affected patients.

DNA, Ribosomal Spacer↗

PubMed2000

Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture.

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in </=4 h after enrichment.

Animals↗

PubMed1990

[The laboratory diagnosis of Borrelia burgdorferi infection].

The multiplicity of the clinical appearance of Lyme disease makes it necessary to confirm the diagnosis by detecting the pathogen or specific antibodies. Isolation of the pathogen from infected tissue or body fluids is difficult, so that, to date, only serology is feasible for routine diagnosis. In view of a lack of standardisation of borrelia serology, the clinician must expect false negative, and--even more so--false-positive, results. Future laboratory diagnosis should be markedly improved by nucleic acid hybridisation for the detection of the pathogen, and the use of specific immunodominant antigens for antibody detection.

Antibodies, Bacterial↗

PubMed1990

Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid.

The polymerase chain reaction was used to detect Treponema pallidum in specimens of cerebrospinal fluid (CSF), as a means of diagnosing syphilis. Segments of the TmpA and 4D genes were amplified to provide an estimated threshold sensitivity of approximately 65 organisms in 0.5 ml. A spectrum of pathogens known to cause meningitis, and several non-pathogenic treponemes were unreactive. Treponema pertenue, and only one of 30 control specimens of CSF were positive. In contrast, 10 of 19 CSFs from patients being evaluated for latent or tertiary syphilis were positive, as were 7 of 28 specimens from HIV-positive patients.

Animals↗