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Results for “Pathogen detection”

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At least 721 records · Page 40Linked to original sources

PubMed1993

Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction.

Amplification of DNA sequences from ribosomal DNA (rDNA) was tested as a specific and sensitive method for the detection of small numbers of Toxoplasma gondii tachyzoite cells. We applied the polymerase chain reaction (PCR) on the basis of detection of the 110-fold repetitive rDNA as a target by using (i) DNA sequences within the small ribosomal subunit known to be universal and conserved in all eukaryotes and (ii) small ribosomal subunit and intergenic spacer rDNA sequences known to be T. gondii species specific. The level of sensitivity obtained from a crude cell lysate allowed the detection of as few as one parasite visualized directly as a specific PCR product in agarose gels. By using a combination of universal and T. gondii species-specific primers, we propose a comultiplex-based PCR approach as a new diagnostic tool. The combination of sensitivity, specificity, and built-in positive and negative PCR controls should make detection of the rDNA sequences by comultiplex PCR a useful clinical test for the diagnosis of toxoplasmosis and for epidemiological studies. Finally, the idea of a built-in positive control to support or counter the T. gondii-specific PCR result is novel and is a notable advance.

Animals↗

PubMed2000

Frequency of low-level bacteremia in children from birth to fifteen years of age.

A single blood culture inoculated with a small volume of blood is still frequently being used for the diagnosis of bacteremia in children because of the continued belief by many that bacteria are usually found in high concentrations in the blood of pediatric patients with sepsis. To determine the importance of both blood volume cultured and the number of culture devices required for the reliable detection of pathogens in our pediatric population, blood from children from birth to 15 years of age and with suspected bacteremia at York Hospital (a 500-bed community hospital) was inoculated into at least a Pediatric Isolator (Wampole Laboratories; 1.5 ml of blood) or a standard Isolator (10 ml of blood) and a bottle of ESP anaerobic broth (Trek Diagnostic Systems; 0.5 to 10 ml of blood). The use of a second Isolator and additional aerobic and anaerobic bottles and the total blood volume recommended for cultures (2 to 60 ml) depended on the weight and total blood volume of each patient. One hundred forty-seven pathogens were recovered from the blood of 137 (3.6%) of 3,829 children for whom culturing was done. Of 121 septic episodes for which the concentration of pathogens in the blood could be determined using Isolators, 73 (60. 3%) represented low-level bacteremia (</=10 CFU/ml of blood), including 28 pathogens (23.1%) which were detected at concentrations of only </=1.0 CFU/ml. Of 144 septic episodes for which two or more culture devices (Isolators and/or bottles) were inoculated, 85 (59%) were associated with false-negative results from one or more of the culture devices. Of the 128 children for whom antibiotic therapy records were complete, therapy was either started or changed for 88 (68.8%) following notification of positive blood cultures. Low-level bacteremia was common in our pediatric population, requiring the culturing of up to 4 or 4.5% of a patient's total blood volume for the reliable detection of pathogens and appropriate, timely changes in empiric therapy.

Adolescent↗

PubMed1996

Detection of pathogen-related oral spirochetes, Treponema denticola, and Treponema socranskii in dental plaque from dogs.

Spirochetes have been observed in dental plaque from dogs, but specific spirochetes have not been identified. In particular, it is not known whether treponemes associated with periodontal diseases in humans also occur in dogs, and whether, like in humans, detection of specific treponemes correlates with periodontal status of dogs. Forty-two dogs were grouped according to the worst periodontal condition in the mouth, as determined by overt signs of inflammation and pocket probing depths. A representative specimen of dental plaque was obtained by pooling subgingival plaque collected from three uniform reference sites, irrespective of periodontal status at selected sites. The presence of pathogen-related oral spirochetes. Treponema denticola, and T. socranskii was determined using specific monoclonal antibodies in an immunocytochemical microscopic assay. All three treponemes were detected in all groups, but a significantly greater proportion of dogs with pocket probing depths > or = 5 mm had detectable treponemes, compared to dogs that were in periodontal health.

Animals↗

PubMed1989

The use of dirty bedding for detection of murine pathogens in sentinel mice.

Sentinel Swiss (CD-1) mice, housed without filter bonnets, were seronegative for mouse hepatitis virus (MHV) for 8 consecutive months in an experimental colony of CD-1 mice. MHV titers had been detected sporadically in sentinel mice housed in this colony during a 2 year period. In an effort to determine whether MHV was still present in the colony, two methods of exposing sentinel mice to an animal room environment were compared under routine husbandry practices. Eight cages (12 mice per cage; 2 cages per rack) of experimental virus antibody free sentinel mice, housed without filter bonnets, were placed on the bottom shelf of 4 of 12 racks in the room. Twice each week, four cages of sentinel mice received a composite sample of dirty bedding (bedding used previously by mice in the room). The remaining four cages of experimental sentinels received fresh non-used bedding. Sentinel mice were bled at monthly intervals for MHV serology. After 4 months, mice from two cages which received dirty bedding seroconverted to MHV and mice from one cage were positive for Myobia musculi (mites). Three weeks later, all four cages of mice which received dirty bedding were positive for MHV and three were positive for mites. In contrast, only two of four cages of mice which received fresh bedding were positive for MHV and all were negative for mites. These findings indicate the importance of exposing sentinel mice to dirty bedding and that MHV and mites may go undetected for several months in a mouse colony when the incidence levels are low where standard sanitation procedures are used.

Animals↗

PubMed2005

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction.

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.

Animals↗

PubMed2006

Signature patterns revealed by microarray analyses of mice infected with influenza virus A and Streptococcus pneumoniae.

We used cDNA microarrays to identify differentially expressed genes in mice in response to infections with influenza virus A/PR/8/34 (H1N1) and Streptococcus pneumoniae. Expression microarray analysis showed up-regulation and down-regulation of many genes involved in the defense, inflammatory response and intracellular signaling pathways including chemokine, apoptosis, MAPK, Notch, Jak-STAT, T-cell receptor and complement and coagulation cascades. We have revealed signature patterns of gene expression in mice infected with two different classes of pathogens: influenza virus A and S. pneumoniae. Quantitative real-time RT-PCR results confirmed microarray results for most of the genes tested. These studies document clear differences in gene expression profiles between mice infected with influenza virus A and S. pneumoniae. Identification of genes that are differentially expressed after respiratory infections can provide insights into the mechanisms by which the host interacts with different pathogens, useful information about stage of diseases and selection of suitable targets for early diagnosis and treatments. The advantage of this novel approach is that the detection of pathogens is based on the differences in host gene expression profiles in response to different pathogens instead of detecting pathogens directly.

Animals↗

PubMed2002

PCR detection of specific pathogens in water: a risk-based analysis.

The relative concentration of pathogens in water samples collected from storm drains and adjacent surfaces was evaluated using established PCR-based protocols. Out of the 58 samples collected from 21 different storm drains, 22% were PCR positive for Escherichia coli ETEC, Salmonella, or adenovirus. The risk of swimming related illnesses associated with detection of E. coli ETEC and Salmonella ranged from 0.39 to 30:100 000 and 0.3-25:1000, respectively. The detection limit corresponding to a negative-PCR result was evaluated in reference to water quality standards developed using a risk-based approach that integrates human dose-response data with acceptable levels of risk promulgated by the U.S. EPA for recreational contact. The percent of samples with an acceptable detection limit ranged from 0% for Giardia lamblia and Shigella to 100% for E. coli ETEC. The principal factor influencing the detection limit of G. lamblia and Shigella was sample volume. The principal factor influencing the detection limit of the remaining bacteria and protozoa, including E. coli O157:H7, Salmonella, and Cryptosporidium parvum, was the presence of inhibitory compounds in the purified nucleic acid extracts. Both recovery and inhibition adversely impacted the detection limit of viruses. Ambient water quality standards based on the occurrence of specific pathogens enumerated with PCR-based assays could serve as a method of evaluating the biological quality of water but only after significant improvements in filtration and purification protocols. The risk-based methodology developed in this study can be used to evaluate future improvements in filtration and purification protocols.

Animals↗

PubMed2005

Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays.

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.

Animals↗

PubMed2004

Detection of infectious pathogenic viruses in water and wastewater samples from urbanised areas.

Regardless of the remarkable improvement in sanitary conditions in urbanised areas, infectious diseases caused by pathogenic viruses in water have been reported year after year. The actual situation for the behaviour of pathogenic viruses in urbanised society should be understood. In this study, infectious enteroviruses and adenoviruses in water and wastewater samples from urbanised areas were investigated. Infectious enteroviruses were continuously detected from sewage and sewage sludge during the summer season. The detection of infectious viruses in sewage and sewage sludge was facilitated by the enzymatic virus elution (EVE) method. The concentration of infectious enteroviruses in positive samples of sewage and sewage sludge was >400 virions/L. Infectious viruses were also detected from water samples at levels >4 virions/L.

Cities↗

PubMed2002

Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA.

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.

Animals↗

PubMed1999

Multiplex detection of four pathogenic retroviruses using molecular beacons.

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Base Sequence↗

PubMed2004

Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay.

At least 18 human genetic diseases are caused by expansion of short tandem repeats. Here we describe a successful application of a fluorescent PCR method for the detection of expanded repeats in FRDA1, SCA10, and SCA12 genes. Although this test cannot give a precise estimate of the size of the expansion, it is robust, reliable, and inexpensive, and can be used to screen large series of patients. It proved useful for confirming the presence of large expansions in the Friedreich ataxia gene following an ambiguous result of long-range PCR, as well as rapid pre-screening for large repeat expansions associated with Friedreich ataxia and SCA10 and the shorter repeat expansions associated with SCA12.

Case-Control Studies↗

PubMed2002

Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid Anthrax Test.

Rapid-cycle real-time polymerase chain reaction has immediate and important implications for diagnostic testing in the clinical microbiology laboratory. In our experience this novel testing method has outstanding performance characteristics. The sensitivities for detecting microorganisms frequently exceed standard culture-based assays, and the time required to complete the assays is considerably shorter than that required for culture-based assays. We describe the principle of real-time polymerase chain reaction and present clinical applications, including the detection of Bacillus anthracis, the causative agent of anthrax. This latter test is commercially available as the result of a collaborative venture between Mayo Clinic and Roche Applied Science, hence the designation The Mayo-Roche Rapid Anthrax Test.

Anthrax↗

PubMed2005

Low-level detection of viral pathogens by a surface-enhanced Raman scattering based immunoassay.

The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.

Buffers↗

PubMed2004

Tick-borne encephalitis virus-specific RT-PCR--a rapid test for detection of the pathogen without viral RNA purification.

Among diseases transmitted by ticks in the Czech Republic, tick-borne encephalitis (TBE) caused by Tick-borne encephalitis virus (TBEV) and Lyme disease caused by Borrelia burgdorferi spirochete are most important. We propose an effective and specific test for detection of TBEV in a single tick or a pool of ticks based on the detection of TBEV RNA using an RT-PCR technique without RNA purification. The method is very sensitive with the detection limit of about 14 fg TBEV RNA in total RNA obtained from brain suspension from suckling mice infected with TBEV per reaction. The primers were derived from the 5'-terminal non-coding region, a highly conserved part of the virus. The method was successfully applied to field-collected ticks in detecting TBEV RNA. This method can be used in studies of several aspects of TBEV: epidemiology, screening of natural foci, circulation and detection of virus genome sequences in clinical materials.

5' Untranslated Regions↗

PubMed2004

Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes.

Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.

Aggregatibacter actinomycetemcomitans↗

PubMed1991

DNA probe detection of periodontal pathogens in HIV-associated periodontal lesions.

Recent studies have shown that an atypical gingivitis and a rapidly progressive periodontal disease may be early-occurring opportunistic infections associated with human immunodeficiency virus (HIV) infection. This study examined the prevalence of selected periodontal pathogens associated with these HIV-related periodontal lesions. Subgingival plaque samples were obtained from both HIV-seronegative and HIV-seropositive homosexual men and from presumably uninfected heterosexual men. DNA probes were used to detect Actinobacillus actinomycetemcomitans, Bacteroides intermedius, Bacteroides gingivalis, Eikenella corrodens and Wolinella recta in the plaque. The healthy sites in both the seronegative and seropositive homosexual groups showed a greater prevalence of all test bacteria, except for E. corrodens, than did the heterosexual group. HIV-associated periodontitis sites showed a microbial profile qualitatively similar to that of conventional periodontitis, except that B. gingivalis was more prevalent in conventional periodontitis. In contrast, HIV-associated gingivitis sites exhibited a greater prevalence of all bacteria tested than conventional gingivitis sites. In fact, HIV gingivitis generally showed a bacterial profile similar to that of the HIV periodontitis lesions, except that W. recta was significantly more prevalent in HIV periodontitis. These data suggest that the HIV gingivitis lesion is a precursor to HIV periodontitis. Thus, early identification and prophylactic treatment of high-risk individuals may prevent the destruction of periodontal tissues.

Adult↗

PubMedDate not supplied

Genetic methods for the detection of microbial pathogens. Identification of enterotoxigenic Escherichia coli by DNA colony hybridization: collaborative study.

Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures of E. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-1 adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.

Autoradiography↗