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Results for “Pathogen detection”

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PubMed2005

Impact of antibiotics on pathogens associated with otitis media with effusion.

OBJECTIVE: To further understand the roles of bacteria and antibiotics in the development of otitis media with effusion (OME). METHODS: Samples of middle-ear effusion (MEE) were collected during the placement of ventilation tubes to treat chronic OME. Children with acute otitis media within the past three months were excluded from this study. We used polymerase chain reaction (PCR) to detect pathogens and to test the susceptibility of Streptococcus pneumoniae to penicillin. RESULTS: Among MEE samples from 52 children, PCR detected bacterial DNA in 32 per cent (24/75) of them. S. pneumoniae was detected more frequently in middle ears that required ventilation tube insertion at least twice compared with those requiring ventilation tube insertion only once (5/15 versus 4/60; p = 0.013). Higher levels of S. pneumoniae were detected in MEE from children with, than without, a long history of antibiotic administration (7/10 versus 2/14; p = 0.0187). The pbp genes of all isolated S. pneumoniae contained mutations. CONCLUSIONS: Long exposure to antibiotics might significantly influence the bacterial genome in MEE.

Anti-Bacterial Agents↗

PubMed2001

Utility of paired BACTEC MYCO/F LYTIC blood culture vials for detection of bacteremia, mycobacteremia, and fungemia.

In previous bloodstream infection studies in Malawi, we inoculated blood from a single venesection into a single BACTEC MYCO/F LYTIC (MFL) vial. Inoculation of one vial, however, would be expected to reduce the sensitivity of bloodstream pathogen detection with MFL vials. To ascertain the degree of this loss of sensitivity, blood was drawn from each of 228 febrile, adult inpatients in Malawi and 5 ml of each blood sample was inoculated into each of two MFL vials. Of 228 paired vials, 51 (22%) were both positive, 172 (75%) were both negative, and 5 (3%) had discordant results. Bloodstream infection would have been detected in 11 (92%) of 12 patients with mycobacteremia and 38 (92%) of 41 patients with bacteremia had only one MFL vial been inoculated. Our study shows that a second MFL vial does not significantly increase diagnostic sensitivity.

Bacteremia↗

PubMed2024

Beyond water and soil: Air emerges as a major reservoir of human pathogens.

Assessing the risk of human pathogens in the environment is crucial for controlling the spread of diseases and safeguarding human health. However, conducting a thorough assessment of low-abundance pathogens in highly complex environmental microbial communities remains challenging. This study compiled a comprehensive catalog of 247 human-pathogenic bacterial taxa from global biosafety agencies and identified more than 78 million genome-specific markers (GSMs) from their 17,470 sequenced genomes. Subsequently, we analyzed these pathogens' types, abundance, and diversity within 474 shotgun metagenomic sequences obtained from diverse environmental sources. The results revealed that among the four habitats studied (air, water, soil, and sediment), the detection rate, diversity, and abundance of detectable pathogens in the air all exceeded those in the other three habitats. Air, sediment, and water environments exhibited identical dominant taxa, indicating that these human pathogens may have unique environmental vectors for their transmission or survival. Furthermore, we observed the impact of human activities on the environmental risk posed by these pathogens, where greater amounts of human activities significantly increased the abundance of human pathogenic bacteria, especially in water and air. These findings have remarkable implications for the environmental risk assessment of human pathogens, providing valuable insights into their presence and distribution across different habitats.

Humans↗

PubMed2006

Detection of viruses: atomic force microscopy and surface enhanced Raman spectroscopy.

This paper demonstrates the capability of atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS) to function effectively as ultra-sensitive readout tools for chip-scale platforms designed for pathogen detection in complex biological media. AFM allows direct (i.e., label-free) visualization and quantification of nanometer-sized viruses captured on a smooth, selective surface. AFM readout led to optimization of a capture substrate for feline calicivirus (FCV), and yielded a limit of detection of 3 x 10(6) FCV/mL. SERS-based detection of FCV, carried out in a sandwich-type assay, requires labelling of the substrate-bound FCV with a selective extrinsic Raman label (ERL). These studies yielded a limit of detection of 1 x 10(6) FCV/mL. The prospects of these two readout methods as additions to the arsenal of tools in bioterrorism prevention are briefly discussed.

Calicivirus, Feline↗

PubMed2006

The additional value of real-time PCR in the quantitative detection of periodontal pathogens.

BACKGROUND AND AIM: For the analysis of subgingival plaque, anaerobic bacterial culture has been the gold standard for many years. Currently, molecular microbial techniques have become available to identify and quantify target organisms with high specificity and sensitivity. The technique of real-time polymerase chain reaction(RT-PCR) provides a new tool to detect oral pathogens both in oral and non-oral human infections. The aim of this study was to compare the RT-PCR and anaerobic culture for detection and quantification of six periodontal pathogens in periodontal health and disease. MATERIAL AND METHODS: Subgingival plaque samples from 259 adult patients with periodontitis and 111 healthy controls were analysed with quantitative anaerobic culture and quantitative RT-PCR for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Micromonas micros and Fusobacterium spp. RESULTS: All species were more frequently isolated from patients than controls with both culture and RT-PCR. P. gingivalis, T. forsythia and M. micros appeared significant markers for disease with both techniques. P. intermedia was significantly associated with periodontitis by RT-PCR only (OR 9.7), whereas A. actinomycetemcomitans showed a significant relationship by culture only. The critical differences between culture and RT-PCR were culture-negative/PCR-positive samples which amounted to 7% for A. actinomycetemcomitans, 3% for P. gingivalis, 7% for T. forsythia, 20% for P. intermedia, 6% for M. micros, and 0.8% for Fusobacterium spp. in periodontitis patients and 12%, 3%, 2%, 35%, 14% and 0%, respectively, in the periodontally healthy group. Furthermore, periodontitis individuals had significantly higher amount of all of the test species in the subgingival plaque samples compared with healthy subjects. CONCLUSION: RT-PCR provides a new rapid diagnostic tool and opens the opportunity to detect small numbers of oral pathogens in clinical specimens, which are under the detection limit by culture technique.

Adult↗

PubMed2026

The Next Step: The Role of Metagenomic Next-Generation Sequencing in Microbial Detection of Culture-Negative Cardiovascular Infections.

Cardiovascular infections, including those that involve native and prosthetic heart valves, implantable cardiac devices, mechanical circulatory assist devices, and vascular grafts, are associated with significant morbidity and mortality risks. Optimal management of these complex infections requires pathogen-directed antimicrobial therapy. However, standard culture-based methods often fail to identify causative organisms due to prior antimicrobial use, infections due to fastidious organisms, or biofilm-associated infections. Emerging evidence suggests that microbial cell-free DNA (mcfDNA) and metagenomic testing can enhance pathogen detection, particularly in culture-negative cases. However, their results require careful clinical interpretation, often necessitating input from infectious diseases specialists. In this review, we examine published evidence regarding metagenomic testing for cardiovascular infections and its impact on patient care. We propose a framework for microbiological adjudication of mcfDNA results, introduce standardized definitions for clinical impact assessment, and provide guidance on integrating mcfDNA testing into diagnostic evaluation of patients with culture-negative cardiovascular infections.

Humans↗

PubMed2005

Effects of clinical mastitis on ovarian function in post-partum dairy cows.

Mastitis-induced ovarian abnormalities were studied in a field trial. At 1-3 day after calving, > or = 2 parity cows not affected with chronic recurrent mastitis and yielding < 400,000/ml somatic cell count (SCC) individual milk in the previous lactation, were enrolled in the study. Thereafter milk samples were collected three times weekly for 95-100 day for progesterone (P4) assay. Individual P4 profiles were used to monitor ovarian cyclicity. When mastitis was diagnosed in the first 80 day post-partum (pp), clinical signs were recorded and scored, and aseptic milk samples were taken to identify the mastitis pathogens. Depending on the isolated pathogens the cows were blocked into one of the three sub-groups affected by either Gram-positive (GP), or Gram-negative (GN) bacteria, or of those with no detected pathogens (NDP). Cows suffering from any type of mastitis between days 15 and 28 (n = 27) showed a delay in the onset of ovarian cyclicity, and estrus was postponed compared to cows affected during the first 14 day pp (n = 59) and controls (n = 175) (38.6 +/- 2.3 vs 33.4 +/- 2.1 and 32.0 +/- 1.0 day, respectively, for onset of ovarian cyclicity and 90.7 +/- 2.5 vs 80.2 +/- 2.8 and 83.9 +/- 2.1 day, respectively, for estrus; both p < 0.05). The percentage of cows ovulating by day 28 was lower in those affected by mastitis between days 14 and 28 compared to cows between days 1 and 14 and controls (22.2% vs 47.5 and 50.3%, respectively; p < 0.05). A significantly higher rate of premature luteolysis was observed in GN + NDP compared to GP mastitis and healthy cows (46.7% vs 8.3 and 2.0%, respectively; p < 0.001). If the mastitis outbreak occurred during the follicular phase, the duration of this cycle segment was lengthened in GN + NDP mastitis compared to GP mastitis and healthy cows (10.8 +/- 0.9 vs 7.9 +/- 0.1 and 7.2 +/- 0.1, respectively; p < 0.001). The results indicate that mastitis can affect the resumption of ovarian activity in pp dairy cows. Mastitis may also impair reproduction also in cyclic cows: this effect can be the consequence of premature luteolysis or a prolonged follicular phase.

Animals↗

PubMed2006

Applications of real-time PCR in the screening of platelet concentrates for bacterial contamination.

Although there have been major improvements over the past few decades in detection methods for blood-borne infectious agents, platelet concentrates are still responsible for most cases of transfusion-transmitted bacterial infections. To date, real-time PCR is an indispensable tool in diagnostic laboratories to detect pathogens in a variety of biological samples. In this article, the applications of this powerful technique in the screening of platelet concentrates for bacterial contamination are discussed. Next to pathogen-specific (real-time) PCR assays, particular attention is directed to the recently developed 16S rDNA real-time PCR. This assay has been proven as a convenient way to detect bacterial contamination of platelet concentrates. The assay is sensitive and enables rapid detection of low initial numbers of bacteria in platelet concentrates. The short turnaround time of this assay allows high-throughput screening and reduction of the risk of transfusion of bacterially contaminated units. As with every method, real-time PCR has its advantages and disadvantages. These and especially limitations inherent to generation of false-positive or -negative results are emphasized. The universal nature of detection of the assay may be suitable for generalized bacterial screening of other blood components, such as red blood cells and plasma. Therefore, it is necessary to adapt and optimize detection in red blood cells and plasma with real-time PCR. Further sophistication, miniaturization and standardization of extraction and amplification methods should improve the total performance and robustness of the assay. Hence, real-time PCR is an attractive method in development as a more rapid screening test than currently used culture methods to detect bacterial contamination in blood components.

Animals↗

PubMed2000

[Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid].

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.

Adhesins, Bacterial↗

PubMed2005

Integrated minimum-set primers and unique probe design algorithms for differential detection on symptom-related pathogens.

MOTIVATION: Differential detection on symptom-related pathogens (SRP) is critical for fast identification and accurate control against epidemic diseases. Conventional polymerase chain reaction (PCR) requires a large number of unique primers to amplify selected SRP target sequences. With multiple-use primers (mu-primers), multiple targets can be amplified and detected in one PCR experiment under standard reaction condition and reduced detection complexity. However, the time complexity of designing mu-primers with the best heuristic method available is too vast. We have formulated minimum-set mu-primer design problem as a set covering problem (SCP), and used modified compact genetic algorithm (MCGA) to solve this problem optimally and efficiently. We have also proposed new strategies of primer/probe design algorithm (PDA) on combining both minimum-set (MS) mu-primers and unique (UniQ) probes. Designed primer/probe set by PDA-MS/UniQ can amplify multiple genes simultaneously upon physical presence with minimum-set mu-primer amplification (MMA) before intended differential detection with probes-array hybridization (PAH) on the selected target set of SRP. RESULTS: The proposed PDA-MS/UniQ method pursues a much smaller number of primers set compared with conventional PCR. In the simulation experiment for amplifying 12 669 target sequences, the performance of our method with 68% reduction on required mu-primers number seems to be superior to the compared heuristic approaches in both computation efficiency and reduction percentage. Our integrated PDA-MS/UniQ method is applied to the differential detection on 9 plant viruses from 4 genera with MMA and PAH of 11 mu-primers instead of 18 unique ones in conventional PCR while amplifying overall 9 target sequences. The results of wet lab experiments with integrated MMA-PAH system have successfully validated the specificity and sensitivity of the primers/probes designed with our integrated PDA-MS/UniQ method.

Algorithms↗

PubMed1999

Use of polymerase chain reaction to detect Proteus mirabilis and Ureaplasma urealyticum in urinary calculi.

In this study, we evaluated the efficacy of the polymerase chain reaction (PCR) in detecting urea-splitting microorganisms in desiccated urinary tract infection stones. Seventy-eight urinary tract stones were tested for the presence of Proteus mirabilis and Ureaplasma urealyticum by means of PCR with species-specific primers. Twenty-seven stone samples were composed of struvite and/or carbonate apatite (infection stone); 40 were calcium oxalate and/or calcium phosphate; seven were mixed, with struvite/carbonate apatite and calcium oxalate; and four were uric acid stones. PCR was performed with DNA extracted from pulverized stone pieces. Initial assays using the pulverized stone specimens spiked with microorganisms showed that PCR could not detect U. urealyticum at densities below 10(3) color changing units (CCU), or P. mirabilis at densities below 10(4) colony-forming units (CFU). PCR was negative for U. urealyticum and P. mirabilis in all metabolic stones from patients. P. mirabilis was detected by PCR in 10 of 34 patients with infection stones. Preoperative urine cultures grew P. mirabilis in three of these 10 patients, and were negative for P. mirabilis in the other seven. U. urealyticum was detected by PCR in stone samples from four patients, two of which were also PCR-positive for P. mirabilis. All four of these patients had infection stones: two had residual stones, and the other two had recurrence of urinary stones after their operations. These results demonstrate that microorganisms in urinary stones can be detected by PCR even when the voided urine culture is negative. Investigations into the role of bacterial infection in stone formation will require further improvements in the sensitivity of PCR assays for pathogen detection.

Adult↗

PubMed2004

Detection of a bovine group C rotavirus from adult cows with diarrhea and reduced milk production.

Only two strains (Shintoku and porcine-like WD534tc) of group C rotavirus (GCR) from cattle have been reported to date. A GCR designated the Yamagata strain was the only pathogen detected in an outbreak of adult cow diarrhea accompanied by a decrease in milk production. The nucleotide sequences of the VP6 and VP7 genes from strain Yamagata were determined. Comparative sequence analysis showed that the sequence identities between strains Yamagata and Shintoku were markedly high in both VP6 gene (98.1%) and VP7 gene (93.5%), and that these strains belonged to the same clusters which were distinguished from GCRs from different host species in phylogenetic trees of these genes. These results suggested strongly that cattle species is one of the natural hosts of GCR infection, and that GCRs are a cause of adult cow diarrhea.

Animals↗

PubMed2004

Direct detection of bacterial pathogens in brain abscesses by polymerase chain reaction amplification and sequencing of partial 16S ribosomal deoxyribonucleic acid fragments.

OBJECTIVE: To evaluate the feasibility of detecting bacterial pathogens directly from the clinical brain abscess specimens by polymerase chain reaction (PCR) amplification and sequencing of bacterial 16S ribosomal deoxyribonucleic acid (rDNA). METHODS: A total of 14 specimens were tested by both culture and PCR amplification, targeting the full-length or a partial region of 16S rDNA. 16S rDNA is known to be conserved in bacteria. Sequencing of partial-length and full-length 16S rDNA was performed. The sequence data were compared with known sequences of 16S rDNA in the National Center for Biotechnology Information GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithm. The species with the best match of similarity were regarded as the pathogenic species in the samples. We also developed a Streptococcus-specific multiplex PCR analysis for identifying members of the Streptococcus species, the most common pathogen of brain abscesses. RESULTS: The 10 culture-positive specimens were all PCR-positive for partial 16S rDNA, but only seven were positive for full-length 16S rDNA amplification. Bacterial DNA was not detected in the remaining four specimens with a negative culture. Species identification by phenotypes from culture was in agreement with that by sequencing results of partial-length (or full-length) 16S rDNA. The Streptococcus-specific PCR analysis could detect Streptococcus species correctly in one step. CONCLUSION: Bacterial 16S rDNA sequences provide reliable clues to the identification of unknown pathogens. PCR analysis of 16S rDNA and sequencing may identify pathogens to the species level directly from brain abscesses. This approach is rapid and is useful especially in the identification of slow-growing and fastidious organisms.

Adolescent↗

PubMed2006

Development and evaluation of methods to detect nucleopolyhedroviruses in larvae of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough).

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r(2) = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.

Animals↗

PubMed1995

Genital tract infections in sexually active women in Barbados.

Ninety-eight women attending three different clinics were prospectively studied for the presence of genital tract infections, including Chlamydia trachomatis. Of these 98 women, 35 were presenting to a polyclinic with symptoms of genital tract infection, 55 were attending an antenatal clinic for their first visit, and 8 referred to a colposcopy clinic because of an abnormal Papanicolaou smear were included. Gonorrhoea was detected in one patient, syphilis in two, and Trichomonas vaginalis in six. Candida albicans and Chlamydia trachomatis were each detected in 18 patients, while the most common condition was bacterial vaginosis, detected in 35 patients. The prevalence of these infections was lowest in patients referred for colposcopy and highest in the women attending the antenatal clinic. Chlamydia trachomatis was the most common sexually-transmitted pathogen detected in this population. These data emphasise the need for an aggressive approach to the diagnosis and treatment of chlamydial infection in females.

Adolescent↗

PubMed2005

Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States.

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United States using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5' nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.

Animals↗

PubMed2001

Monitoring of microbial hazards at farms, slaughterhouses, and processing lines of swine in Korea.

This study was executed to investigate microbiological hazards at swine farms, slaughterhouses, dressing operations, and local markets for the application of the hazard analysis critical control point system in Korea by analyzing total aerobic plate count (APC) and presence of pathogens. Six integrated pig farms and meat packers were selected from six different provinces, and samples were collected from pig carcasses by swabbing and excision methods at the slaughterhouses, processing rooms, and local markets, respectively. APCs of water in water tanks were relatively low, 1.9 to 3.1 log10 CFU/ml; however, they were increased to 4.6 to 6.9 log10 CFU/ml when sampled from water nipples in the pigpen. APCs of feeds in the feed bins and in the pigpens were 4.4 to 5.4 and 5.2 to 6.7 log10 CFU/g, respectively. Salmonella spp., Staphylococcus aureus, and Clostridium perfringens were detected from water and feed sampled in pigpens and pigpen floors. S. aureus was the most frequently detected pathogenic bacteria in slaughterhouses and processing rooms. Listeria monocytogenes and Yersinia enterocolitica were also detected from the processing rooms of the Kyonggi, Kyongsang, and Cheju provinces. Even though APCs were maintained at the low level of 3.0 log10 CFU/g during slaughtering and processing steps, those of final pork products produced by the same companies showed relatively high numbers when purchased from the local market. These results indicated that the cold chain system for transporting and merchandising of pork products was deficient in Korea. Water supply and feed bins in swine farms and individual operations can be identified as critical control points to reduce microbiological hazards in swine farms, slaughterhouses, and processing plants.

Abattoirs↗

PubMed2006

DAMPs, PAMPs and alarmins: all we need to know about danger.

Multicellular animals detect pathogens via a set of receptors that recognize pathogen-associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term "alarmin" is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage-associated molecular patterns (DAMPs).

Animals↗