SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
We have developed a method for genomic representation using Type IIB restriction endonucleases. Representation by concatenation of restriction digests, or RECORD, is an approach to sample the fragments generated by cleavage with these enzymes. Here, we show that the RECORD libraries may be used for digital karyotyping and for pathogen identification by computational subtraction.
Flavobacterium meningosepticum, because of its unusual susceptibility patterns and predilection for debilitated or immunocompromised hosts, can be a dangerous pathogen. Identification of the organism and proper susceptibility studies, which are essential in designing antibiotic therapy, helped in the selection of a regimen that proved curative in this case.
BACKGROUND: The origins of asthma and allergic disease begin in early life for many individuals. It is vital to understand the factors and/or events leading to their development. METHODS: The Childhood Origins of Asthma project evaluated children at high risk for asthma to study the relationships among viral infections, environmental factors, immune dysregulation, genetic factors, and the development of atopic diseases. Consequently wheezing illnesses, viral respiratory pathogen identification, and in vitro cytokine response profiles were comprehensively evaluated from birth to 3 years of age, and associations of the observed phenotypes with genetic polymorphisms were investigated. RESULTS: For the entire cohort, cytokine responses did not develop according to a strict T helper cell 1 or T helper cell 2 polarization pattern during infancy. Increased cord blood mononuclear cell phytohemagglutin-induced interferon-gamma responses of mononuclear cells were associated with decreased numbers of moderate to severe viral infections during infancy, especially among subjects with the greatest exposure to other children. In support of the hygiene hypothesis, an increased frequency of viral infections in infancy resulted in increased mitogen-induced interferon-gamma responses at 1 year of age. First year wheezing illnesses caused by respiratory viral infection were the strongest predictor of subsequent third year wheezing. Also, genotypic variation interacting with environmental factors, including day care, was associated with clinical and immunologic phenotypes that may precede the development of asthma. CONCLUSIONS: Associations between clinical wheezing, viral identification, specific cytokine responses and genetic variation provide insight into the immunopathogenesis of childhood asthma and allergic diseases.
Bayes factors comparing two or more competing hypotheses are often estimated by constructing a Markov chain Monte Carlo (MCMC) sampler to explore the joint space of the hypotheses. To obtain efficient Bayes factor estimates, Carlin and Chib (1995, Journal of the Royal Statistical Society, Series B57, 473-484) suggest adjusting the prior odds of the competing hypotheses so that the posterior odds are approximately one, then estimating the Bayes factor by simple division. A byproduct is that one often produces several independent MCMC chains, only one of which is actually used for estimation. We extend this approach to incorporate output from multiple chains by proposing three statistical models. The first assumes independent sampler draws and models the hypothesis indicator function using logistic regression for various choices of the prior odds. The two more complex models relax the independence assumption by allowing for higher-lag dependence within the MCMC output. These models allow us to estimate the uncertainty in our Bayes factor calculation and to fully use several different MCMC chains even when the prior odds of the hypotheses vary from chain to chain. We apply these methods to calculate Bayes factors for tests of monophyly in two phylogenetic examples. The first example explores the relationship of an unknown pathogen to a set of known pathogens. Identification of the unknown's monophyletic relationship may affect antibiotic choice in a clinical setting. The second example focuses on HIV recombination detection. For potential clinical application, these types of analyses must be completed as efficiently as possible.
In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.
Accurate diagnosis of pneumococcal pneumonia in the acute-care setting remains a challenge due to the inadequate sensitivity of conventional diagnostic tests. Sputum cultures, which are likely to have the highest diagnostic yields of all specimen types, have been considered unreliable, due to their inability to differentiate colonization from infection. Our objective was to evaluate the potential clinical utility of a rapid quantitative real-time PCR assay using sputum samples for Streptococcus pneumoniae in adult patients with community-acquired pneumonia (CAP). A prospective clinical observational study of consecutively enrolled emergency department patients with CAP was performed; only those patients with excess good-quality sputum samples were included for evaluation. Sputum samples were tested for the presence of S. pneumoniae by using a quantitative PCR that targets the pneumolysin gene. PCR findings were compared with those of a composite reference standard comprising Gram staining of sputum samples and sputum/blood cultures. The area under the curve (AUC) and a log-transformed threshold, which provides the maximal sensitivity and specificity, were calculated. Of 487 subjects enrolled, 129 were evaluable. Receiver operating characteristic curve analysis demonstrated an AUC of 0.87. Sensitivity and specificity were 90.0 percent and 80.0 percent, respectively; positive and negative predictive values were 58.7 percent and 96.2 percent, respectively. We have demonstrated that a quantitative rapid pneumolysin PCR assay has favorable accuracy for diagnosis of pneumococcal pneumonia in adult patients with CAP; this assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute-care setting, where rapid pathogen identification may assist in selection of the most appropriate antibiotics.
Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed.
BACKGROUND: Indiscriminate antibiotic use remains common in febrile patients with acute necrotizing pancreatitis (ANP), particularly during the early phase. Metagenomic next‑generation sequencing (mNGS) has shown diagnostic utility for infected pancreatic necrosis (IPN) and may offer a means to guide antimicrobial therapy. We aimed to explore whether mNGS could potentially improve the appropriateness of antibiotic use in ANP patients presenting with early fever. METHODS: This prospective multicenter cohort study was conducted at five hospitals in China, enrolling ANP patients who developed fever within two weeks of symptom onset. Antibiotic susceptibility was defined per local microbiology laboratory reports. The hypothetical impact of mNGS on reducing inappropriate antibiotic use was evaluated through a retrospective simulation using predefined criteria from the BGI China antimicrobial drug usage card, as mNGS results were not disclosed to the treating teams during the actual clinical course. RESULTS: Between May 2023 and December 2024, 125 ANP patients with early fever were enrolled. Antibiotics were administered to 91.2% (114/125) of patients, whereas only 23.2% (29/125)were eventually confirmed to have IPN, and the rate of appropriate antibiotic use was 14.5% (17/117) based on conventional culture. In our simulated model, if therapy had been guided by plasma mNGS results, the estimated rate of appropriate antibiotic use could have increased to 71.8%. CONCLUSIONS: Plasma mNGS facilitates rapid pathogen identification and shows potential for improving antibiotic appropriateness in ANP patients with early fever.
AIM: To investigate epidemiology and pathogenic mite species of intestinal and urinary acariasis in individuals with different occupations. METHODS: A total of 1994 individuals were tested in this study. History collection, skin prick test and pathogen identification were conducted. The mites were isolated from stool and urine samples by saturated saline flotation methods and sieving following centrifugation, respectively. RESULTS: Among the 1994 individuals examined, responses to the skin prick test of "+++", "++", "+","+/-" and "-" were observed at frequencies of 3.96 % (79), 3.21 % (64), 2.31 % (46), 1.25 % (25) and 89.27 % (1780), respectively. A total number of 161 (8.07 %) individuals were shown to carry mites, with 92 (4.61 %) positive only for stool samples, 37 (1.86 %) positive only for urine samples and 32 (1.60 %) for both. The positive rate of mites in stool samples was 6.22 % (124/1994), being 6.84 % (78/1140) for males and 5.39 % (46/854) for females. No gender difference was observed in this study (chi(2)=1.77, P>0.05). The mites from stool samples included Acarus siro, TyroPhagus putrescentiae, Dermatophagoides farinae, D. pteronyssinus, Glycyphagus domesticus, G. ornatus, Carpoglyphus lactis and Tarsonemus granaries. The positive rate of mites in urine samples was 3.46 % (69/1994). The positive rates for male and female subjects were found to be 3.95 % (45/1140) and 2.81 % (24/854) respectively, with no gender difference observed (chi(2)=1.89, P>0.05). Mites species in urine samples included Acarus siro, Tyrophagus putrescentiae, T. longior, Aleuroglyphus ovatus, Caloglyphus berlesei, C. mycophagus, Suidasia nesbitti, Lardoglyphus konoi, Glycyphagus domesticus, Carpoglyphus lactis, Lepidoglyphus destructor, Dermatophagoides farinae, D. pteronyssinus, Euroglyphus magnei, Caloglyphus hughesi, Tarsonemus granarus and T. hominis. The species of mites in stool and urine samples were consistent with those separated from working environment. A significant difference was found among the frequencies of mite infection in individuals with different occupations (chi(2)=82.55, P<0.001), with its frequencies in those working in medicinal herb storehouses, those in rice storehouse or mills, miners, railway workers, pupils and teachers being 15.89 % (68/428), 12.96 % (53/409), 3.28 % (18/549), 2.54 % (6/236), 5.10 % (13/255) and 2.56 % (3/117), respectively. CONCLUSION: The prevalence of human intestinal and urinary acariasis was not associated with gender, and these diseases are more frequently found in individuals working in medicinal herb, rice storehouses or mills and other sites with high density of mites. More attention should be paid to the mite prevention and labor protection for these high-risk groups.
A preparation of Bacillus anthracis (Sterne strain) spores was used to evaluate commercially available reagents and portable equipment for detecting anthrax contamination by using real-time PCR and was used to assess the fate of spores added directly to bulk tank milk. The Ruggedized Advanced Pathogen Identification Device (RAPID) was employed to detect spores in raw milk down to a concentration of 2,500 spores per ml. Commercially available primers and probes developed to detect either the protective antigen gene or the lethal factor gene both provided easily read positive signals with the RAPID following extraction from milk with a commercially available DNA extraction kit. Nucleotide sequence analysis of the vrrA gene with the use of DNA extracted from spiked milk provided molecular data that readily identified the spores as B. anthracis with a 100% BLAST match to the Sterne and Ames strains and easily distinguished them from B. cereus. Physical-fate and thermal-stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction of whole milk. A single treatment at standard pasteurization temperatures, while 100% lethal to vegetative cells, had no effect on spore viability even 14 days after the treatment. Twenty-four hours after the first treatment, a second treatment at 72 degrees C for 15 s reduced the viability of the population by ca. 99% but still did not kill all of the spores. From these studies, we conclude that standard pasteurization techniques for milk would have little effect on the viability of B. anthracis spores and that raw or pasteurized milk poses no obstacles to the rapid detection of the spores by molecular techniques.
Clostridium perfringens is one of the etiologic agents of gas gangrene that can occur when a wound is contaminated with soil. Type A C. perfringens can cause foodborne and nonfoodborne gastrointestinal illnesses due to an enterotoxin (CPE) produced by some strains during sporulation. We developed a quantitative real-time PCR assay based on fluorescence resonance energy transfer hybridization chemistry that targets the C. perfringens-specific phospholipase C (plc) gene and the enterotoxigenic gene (cpe) with the LightCycler and the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). The assay can detect as few as 20 copies of target sequences per PCR. The total assay time, from extraction to PCR analysis, is 90 min. This assay is rapid, sensitive, and specific and will allow direct detection of C. perfringens in water, food, and stool samples. It should prove helpful in investigating foodborne illnesses due to C. perfringens and can be used as a tool to ensure the safety of food and water supplies.
In 1998, the United States National Research Council prepared a report assessing the viability of potable reuse. The report concludes that planned indirect potable reuse is a viable application of reclaimed water but should be considered for implementation only after other alternative measures, such as development of new water sources, nonpotable reuse, and water conservation, have been evaluated and determined not to be economically or technically feasible. Although health-effects research has not indicated that there have been any adverse health effects resulting from drinking highly treated reclaimed water, those data are sparse. There are uncertainties associated with assessing the potential health risks of drinking reclaimed water. The report acknowledges that several issues remain unresolved regarding reclaimed water treatment and reliability, microbial and chemical constituents of concern, and water quality and health effects monitoring. Research needs include methods to detect emerging pathogens, identification and quantification of trace organic compounds, development of suitable in vivo toxicological methods, and evaluation of the effectiveness and reliability of multiple treatment barriers. The report also concludes that direct potable reuse is not a viable option to consider at this time.
Neutropenic patients with cancer are a heterogeneous group of patients who carry a variable risk for infection. When such patients present with fever, appropriate empiric antibiotic therapy is initiated and continued until clinical improvement or clinical or microbiologic data direct a modification in treatment. As the duration of neutropenia increases, so does the need for antimicrobial modifications. Changes in therapy may include antimicrobials directed against gram-positive bacteria, resistant gram-negative bacteria, or fungi. Because of the high risk for colonization by vancomycin-resistant enterococci, vancomycin use is restricted as first-line empiric therapy unless the patient is at a high risk for serious gram-positive infection. Usually in the setting of neutropenia, gram-positive infections are of low virulence. Prophylactic antibiotic therapy may increase the selection of resistant strains and should be avoided. Antibiotic therapy should always be combined with prudent infection-control measures, such as aseptic practices, barrier isolation, handwashing, removal of infected catheters, and infection monitoring. In the immunocompromised patient with cancer and neutropenia, all infections should be treated, with the extent, duration, and site of treatment being directed by risk stratification and specific pathogen identification. Patients with neutropenia are at risk for severe morbidity and mortality related to infection.
The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.
The immune system needs to recognise target protein antigens from pathogens residing in both extracellular and intracellular locations. Intricate proteolytic processing events that follow antigen/ pathogen encounter provide the immune system with a complex display of a heterogeneous peptide mix, instrumental in the initiation of T cell immune responses, and allow the separation of extracellular and intracellular pathogen identification. However, recent evidence shows that this conventional dimorphism in the proteolytic processing of endogenous versus internalised antigen is less restrictive than originally recognized. The events that constitute the conventional major histocompatibility complex (MHC)-restricted processing pathways are accompanied by interesting deviations that provide novel adjuncts for the processing machinery to gain access to antigen in varied intracellular locations. This review discusses these aspects of classical and non-classical processing pathways for MHC-restricted protein presentation, which play significant roles in both optimising and diversifying the peptide repertoire available for immune recognition.
The occurrence of coligranulomatosis in a Brazilian flock of 1500 8-to-12-month-old coturnix quail is described. The disease caused an 85% drop in egg production and 15% mortality. Lesions were located on the mesentery, intestines, gizzard, heart, oviducts, ovaries, and liver. Samples of these organs were submitted for bacteriological culture, isolation, identification, pathogenicity assay by inoculation of diluted cultures into healthy hens, and histopathology.
In this paper we describe a case of bilateral broncho-pneumonia due to invasion by mycoplasma pneumoniae, leading to ARDS. A high serum level of cold agglutinins, a mild haemolysis and a rash resembling that of an erythema multiforme were noted. The initial treatment was non-specific. After pathogen identification tetracycline therapy was initiated. Bacterial superinfections were treated with appropriate antibiotic schemas. The observed biphasic clinical course could be explained as an immunological phenomenon.
One hundred fifty-one patients with presumed aerobic-anaerobic mixed peritoneal infections were treated in a prospective, randomized trial with either cefotaxime alone (76) or the combination of gentamicin-clindamycin (75). Primary and complicating foci of sepsis were cultured for both aerobic and anaerobic pathogen identification and antibiotic susceptibility. In vitro aerobic disk sensitivities (114 isolates) to cefotaxime were 82% and to gentamicin, 88%; anaerobic agar-diffusion sensitivities (227 isolates) to cefotaxime were 87% and to clindamycin, 98%. Only enterococci and Pseudomonas sp were consistently resistant to cefotaxime. Infection was eliminated in 82% of those treated with cefotaxime and in 87% of those treated with the gentamicin-clindamycin combination, yet sepsis recurred in 11% of those treated with cefotaxime and in 13% for those given gentamicin-clindamycin. Five patients (7%) demonstrated nephrotoxicity for gentamicin. (Serum creatinine increased greater than 1.5 mg/100 ml over pretreatment levels.) Otherwise, incidence and severity of adverse reactions were identical for the two groups and consisted primarily of phlebitis and diarrhea. One patient in each treatment group died of uncontrolled sepsis. Although results suggested a laboratory superiority of gentamicin-clindamycin, there was a clinical equality in therapeutic benefit and a greater safety following the use of cefotaxime alone.