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PubMed2005

Polyaniline synthesis and its biosensor application.

In this study, five polyaniline compounds were synthesized using different protonic acids and incorporated into a conductometric biosensor used for bovine viral diarrhea virus detection. The biosensor was developed and evaluated by the authors for bacterial pathogen detection in previous studies. The biosensor consisted of two parts: the immunosensor and the electronic data collection system. Liquid sample moved through the immunosensor surface by capillary action. The specificity of the biosensor was based on the unique binding characteristics of the polyclonal and monoclonal antibodies immobilized on the immunosensor. Polyaniline was used in the biosensor architecture as the transducer due to its electronic and bio-molecular properties. Results showed that the biosensor was sensitive at a concentration of 10(3) cell culture infective dose per milliliter (CCID/ml) of BVDV antigens. The promising results on the BVDV detection demonstrated that the conductometric biosensor was interchangeable for different target molecules of detection. Further modification could be implemented to evaluate the biosensor as a rapid diagnostic device to detect other infectious disease outbreaks in livestock population.

Aniline Compounds↗

PubMed2006

The real-time polymerase chain reaction.

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

Animals↗

PubMed2006

Testing for heparin-induced thrombocytopenia antibodies.

Heparin-induced thrombocytopenia (HIT) has a distinct clinical profile and unique pathogenesis. It is caused by platelet-activating IgG antibodies that recognize multimolecular complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions. Although an immune response to PF4/heparin associated with heparin treatment is very common, clinical HIT occurs only among the minority of patients whose antibodies are capable of strongly activating platelets. This explains why certain platelet activation assays and anti-PF4/polyanion immunoassays have high sensitivity for HIT and why diagnostic specificity is highest for those assays that preferentially detect pathogenic antibodies, such as the washed platelet activation assays or immunoassays that detect only IgG antibodies. Negative results obtained in a solid-phase PF4/polyanion immunoassay generally exclude HIT (high negative predictive value), especially in a setting of a low pretest probability. In addition, because the magnitude of a positive test result correlates with greater likelihood of HIT, a Bayesian diagnostic approach that combines pretest probability and the magnitude of a positive test result is recommended. Recent studies suggest that presence of anti-PF4/polyanion antibodies in certain clinical settings confers an adverse prognosis, even without clinically evident HIT. Whether such antibodies impart "forme fruste" HIT or are simply a surrogate marker for a non-HIT adverse risk factor such as inflammation is unresolved.

Anticoagulants↗

PubMed1999

An investigation of the presence of ultramicrocells in natural mineral water.

The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated. Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%). Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1. Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells'. Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long. Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present. If pathogens are able to form ultramicrocells, these may go undetected.

Cellulose↗

PubMed2026

Diverse haplotypes at a complex Solanum americanum locus confer resistance to Phytophthora infestans and P. capsici.

Plants encounter diverse pathogens and have evolved a two-layered innate immune system to detect pathogen molecules and activate defense mechanisms that restrict infection. Most cloned plant Resistance (R) genes encode NLR immune receptors. NLR genes are often found in clusters of paralogs with sequence and copy number variation; whether these NLR clusters evolve in response to single or multiple pathogens has been unclear. We report here the isolation of a Phytophthora capsici resistance gene, Rpc2, along with a novel P. infestans resistance gene, Rpi-amr5, from two Solanum americanum accessions. These orthologous genes reside in the Rpi-amr1 cluster, which has previously been associated with resistance to P. infestans. By screening RXLR effector libraries of P. infestans and P. capsici, we identified multiple effectors recognised by both NLRs. Our findings highlight the complexity of NLR clusters and evolution driven by interactions with multiple pathogens. This work will underpin efforts to elevate resistance against Phytophthora pathogens and enhances our understanding of NLR evolution.

Journal Article↗

PubMed1990

Gene-for-gene complementarity in plant-pathogen interactions.

The cloning of avirulence genes has greatly aided our understanding of plant-pathogen specificity. It has proven that the gene-for-gene relationship first noted by Flor is correct--single avirulence gene encoding single protein products indeed are the genetic elements that interact with plant disease resistance genes. Furthermore, firm genetic evidence has provided insight into how two cloned avirulence genes (the TMV coat gene and avrD) cause the HR. The differences in structure of pathogen elicitors also indicates that plants have evolved diverse recognitional mechanisms to detect pathogens. It is appealing to speculate, therefore, that elicitors represent the plant equivalent of antigens in vertebrates. Another consequence of these results has been the establishment of firm genetic and biochemical evidence supporting the elicitor-receptor model for recognition of incompatible pathogen races by plants. In both TMV and bacterial pathogens, we are also beginning to understand how avirulence genes are altered to confound plant recognition of the pathogen. The next few years should yield additional information on avirulence gene structure as well as the important questions of their function in the pathogen and the molecular mechanisms whereby plant recognition occurs. The marked successes in cloning avirulence genes underscore only more forcefully the pressing need to clone and characterize plant disease resistance genes. Certainly an understanding of these genes is required to further our basic knowledge of active defense in plants and to permit their manipulation for improved control of plant diseases in practical agriculture.

Cloning, Molecular↗

PubMed2003

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coil O157:H7, Salmonella, and Listeria with BAX, and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples.

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

Agaricales↗

PubMed2007

Combination of immunosensor detection with viability testing and confirmation using the polymerase chain reaction and culture.

Rapid and accurate differential determination of viable versus nonviable microbes is critical for formulation of an appropriate response after pathogen detection. Sensors for rapid bacterial identification can be used for applications ranging from environmental monitoring and homeland defense to food process monitoring, but few provide viability information. This study combines the rapid screening capability of the array biosensor using an immunoassay format with methods for determination of viability. Additionally, cells captured by the immobilized antibodies can be cultured following fluorescence imaging to further confirm viability and for cell population expansion for further characterization, e.g., strain identification or antibiotic susceptibility testing. Finally, we demonstrate analysis of captured bacteria using the polymerase chain reaction (PCR). PCR results for waveguide-captured cells were 3 orders of magnitude more sensitive than the fluorescence immunoassay and can also provide additional genetic information on the captured microbes. These approaches can be used to rapidly detect and distinguish viable versus nonviable and pathogenic versus nonpathogenic captured organisms, provide culture materials for further analysis on a shorter time scale, and assess the efficacy of decontamination or sterilization procedures.

Bacillus subtilis↗

PubMed2026

Detection rate of pathogenic variants by postmortem genetic testing for sudden cardiac death among children and young adults: systematic review and meta-analysis.

PURPOSE: Postmortem genetic testing (PMGT) can clarify the causes of sudden cardiac death (SCD) in children and young adults and provide preventive care for relatives. We systematically reviewed studies to estimate the detection rate of pathogenic variants identified by PMGT in SCD cases aged 1-50 years and examined factors influencing detection rates. METHODS: Ovid MEDLINE and Ovid Embase were searched for observational studies on PMGT in cases of SCD, records in duplicate were screened, and study- and variant-level data were extracted. Risk of bias was assessed using the Joanna Briggs Institute checklist. The pooled detection rates were estimated using random-effects meta-analysis, and heterogeneity was explored based on subgroup and meta-regression analyses. RESULTS: Sixty-six studies (4,452 cases from 23 countries) were included. The pooled detection rate was 19% (95% confidence interval, 15% to 24%). Among the detected pathogenic variants, 76% were found in genes included on the ACMG Secondary Findings list. Higher detection rates were associated with earlier publication years, lower mean age, and lower risk of bias. Substantial between-study heterogeneity persisted (I2 = 91%) despite the subgroup and meta-regression analyses. CONCLUSION: PMGT can be used to identify pathogenic variants in young SCD cases, however, there is considerable heterogeneity in study conditions.

Forensic genetics↗

PubMed2003

Pathogen discovery from human tissue by sequence-based computational subtraction.

We have recently reported a new pathogen discovery approach, "computational subtraction". With this approach, non-human transcripts are detected by sequencing cDNA libraries from infected tissue and eliminating those transcripts that match the human genome. We show now that this method is experimentally feasible. We generated a cDNA library from a tissue sample of post-transplant lymphoproliferative disorder (PTLD). 27,840 independent cDNA sequences were filtered by computational subtraction against the known human sequence to identify 32 nonmatching transcripts. Of these, 22 (0.1%) were found to be amplifiable from both infected and noninfected samples and were inferred to be human DNA not yet contained in the available human genome sequence. The remaining 10 sequences could be amplified only from Epstein-Barr virus (EBV)-infected tissues. All 10 corresponded to the known EBV sequence. This proof-of-principle experiment demonstrates that computational subtraction can detect pathogenic microbes in primary human-diseased tissue.

DNA, Complementary↗

PubMed2006

Pattern recognition of proteins based on an array of functionalized porphyrins.

A practical protein-detecting array is desirable for its potential application in proteomics, medical diagnostics, and pathogen detection. Here, we report a novel protein-detecting array based on porphyrins containing peripheral amino acids as protein surface receptors. The array of porphyrin receptors showed a unique pattern of fluorescence change upon interaction with certain protein samples. Both metal and nonmetal-containing proteins and mixtures of proteins gave distinct patterns, allowing their unambiguous identification. The composite pattern for each sample was subjected to principal component analysis (PCA) to generate a clustering map for more practical visualization. Increasing the number of porphyrin receptors from eight to sixteen gave improved resolution, suggesting that this array is expandable to give satisfactory resolution for any given sample system by carefully maximizing the chemical diversity of the receptors.

Animals↗

PubMed1997

Prevalence and characteristics of severe rotavirus infections in Nicaraguan children.

We analyzed the prevalence of rotavirus in 296 children age between 3 and 36 months who were hospitalized in 1994 with severe gastro-enteritis at two health centres for diarrhoea treatment in León, Nicaragua. Enteric viruses were detected in 96 (32.4%) of the children and rotaviruses were the most common pathogens detected in 84 (28%). The majority of rotavirus infections occurred in children less than 1 year old and all strains isolated belonged to subgroup II and had 'long' RNA patterns. Molecular epidemiology of 55 rotavirus strains revealed that all had the same RNA migration pattern and serotyping of 37 strains by PCR technology revealed that all isolates belonged to serotype 3. A significant observation was that only one electropherotype of rotavirus circulated. No non-group A rotaviruses were found by RNA gel electrophoresis. Adenoviruses were found by ELISA in 14 of 265 (5%) children and were most frequently detected during the 1st year of life. Of 103 faecal samples analyzed by electron microscopy, four contained small round structured viruses.

Adenovirus Infections, Human↗

PubMed1994

Identification of pathogenic leptospires by recombinant DNA probes.

Early diagnosis of leptospirosis of pulmonary diffuse hemorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific method for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector pUC9. Recombinant plasmids which have homologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid, designated pCX7, could detect 1.7 kb fragment of strain 017, 9.0 kb of strain 601 and 30.0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and Leptonema illini. The recombinant plasmid pCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.

Blotting, Southern↗

PubMed1996

[The etiology of acute lower respiratory tract infections in infants].

The etiology of acute lower respiratory tract infections (ALRI) was studied in pediatric inpatients under 2 years of age admitted to Chiba Municipal Hospital between June 1994 and March 1995. Eighty-seven patients, 99 episodes were investigated for bacterial infection with the use of blood culture and washed sputum culture, for viral infection with the use of virus isolation, antigen detection and antibody assays, for Mycoplasma pneumoniae infection with the use of antibody assay and for Chlamydia infection with the use of antigen detection. Pathogens were identified in 71 (71%) of the 99 episodes. Evidence of bacterial infection was detected in 43 episodes (43%), viral infection in 37 episodes (37%), Mycoplasma pneumoniae infection in 4 episodes (4%) and Chlamydia infection 3 episodes (3%). The major bacterial pathogens were H. influenzae, M. (B) catarrhalis and S. pneumoniae. RS virus and influenza virus epidemics occurred during the winter. A mixed bacterial and viral infection was documented in 13 episodes (13%). RS virus infection was common in infants up to 6 months old. Mixed bacterial and influenza virus infections were common in 1 or more year old children. Virus isolation was useful for the grasp of the viral epidemic. Bacterial associated infections were common in children under 2 years of age with ALRI. Washed sputum culture and sputum gram stains' were useful for the treatment of infant ALRI.

Acute Disease↗

PubMed2004

Microbiology of early CF lung disease.

Recent bronchoscopy studies using assays to measure inflammation and molecular typing techniques have facilitated an increased understanding of the early events that occur within the lungs of young children with cystic fibrosis and provided additional insights into the natural history of lung disease in children. In 2000, the US CF National Patient Registry data showed that among 1000 infants <2 years of age, the first bacterial pathogens detected are Haemophilus influenzae, Staphylococcus aureus, and Pseudomonas aeruginosa and the prevalence of these pathogens in these young infants are 19%, 42%, and 29%, respectively. In addition, 7% harbour Stenotrophomonas maltophilia and <1% harbour Burkholderia cepacia complex. Several investigators have performed bronchoscopy studies on young infants to further examine the natural history of lung disease. In one such study of 40 CF infants, 65%, 63% and 70% of children at 1, 2, and 3 years of age harboured at least one CF pathogen. H. influenzae was most common (38%) in infants at 1 year of age, and S. aureus was most common in 2 (37%) and 3 (36%) year olds. P. aeruginosa increased from 18% at 1 year of age to 33% at 3 years of age and was usually present in high numbers, i.e., > or =10(5) CFU/ml of BAL fluid. Investigators have studied the microbiology of young CF infants using specimens derived from the upper airway (deep throat) compared with the lower airway (broncheoalveolar lavage specimens) to determine if the upper airway is predictive of pathogens in the lower airway. In general, these studies have shown that a negative oropharyngeal culture indicated that isolation of P. aeruginosa from the lower airway was unlikely, but a positive culture did not predict lower airway infection. Similar findings were noted for H. influenzae and S. aureus.

Bronchoscopy↗

PubMed1992

Aetiological studies on hospital inpatients with secretory diarrhoea in Calcutta.

Twenty-five cases of all age groups with secretory diarrhoea admitted to the Infectious Diseases Hospital, Calcutta were studied. Bacterial enteropathogens were detected by bacteriological examination of the faeces, in each and every case. The main pathogens detected were Escherichia coli and Vibrio cholerae. V cholerae was more commonly associated with severe illness except in the very young, while Esch coli was equally associated with moderate and severe illness. Multiple isolates were more commonly associated with severe illness and single isolates were more common in illness of moderate severity.

Adolescent↗

PubMed2006

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Biosensing Techniques↗

PubMed1991

Etiology of childhood diarrhea in Beijing, China.

To determine the role of recently recognized enteropathogens in childhood diarrhea in China, 221 children with diarrhea and 108 controls seen at the Beijing Children's Hospital were studied during April and May 1989. Stools were examined for ova, parasites, and rotavirus, cultured for bacterial pathogens, and probed for enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteropathogenic adherence factor-positive (EAF+) E. coli. Pathogens were identified in 56.5% of children with diarrhea and 43.5% of controls (P = 0.04). Detection of enteropathogens was significantly greater in patients examined within 1 week of symptom onset (65%) than in patients examined later (39%; P = 0.01). ETEC was the most frequently detected pathogen in children with diarrhea, accounting for 20% of the cases. Other agents identified in patients included the following: salmonellae, 12%; rotavirus, 7%; EIEC, 7%; EHEC, 7%; members of the Aeromonas hydrophila group, 6%; EAF+ E. coli, 5%; Ascaris lumbricoides, 3%; shigellae, 3%; campylobacters, 2%; and Vibrio spp., 0.5%. The isolation rates of salmonellae (P = 0.02), EAF+ E. coli (P = 0.04), and mixed pathogens (P = 0.05) were significantly greater for diarrhea patients than for controls. Resistance to multiple antimicrobial agents occurred in 39% of the Salmonella isolates, 22% of the Aeromonas isolates, and 17% of the Shigella isolates. Multiresistant salmonellae (P = 0.05) and shigellae were recovered from diarrheal stools only. Ciprofloxacin, cefotaxime, and imipenem were the only agents tested to which all bacterial isolates were susceptible in vitro. These results suggest that both traditional and newly recognized agents are important causes of childhood diarrhea in Beijing and that therapy may be complicated by indigenous antimicrobial resistance.

Child, Preschool↗