Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis.

AIMS: The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. METHODS AND RESULTS: The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons. CONCLUSIONS: All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.

Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates.

AIMS: To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates. METHODS AND RESULTS: Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (µl) of each culture suspension (1 × 10(8) CFU ml(-1) ) were added to 20 µl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates. CONCLUSIONS: Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities ≥ 97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.

Imaging doses from the Elekta Synergy X-ray cone beam CT system.

The Elekta Synergy is a radiotherapy treatment machine with integrated kilovoltage (kV) X-ray imaging system capable of producing cone beam CT (CBCT) images of the patient in the treatment position. The aim of this study is to assess the additional imaging dose. Cone beam CT dose index (CBDI) is introduced and measured inside standard CTDI phantoms for several sites (head: 100 kV, 38 mAs, lung: 120 kV, 152 mAs and pelvis: 130 kV, 456 mAs). The measured weighted doses were compared with thermoluminescent dosimeter (TLD) measurements at various locations in a Rando phantom and at patients' surfaces. The measured CBDIs in-air at the isocentre were 9.2 mGy 100 mAs(-1), 7.3 mGy 100 mAs(-1) and 5.3 mGy 100 mAs(-1) for 130 kV, 120 kV and 100 kV, respectively. The body phantom weighted CBDI were 5.5 mGy 100 mAs(-1) and 3.8 mGy 100 mAs(-1 )for 130 kV and 120 kV. The head phantom weighted CBDI was 4.3 mGy 100 mAs(-1) for 100 kV. The weighted doses for the Christie Hospital CBCT imaging techniques were 1.6 mGy, 6 mGy and 22 mGy for the head, lung and pelvis. The measured CBDIs were used to estimate the total effective dose for the Synergy system using the ImPACT CT Patient Dosimetry Calculator. Measured CBCT doses using the Christie Hospital protocols are low for head and lung scans whether compared with electronic portal imaging (EPI), commonly used for treatment verification, or single and multiple slice CT. For the pelvis, doses are similar to EPI but higher than CT. Repeated use of CBCT for treatment verification is likely and hence the total patient dose needs to be carefully considered. It is important to consider further development of low dose CBCT techniques to keep additional doses as low as reasonably practicable.

Managing aggression in a psychiatric hospital using a behaviour plan: a case study.

This paper focuses on the critical role of nursing in implementing a behaviour plan in a psychiatric hospital. The plan was implemented with a 40-year-old man with a long history of aggression towards others and self. The study used a single-subject research design with baseline and intervention phases (AB Design). Data were collected on (1) frequency of incidents of aggression towards others and self; (2) use of restrictive interventions to manage aggression (i.e. restraints, pro re nata medication, 1:1 special observation); and (3) frequency of staff injury. The data show a decrease in frequency of aggression towards others and self, a concurrent reduction in the use of restrictive interventions to manage aggression, and a decrease in incidents of staff injury. The behaviour plan helped staff maintain a safe and therapeutic milieu. The behaviour plan has given the patient an opportunity to learn positive replacement behaviours and skills, and the opportunity eventually to leave the hospital to live in a less restrictive community home.

Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping.

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.

[Cyclic vomiting with ketosis as a cause of acute kidney dysfunction: own clinical experience]. / Wymioty cykliczne z ketonemia jako przyczyna ostrych zaburzen czynnosci nerek--obserwacje wlasne.

The aim of the study was to evaluate renal activity during cyclic vomiting with ketosis. The clinical material was obtained from 50 cases of children hospitalized in Department of Pediatrics Military Medical University within 1993-1999 what makes about 1% of all patients. The examined group consisted of 26 boys (52%) and 24 girls (48%). Three of the children were repeatedly hospitalized (3 to 8 times) because of acetonemic vomiting. The special attention during the laboratory studies was paid to evaluation of renal activity. Vomiting with ketosis were associated with temporary kidneys acute dysfunction in 46% of cases. In 98% of cases the parenteral hydration was necessary. Ketonemic vomiting with kidneys dysfunction was observed mainly with the children in pre-school age.

Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk.

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

Diffraction-based cell detection using a microcontact printed antibody grating.

An optical detector has been fabricated that is specific for targeted bacterial cells, by stamping an antibody grating pattern on a silicon surface. The antibody grating alone produces insignificant optical diffraction, but upon immunocapture of cells, the optical phase change produces a diffraction pattern. This technique eliminates much of the surface modifications and the secondary immunochemical or enzyme-linked steps that are common in immunoassays. Microcontact printing provides an alternative to previously reported photolithographic-mediated antibody patterning processes and uses a photolithographic process simply to produce the elastomeric stamp. We have stamped antibodies directly onto clean native oxide silicon substrates with no other chemical surface treatments. Direct binding of the antibodies to the silicon occurs in a way that still allows them to function and selectively bind antigen. The performance of the sensor was evaluated by capturing Escherichia coli O157:H7 cells on the antibody-stamped lines and measuring the intensity of the first-order diffraction beam resulting from the attachment of cells. The diffraction intensity increases in proportion to the cell density bound on the surface.

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider.

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10 g of ground beef when samples were enriched in modified EC broth for 7 h at 37 degrees C. The minimum detectable number of cells for the apple cider samples was calculated to be approximately 0.5 cfu ml-1. The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.

Sensitometric and image quality performance of "rapid" intraoral film processing techniques.

A number of products are available to dentists for the rapid production of intraoral radiographic films but there is little information on their relative merits. This study evaluated the performance of five "rapid" film processing products commonly used by British dentists in comparison with standard Kodak manual processing. Two Perspex contrast-detail test objects were made in order to investigate threshold contrast. Film speed, film gradient, limiting resolution and threshold contrast results are presented. Rapid processing systems possessed lower film speed when compared with Kodak Ektaspeed film and standard Kodak processing. The speed of E-speed film was found to be lower than that of D-speed film when used with Westone "Rapid X-ray" processing. Overall image quality was generally similar for all film/processing combinations evaluated, with the exception of Nix QP which gave markedly poorer image quality.

An in vitro comparison of 10 radiographic methods for working length estimation.

The aim of this in vitro study was to assess the efficacy of 10 currently available methods of producing radiographic images, two conventional and eight rapid, in working length estimation. Thirty extracted teeth comprising 42 root canals were mounted in blocks of poly(methylmethacrylate) and access cavities prepared. A size 15 Hedstrom file was inserted into each root canal and sealed into position. Each tooth was imaged by 10 methods: combinations of conventional (D- and E- speed) film processed with conventional manual chemistry of two proprietary brands of rapid processing chemistry (Westone Rapid Dental and Kodak 'Rapid Access'), Super X30 packet processed film, Hanshin Hi-Fi and Nix NF45-100 films processed using their respective manufacturers' monobath solutions, and the Sens-A-Ray digital X-ray system. Comparisons of length of file visible were made between the D-speed films (conventionally processed using Kodak chemistry) and the nine other radiographic methods. No significant differences in percentage length of file seen were found between D-speed film processed with conventional chemistry and E-speed film processed with the same chemistry or between D-speed film processed with with conventional chemistry and six of the rapid imaging systems studied. The percentage length of the file visible was significantly less for Sens-A-Ray (P=0.02) and for Nix NF45-100 film (P<0.005) when compared with D-speed film processed with conventional chemistry. This difference in file length is probably not of clinical importance for the Sens-A-Ray images but may be so for the Nix images.

Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified polymorphic DNA patterns.

Food and clinical isolates of Listeria monocytogenes recovered from four different outbreaks of listeriosis were analyzed by their PCR-based randomly amplified polymorphic DNA (RAPD) patterns to verify their causal relationships. The generation of DNA fingerprints by PCR-based RAPD analysis is a fast and sensitive method for the epidemiological tracking and identification of bacteria implicated in food poisoning outbreaks. The L. monocytogenes strains used in the study were obtained from the following four outbreaks: California, 1985, Mexican-style cheese; Canadian Maritime Provinces, 1981, coleslaw; Canada, 1989, brie cheese; and Canada, 1989, alfalfa tablets. RAPD profiles were generated by using random 10-mer primers for at least one food and one clinical isolate recovered from each outbreak. Identical profiles for 20 different primers were observed for each pair of food and clinical isolates from two of the four outbreaks. Isolates from the outbreak involving alfalfa tablets exhibited identical patterns for 19 primers; however, primer OPA-1 produced one additional 1.8-kb fragment, designated OPA-1-1.8, that was found in the food isolate but not in the corresponding clinical isolate. Hybridization analysis revealed that the absence of the OPA-1-1.8 polymorphic fragment in the clinical isolate was due to a deletion of at least 1.8 kb. Loss of the OPA-1-1.8 polymorphic fragment could not be induced by infective passage of the L. monocytogenes isolate from the alfalfa tablet through a mouse or by growth of this isolate under selective conditions. This suggests that the isolate recovered from the food was not identical to the isolate recovered from the patient. The ability to produce unique RAPD patterns allows for the discrimination between isolates even if they are of the same serotype and multilocus enzyme electrophoretic type.

Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants.

Two outbreaks of epizootic listerial encephalitis, one in sheep and one in goats, were investigated through pathology, microbiology, and DNA amplification-based techniques. Efforts were made to survey the diversity of Listeria monocytogenes strains in the silage consumed by affected animals and to verify the causal relationship between silage and disease outbreak. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue samples. Random amplified polymorphic DNA patterns revealed two distinct L. monocytogenes strains, one of which was identical to the sheep brain isolate, in the silage associated with the outbreak in sheep. Three brain isolates and one silage isolate, all of which had different random amplified polymorphic DNA patterns, were found in the outbreak involving goats. All isolates from both outbreaks were indistinguishable in an in vitro assay for cell-to-cell spread and growth in macrophages. All brain isolates from the goat outbreak had identical intracellular ActA patterns, which were different from the pattern for the silage isolate. While the sheep brain isolate had an ActA pattern different from that of the corresponding silage isolate, the patterns for the brain isolates from the two outbreaks were not identical. This survey demonstrates the diversity of L. monocytogenes in silage and suggests the existence of one or more selective processes by which certain strains are more prone to give rise to disease.

Technical note: measurement of computed tomography scanner slice widths.

The slice width of a computed tomography scanner can be considered as the full width at half maximum (FWHM) of the slice dose profile. A method is described which uses dental film to image the slice and allows the FWHM of the dose profile to be determine directly from the digitization and analysis of the film image.

Panel analysis of the moderating effects of commitment on job satisfaction, intent to quit, and health following organizational change.

The authors examined the moderating effects of organizational commitment on the relationship of stress with job satisfaction, intent to quit, and health during organizational turmoil. Panel data were provided by hospital employees surveyed before and after a major divisional consolidation. Findings indicated that commitment buffered the relationship between stress and job displeasure (a canonically derived variate combining residualized job dissatisfaction, intent to quit, and irritation). Stress increased job displeasure only when commitment was low.

Use of 'rapid' processing techniques by a sample of British dentists.

Knowledge of current practices amongst general dental practitioners (GDPs) is important in planning postgraduate and undergraduate teaching curricula and in ensuring the relevance of clinical research. A number of methods are available to dentists for the rapid production of intraoral radiographic images, including concentrated and monobath chemistry. No current data exists about the use of such 'rapid' systems in the UK. The aim of this study was to obtain this data, including the relative popularity of the different systems. A questionnaire on 'rapid' processing of radiographs was distributed to all 855 GDPs in four Family Health Services Authorities in North West England. Responses were received from 326 GDPs (38.1%). 29.4% of GDPs used some kind of 'rapid' system, with 'packet processed' films being the most popular. 6.1% of all GDPs used 'rapid' systems routinely, while the remainder used them for specific situations, typically emergencies and endodontics.

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.

Quantitative assessment of a new dental imaging system.

The "Radiovisiography" dental imaging unit (Trophy UK Ltd) is a digital system using an intensifying screen and charge-coupled device in an intra-oral sensor. This paper presents a description of the system and an assessment of the original model in terms of patient dose (relative to film systems), resolution (limiting resolution and modulation transfer function), distortion and image noise (amplitude). The system does offer the possibility of reduced patient exposure and minimal distortion, although resolution and latitude are inferior to standard dental film.