Prognostic significance of hypertension and albuminuria for early mortality after acute myocardial infarction

The motile enterococci with the vanC gene have intrinsic low-level resistance to vancomycin, but have not been implicated in a nosocomial outbreak. We determined the colonization rate of motile enterococci in hospitalized and nonhospitalized patients. Perianal or stool specimens were cultured in Enterococcosel broth supplemented with 6 micrograms of vancomycin per mL. Rapid motility and pigment tests were performed on all enterococci isolated. A total of 82 motile and/or pigmented enterococci were isolated from 679 patients for a colonization rate of 12.1%. There were 43 Enterococcus gallinarum, 32 Enterococcus casseliflavus, 4 Enterococcus flavescens, and 3 Enterococcus mundtii identified. The E. gallinarum vancomycin MIC90 was 32 micrograms/mL and the E. casseliflavus vancomycin MIC90 was 8 micrograms/mL.     

Polyarthritis with perinuclear antineutrophil cytoplasmic antibody inaugurating microscopic polyangiitis. Report of a case

The prevalence of, and clinical risk factors associated with, vancomycin-resistant enterococcal colonization were investigated in patients suspected of having Clostridium difficile infection. Stools submitted for C difficile cytotoxin testing were screened for vancomycin-resistant enterococci (VRE). Isolates were speciated and characterized further by antibiotic susceptibility testing, DNA fingerprinting, and DNA:DNA hybridization for detection of specific vancomycin resistance genes. Of the 79 evaluable patients identified during a 3-month period, 16.5% were VRE-positive. The VRE isolates were genetically heterogeneous, although all carried the vanA gene. DNA fingerprinting data suggest that patient-to-patient transmission occurred, implicating colonized patients as potential reservoirs for VRE transmission. A positive C difficile cytotoxin assay and diabetes mellitus were the only identifiable risk factors associated with VRE colonization. Patients at risk for C difficile infection therefore may serve as reservoirs for VRE.     

The importance of urinary protein excretion during conservative management of severe preeclampsia

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.     

Effects of dopaminergic drugs on food- and cocaine-maintained responding. IV: Continuous cocaine infusions

We modified and optimized a new microplate hybridization assay to detect the varciella-zoster virus (VZV) PCR product, and studied cerebrospinal fluid (CSF) samples of 287 patients with meningitis, encephalitis or other neurological diseases or symptoms. Specific antibodies to VZV and reference antigens were determined by enzyme immunoassay from serum and CSF, they were then compared with clinical findings and with the results obtained by VZV-PCR using different detection methods for VZV-specific amplified DNA. VZV DNA was found in the CSF of 25 patients using the microplate hybridization assay and chemiluminescence detection for amplified DNA. All 25 CSF samples were also positive in Southern blotting. Among the patients, 10 had chickenpox, 4 had shingles, and 11 had no rash at all. The detection rate of VZV-specific DNA by microplate hybridization was 30% higher than that obtained by conventional agarose gel electrophoresis. In most patients the diagnosis was confirmed by demonstrating specific intrathecal antibody production to VZV but not to other viruses. These results indicate the presence of VZV in the central nervous system (CNS) in many patients with chickenpox or shingles, and even in patients without a rash. The microplate hybridization assay based on chemiluminescence detection improves considerably the detection rate of the VZV-PCR product compared to agarose gel electrophoresis and will add to the list of recognized VZV infections in the CNS. It is especially useful in cases where there is no cutaneous manifestation.     

Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.     

Early increases in ischaemic heart disease mortality dissociated from and later changes associated with respiratory mortality after cold weather in south east England

Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV+ patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae. The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV+ patients.     

Lipoid proteinosis of the oral mucosa: case report and review of the literature

High-level resistance (minimum inhibitory concentration, MIC > 1,000 micrograms/ml) to gentamicin (HLGR) in enterococci is common in Taiwan. In this study, we investigated the distribution of gentamicin resistance elements in enterococci isolated at National Taiwan University Hospital in a 1-year period, and also examined the transfer and the genetic variability of the resistance elements of different isolates. Among 109 isolates tested, 43 (39%) HLGR isolates were identified. HLGR was most common in Enterococcus faecium isolates (7/15, 47%), followed by Enterococcus faecalis (34/80, 43%), Enterococcus avium (1/5, 20%), and Enterococcus casseliflavus (1/9, 11%). To understand the mechanism of resistance transfer, four isolates of E. faecalis and five isolates of E. faecium showing HLGR were studied. Transfer of resistance markers to a plasmid-free recipient strain of E. faecalis JH2-7 was observed, with transfer frequencies ranging from 10(-2) to 10(-8). All of the transconjugants contained plasmids, with sizes ranging from 45 kb to larger than 70 kb. At least three plasmid patterns were observed on digestion with HaeIII. Hybridization with a probe specific for the aac6'aph2" gentamicin resistance gene confirmed that all of these HLGR isolates carried a Gm(r) determinant, though the hybridization patterns of the plasmids from E. faecalis and E. faecium were different. Although many similarities exist among enterococcal Gm(r) determinants, the results suggest heterogeneity may occur in the flanking regions of resistance elements.     

A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology

A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.     

DNA optical sensor: a rapid method for the detection of DNA hybridization

A DNA optical sensor system is proposed based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for rapid detection of DNA hybridization. Bacterial alkaline phosphatase (phoA) gene and Hepatitis B virus (HBV) DNA were used as target DNA. A biotinylated DNA probe was used to capture the target gene onto the streptavidin-coated magnetic beads and a calf intestine alkaline phosphatase (CAP)-labelled DNA probe was used for subsequent enzymatic chemiluminescence detection. The detection cycle was less than 30 min, excluding the DNA hybridization time, which was about 100 min. Both the phoA gene and HBV DNA could be detected at picogramme or femtomole level. No response signal was obtained when target DNA did not exist in the sample. Successive sample detection could be made by removing the magnetic field and a washing step.     

Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for vanA, vanB, vanC1, and vanC2 or vanC3 in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant Enterococcus faecium and E. faecalis (VRE). The predominant genotype from human clinical E. faecium isolates was vanB. The PCR van genotype was consistent with the resistance phenotype in all but six cases. One vanA E. faecalis isolate had a VanB phenotype, one vanB E. faecium isolate had a VanA phenotype, and four E. faecalis isolates were consistently negative for vanA, vanB, vanC1, and vanC2 or vanC3, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of vanD by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.     

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for various lengths of time (2-42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.     

Comparison between microwell and bead supports for the detection of human cytomegalovirus amplicons by sandwich hybridization

In this study, we compared the efficiency of capture DNA probes covalently bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene supports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times higher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes were optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target, with faster kinetics of hybridization obtained on beads than on microplates. Sensitivity of the hybridization assay was then determined with a nonisotopic method and the detection limit found was 30 amol of HCMV amplicons on both supports. HCMV DNA extracted from clinical samples were amplified by PCR. The resulting amplicons were then analyzed using the optimized sandwich hybridization assay discussed here. The results perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel.     

Proof of hepatitis A virus negative-sense RNA by RNA/DNA-hybrid detection: a method for specific detection of both viral negative- and positive-strand RNA species

The detection of hepatitis A virus (HAV) negative-strand RNA, which is synthesized during replication of the positive-strand RNA genome, proved to be difficult. We developed a method for the specific detection of HAV negative-strand RNA by RNA-DNA hybridization and luminescence detection using an anti-RNA:DNA hybrid antibody. This method, which is also applicable for the specific detection of positive-strand RNA, offers a simple, yet relatively rapid and certain means of detecting low amounts of RNA such as HAV negative-strand RNA. By using appropriate hybridization DNA probes, the method should be applicable for the detection of single-stranded RNA species of different viruses in general.     

Wound infection: a prospective study of 7519 operations

Wound infection was prospectively studied in 7,519 consecutive operations after preoperative classification as clean, clean-contaminated, and infected. The overall infection rate was 3.9 per cent. Clean, 3.2 per cent; clean-contaminated, 4.4 per cent; contaminated, 12.4 per cent; infected, 16.2 per cent. Wound infection was not seasonally related or dependent on changes in house staff. In clean cases, the predominant role of Staphylococcus aureus (37%) has been superceded by enterococci (44%). In clean-contaminated cases, enterococci (43.5%) were the most common, followed by Escherichia coli (40.0%). In contaminated wounds, E. coli was most common (40.0%). The infected case category grew mixed flora (E. coli, 82 per cent; enterococci, 54 per cent, and Pseudomonas aeruginosa, 43 per cent). Nosocomial organisms were important only in the contaminated (14%) and infected (43%) categories. Antibiotic therapy before cultures are available should include agents with activity against enterococci as well as S aureus, and E. coli in clean cases.     

Blood pH in the umbilical artery at birth: an analysis of data from patients delivered in Hesse between 1986 and 1989

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.     

Prognosis of operable squamous cell carcinoma of the esophagus. Relationship with clinicopathologic features and DNA ploidy

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Isolation of bacterial endotoxins from Pseudomonas putida 36 and Escherichia coli (vulgaris)

A prospective analysis of 69 patients who had been treated for nasopharyngeal carcinoma (NPC) by external radiotherapy was carried out. Biopsies from the posterior nasopharynx were performed and analyzed by in situ hybridization using an antisense Epstein-Barr Early RNA (EBER) radio-labelled riboprobe. None of the patients had evidence of disease in the nasopharynx. One patient was found to have nasopharyngeal carcinoma detected only by in situ hybridization. In the subsequent 18-month follow-up of these clinically- and biopsy-negative patients, only one patient developed relapse in the nasopharynx. In situ hybridization is a valuable tool for the detection of NPC and should be routinely available in histopathology laboratories where NPC is regularly diagnosed.