Radiofrequency transmission to monitoring devices in the operating room: a simulation study.

We evaluated radiofrequency (RF) transmission to various monitoring devices using circuits that simulated potentially hazardous conditions for patients in the operating room. Right heart ejection fraction (REF) pulmonary artery catheters, transesophageal atrial pacing stethoscopes, and temperature-sensing esophageal stethoscopes were subjected to RF transmission from an electrosurgery unit. Peak voltage and spark intensity were measured in circuits between the electrocautery dispersive pad and conductive elements of the various medical devices. All monitoring devices with an exposed conductive surface were found to have induced voltages and even spark generation. The ranking for peak voltage from least to most was as follows: disrupted esophageal stethoscope (620 volts), the transesophageal pacemaker (640 volts), and the REF pulmonary artery catheter (PAC) (680 volts). Peak voltage measurements of the REF PAC significantly decreased from 388 +/- 23 to 142 +/- 22 volts (P < 0.0001, Student's t-tests) in a fluid medium compared to air. In a fluid medium, peak voltage significantly decreased from 142 +/- 22 to 85 +/- 15 volts (P < 0.0001, Student's t-tests) when the REF PAC was connected to the cardiopulmonary monitor.

Use of a batch-stirred reactor to rationally tailor biocatalytic polytransesterification.

Despite favorable thermodynamics, high-molecular weight and low-dispersity polyesters are difficult to synthesize biocatalytically in organic solvents. We have reported previously that the elimination of solvent can improve the kinetics and apparent equilibrium significantly (Chaudhary et al., 1997a). We now present the design and use of a batch-stirred enzyme reactor to control the biocatalytic polymerization. Using the reactor, polyester having a molecular weight of 23,400 Da and a polydispersity of 1.69 was synthesized in only 1 h at 60 degrees C. Additional factors like enzyme-deactivation kinetics, enzyme specificity, and initial exothermicity were investigated to develop a better understanding of this complex reaction system.

In situ hybridization in paracoccidioidomycosis.

In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence. When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled. When the complementary probe was used, labelling was higher, reaching the 3% level.

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA.

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates.

The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.

Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA.

Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.

DNA sequence resembling vanA and vanB in the vancomycin-resistant biopesticide Bacillus popilliae.

The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

Assessment of invasion frequencies of cultured HEp-2 cells by clinical isolates of Helicobacter pylori using an acridine orange assay.

AIMS: Recent studies suggest that Helicobacter pylori is an invasive enteropathogen. However, the efficiency with which this pathogen invades mammalian cells remains unknown. Therefore, this study was designed to investigate the invasion frequencies of HEp-2 cells by clinical strains of H pylori. METHODS: An acridine orange assay and cultured HEp-2 cell monolayers were used to determine the HEp-2 cell penetration frequencies of 17 clinical isolates and one American Type Culture Collection (ATCC) strain of H pylori, and single clinical strains of Yersinia enterocolitica, Shigella flexneri, and a non-invasive ATCC Escherichia coli strain. RESULTS: The acridine orange assay demonstrated that invasion frequencies of HEp-2 cells by all H pylori isolates were significant and, in most instances, exceeded those for the S flexneri strain and equalled those for the Y enterocolitica strain. The assay also showed that internalised H pylori organisms remained viable for at least six hours, the maximum time that bacteria and HEp-2 cells were co-incubated. CONCLUSIONS: These results may have important implications for treatment and prevention strategies for this gastric pathogen. Furthermore, the acridine orange assay may be useful for assessing, in vitro, the ability of conventional and newer antibiotics, alone or in combination, to kill intracellular H pylori organisms.

DNA sequence variation within vanA, vanB, vanC-1, and vanC-2/3 genes of clinical Enterococcus isolates.

We studied the DNA sequence variation of van genes of 34 isolates of Enterococcus spp. The isolates containing the vanB gene exhibited between 0 and 41 base pair changes per 801 bp studied when the vanB sequences were compared to that of the reference strain Enterococcus faecalis V583. The isolates carrying the vanC-2 gene exhibited between 0 and 23 base pair changes per 346 bp studied when the vanC-2 sequences were compared to that of the reference strain E. casseliflavus ATCC 25788. Little variation was noted in the vanA and vanC-1 genes.

Recombinant Mycobacterium tuberculosis KatG(S315T) is a competent catalase-peroxidase with reduced activity toward isoniazid.

The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.

Molecular detection and identification of Paracoccidioides brasiliensis.

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

Use of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis to detect a point mutation in the catalase-peroxidase gene (katG) of Mycobacterium tuberculosis.

We have previously reported that a significant percentage (44%) of isoniazid-resistant Mycobacterium tuberculosis strains carry an arginine to leucine mutation in codon 463 (R463L) in the catalase-peroxidase gene (katG). For the current study, we compared the utility of one mutation screening method, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, with a reference method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to detect this mutation. The PCR-SSCP method detects mutations by electrophoretic mobility shifts of single-stranded DNA in nondenaturing polyacrylamide gels. The RFLP method detects a loss in an Mspl restriction site which occurs when the R463L is present. Eighty one M. tuberculosis strains, including the wild type strain H37Rv, with isoniazid susceptibility in the range < 0.12 to > 32 micrograms ml-1 were evaluated. The results for the PCR-SSCP method were in complete agreement with the PCR-Mspl RFLP reference method. Of 81 M. tuberculosis strains analysed, 13 showed mobility shifts by the PCR-SSCP method and all of those strains carried the R463L as detected by the PCR-Mspl RFLP method. All of the remaining 54 strains had PCR-SSCP and PCR-Mspl RFLP results identical to the wild type (R463) M. tuberculosis strain, H37Rv. It is concluded that the described PCR-SSCP is a reliable method for screening M. tuberculosis strains for the katG R463L mutation.

Prospective evaluation of the utility of molecular techniques for diagnosing nosocomial transmission of multidrug-resistant tuberculosis.

OBJECTIVE: To compare molecular techniques with conventional diagnostic methods for evaluating nosocomial transmission of multidrug-resistant tuberculosis (MDR-TB). DESIGN: We conducted a 12-week postexposure inception cohort study of health-care personnel who had been exposed to a patient with MDR-TB. MATERIAL AND METHODS: In addition to baseline and follow-up tuberculin skin tests and chest roentgenography, weekly pulmonary specimens were evaluated by (1) auramine-rhodamine fluorescent staining, (2) culture for mycobacteria, and (3) polymerase chain reaction (PCR) to amplify IS6110, a nucleic acid insertion sequence unique to the Mycobactrium tuberculosis complex. RESULTS: The index patient's isolate of M. tuberculosis showed a mutation in codon 531 of the RNA polymerase beta subunit (rpoB) gene of M. tuberculosis, which is associated with rifampin resistance and considered a marker for this MDR-TB strain. All pulmonary and gastric specimens from study participants had negative auramine stains and cultures for mycobacteria, One person, however, had separate specimens with repeatedly positive PCR results for IS6110 sequences, but the specimens contained a wild-type M. tuberculosis rpoB codon 531 dissimilar from the index patient's strain. CONCLUSION: Although both molecular and conventional testing showed that no exposed person was infected with the MDR-TB strain, molecular test results were available sooner and seemed more sensitive for detecting M. tuberculosis in one exposed person, presumably in a preinfection or "colonized" stage. Molecular methods provided information that helped distinguish this person's M. tuberculosis strain from the index patient's MDR-TB strain. Additional prospective studies should assess the value of these molecular techniques in similar clinical settings.

Molecular probes for diagnosis of fungal infections.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance.

The complete catalase-peroxidase (katG) gene DNA sequence was determined for 15 strains of Mycobacterium tuberculosis with a wide range of susceptibility to isoniazid. Five of 9 strains with isoniazid MICs > or = 1.0 microgram/mL had one or more missense mutations and all 5 strains had a common G-->T transversion in codon 463, causing the replacement of arginine with leucine and the loss of an NciI or MspI restriction site. None of 6 strains with an isoniazid MIC < 1.0 microgram/mL had mutations affecting codon 463. Restriction analysis of 43 strains with isoniazid MICs > or = 1.0 microgram/mL showed that 19 (44.2%) had lost the NciI-MspI restriction site at the locus of codon 463 while only 1 of 32 strains with isoniazid MICs < or = 1.0 microgram/L had this restriction polymorphism. These results indicate that the mutation arginine-->leucine in codon 463 of the catalase-peroxidase gene occurs in a significant fraction (44.2%) of M. tuberculosis strains with isoniazid MICs > or = 1.0 microgram/mL.

A comparison of 16S ribosomal DNA sequences from five isolates of Helicobacter pylori.

Other workers have found that clinical isolates of Helicobacter pylori exhibit very extensive DNA sequence polymorphisms when they are examined by ribotyping or some other genomic sequence characterization technique. In fact, it is rare to find similar clones, much less identical ones, among isolates. We found that the levels of divergence between the 16S ribosomal DNA sequences of individual organisms and the consensus sequence of the five isolates which we examined ranged from 0.2 to 0.5%. In contrast, other workers have shown that levels of divergence between the 16S ribosomal DNA sequence of H. pylori and the 16S ribosomal DNA sequences of four other Helicobacter species range from 2.7 to 8.0%. Our results show that the H. pylori 16S ribosomal DNA is not very polymorphic and support the conclusion that H. pylori is a unique species.

Spaceflight results in reduced mRNA levels for tissue-specific proteins in the musculoskeletal system.

The purpose of the present study in growing rats was to investigate the effects of short-term spaceflight on gene expression in bone and muscle and on cortical bone histomorphometry. Two experiments were carried out; Physiological Systems Experiments 1 and 2 were 4- and 10-day flights, respectively. Radial bone growth in the humerus was unchanged during the 4-day flight and decreased during the 10-day flight. Expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase was unchanged in biceps, calvarial periosteum, and long-bone periosteum after spaceflight. Similarly, no changes in ribosomal RNA levels were observed in long-bone or calvarial periosteum after spaceflight. In contrast, spaceflight decreased steady-state mRNA levels for actin in muscle (4-day flight). Osteocalcin (both spaceflights) and the prepro-alpha 2[I] chain of type I precollagen (10-day flight) mRNA levels were decreased in long-bone and calvarial periosteum after spaceflight. These results indicate that the effects of spaceflight on the musculoskeletal system include decreased expression of some muscle- and bone-specific genes as well as decreased bone formation. Interestingly, detectable reductions in gene expression for bone matrix proteins preceded histological evidence for decreased bone formation.

Temperature, pH and agitation rate as dissolution test discriminators of zofenopril calcium tablets.

Comparative in vitro dissolution studies were performed on several tablet batches of zofenopril calcium, an ACE inhibitor, to determine if they could be differentiated on the basis of their release rates. The samples included six batches produced at Site 1 and one batch produced at Site 2. Using regular dissolution conditions (USP paddle method at a 50-rpm agitation speed in phosphate buffer, pH 7.5, at 37 degrees C), release rates of all the tablet batches were similar. By independently altering one of the dissolution test parameters, either a lower pH or a slower agitation rate, discrimination between the Site 1 and Site 2 tablets was enhanced. Discrimination was only slightly enhanced when a lower dissolution medium temperature was used. Tablets made from different polymorphs of zofenopril calcium could not be differentiated by their dissolution profiles, even with the more discriminating conditions. The dissolution profiles of certain other zofenopril calcium tablets (including film-coated vs uncoated tablets, and tablets made with micronized vs unmicronized drug particles) were indistinguishable using a 50-rpm agitation rate, but they could be clearly differentiated using a 40-rpm agitation rate.

Gene identification by arrested primer extension.

This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identified by the length of the extension products on gel electrophoresis. This simple procedure eliminates the much-demanding process of sequencing. We term this process Arrested Primer Extension (APE). As a demonstration of the feasibility of this method, we have used APE to speciate a known set of cultured mycobacteria. There should be many other applications of this method.