Efficacy of high-level disinfectants for reprocessing GI endoscopes in simulated-use testing.

BACKGROUND: There has been recent public concern regarding the adequacy of current practices for flexible endoscope reprocessing. High-level disinfection is defined by the Food and Drug Administration (FDA) as a minimum of 6-log reduction of mycobacteria under a worst-case scenario. Several agents are currently approved by the FDA, but published data on their relative efficacies against mycobacteria are lacking. The objective of this study was to determine the efficacy of these agents for high-level disinfection. METHODS: In simulated-use testing, video endoscopes (5 colonoscopes and 5 duodenoscopes) were each inoculated with 9.0 x 10(7) colony-forming units of Mycobacterium chelonae. Cleaning was performed by using a standardized protocol. Each endoscope was then subjected to chemical disinfection with Cidex (2.0% glutaraldehyde) at 20 degrees C for 20 minutes, Sporox (7.5% hydrogen peroxide) at 20 degrees for 30 minutes, and Steris 20 (0.2% peracetic acid) at 50 degrees C to 56 degrees C for 12 minutes using the Steris System 1 processor. Although not FDA-approved, tests were also conducted by using 70% isopropyl alcohol at 20 degrees C for 20 minutes. These results were compared with disinfection with ethylene oxide gas. All channels were sampled for M chelonae before and after manual cleaning and after disinfection. RESULTS: Cleaning alone resulted in an average log reduction of 3. Cidex, Sporox, Steris 20, ethylene oxide gas, and isopropyl alcohol, in combination with manual cleaning, each achieved a 6-log or greater reduction of the mycobacterial inoculum. No organisms were recovered from any channel after reprocessing with ethylene oxide and Steris 20. CONCLUSIONS: Commercially available high-level disinfectants are equally efficacious for reprocessing flexible GI endoscopes when used in conjunction with cleaning and in accordance with recommended guidelines.

High-level disinfection of gastrointestinal endoscopes: are current guidelines adequate?

OBJECTIVE: For a germicide to obtain a high level disinfection (HLD) claim, FDA requires demonstration of a 6-log reduction of mycobacterial inoculum under worst case conditions. The purpose of this study was to assess the adequacy of current guidelines for high level disinfection of GI endoscopes using alkaline glutaraldehyde in simulated-use testing. METHODS: Various gastrointestinal endoscopes were contaminated with Mycobacterium chelonae in 46 experiments. Quantitative cultures were obtained from each endoscope channel separately after each step: inoculation, standardized manual cleaning, immersion in 2% glutaraldehyde (Cidex) for 10, 20, or 45 min at room temperature, 70% isopropanol rinse, and drying. RESULTS: Manual cleaning alone achieved a 4-log reduction. After 10 min of glutaraldehyde exposure, but before alcohol rinse, two of 10 experiments failed to achieve a 6-log reduction. However, after alcohol rinse, all 10 experiments achieved HLD. After 20 min of glutaraldehyde exposure, but before alcohol rinse, one of 18 experiments failed to achieve a 6-log reduction. After alcohol rinse, all 18 experiments achieved HLD. After 45 min of glutaraldehyde exposure, but before alcohol rinse, one of 18 experiments failed to achieve a 6-log reduction. After alcohol rinse, all 18 experiments achieved HLD. Thus, if the entire reprocessing protocol including manual cleaning, glutaraldehyde exposure, alcohol rinse, and drying was taken into account, the required 6-log reduction of mycobacteria was achieved with a minimum of 10 min of glutaraldehyde exposure at room temperature. CONCLUSIONS: Current guidelines for high level disinfection using glutaraldehyde are appropriate. Alcohol rinse is a valuable adjunctive step for drying and for its bactericidal effects.

Successful reversal of Barrett's esophagus with multipolar electrocoagulation despite inadequate acid suppression.

BACKGROUND: Barrett's epithelium is a risk factor for esophageal cancer. In this study we attempted to reverse Barrett's epithelium in an acid-reduced environment by using multipolar electrocoagulation. METHODS: All patients had specialized columnar epithelium of at least 2 cm in length. Patients with dysplasia on successive screening examinations were excluded. Esophageal motility and 24-hour pH studies were performed before therapy and at 9 and 18 weeks. All patients received lansoprazole 30 mg twice a day regardless of pH study results. Multipolar electrocoagulation was applied to one side of the esophagus (2 to 3 cm per session) with the contralateral side serving as control. Biopsies were obtained from each 2 cm length at each endoscopy and reviewed by a single, blinded pathologist. At the 9-week evaluation of the treatment side, all patients elected to undergo multipolar electrocoagulation therapy for the control side. RESULTS: Twenty-seven patients completed the study: 21 men and 6 women, ages 33 to 81 years, length of specialized columnar epithelium 2 to 10 cm. Endoscopy at the 18-week follow-up showed normal mucosa (n = 16), residual small (< 3 mm) islands or tongue-shaped extensions of apparent specialized columnar epithelium (n = 7), untreated distal strips (< 5 mm) of apparent specialized columnar epithelium at the gastroesophageal junction (n = 3), and persistent specialized columnar epithelium (n = 1). Histologically, only 5 of 27 patients had residual specialized columnar epithelium at week 18. Of 16 patients with a normal endoscopic appearance, one still had specialized columnar epithelium on biopsy. While on lansoprazole, pH studies were normal in 15 of 26 patients at 9 weeks. Of 22 patients with histologic evidence of specialized columnar epithelium reversal, 10 had persistently abnormal 24-hour pH studies (pH < 4, 6.1% to 33.7% of total time). Four of five patients with residual specialized columnar epithelium on biopsy had persistent reflux. Side effects of multipolar electrocoagulation were transient, and treatment did not adversely alter motility patterns. CONCLUSIONS: Multipolar electrocoagulation therapy can induce re-epithelialization with normal squamous mucosa in most patients with specialized columnar epithelium and does not adversely affect esophageal motility. Adequate acid suppression is not mandatory for therapy to be effective in the short term.

Efficacy of various disinfectants in killing a resistant strain of Pseudomonas aeruginosa by comparing zones of inhibition: implications for endoscopic equipment reprocessing.

OBJECTIVE: Previous studies have shown that high-level disinfection of GI endoscopes may not be reliably achieved using glutaraldehyde at room temperature. In our laboratory, we have isolated a strain of Pseudomonas aeruginosa that is resistant to disinfection with glutaraldehyde. We compared the bactericidal activity of various disinfectants against this organism. METHODS: One hundred microliters of an overnight culture of this organism was spread onto blood agar plates. Twenty microliters of a disinfectant was placed on a sterile 7-mm filter paper, placed on the blood agar plate, and incubated overnight at 37 degrees C to determine the zone of inhibition for each disinfectant tested. Disinfectants included Cidex, Dispatch, Virahol, OMNI II, Lysol, IodoFive, Lysol I.C. Spray, and Chlorox. The zone of inhibition (i.e., clearing) roughly correlates with the bactericidal strength of the disinfectant. RESULTS: Compared with the glutaraldehyde-containing solution Cidex, the alcohol-containing disinfectants Lysol I.C. Spray and Virahol had the largest mean zones of inhibition (11.33 vs 20.60 and 20.55 mm; p = 0.0001). The hypochlorite compounds Chlorox (1:10 dilution) and Dispatch had mean zones of inhibition similar to that of Cidex (11.08 and 11.25 mm vs 11.33 mm; p = not significant). The phenolic compounds OMNI II and Lysol had mean zones of inhibition smaller than that of Cidex (10.50 and 10.35 mm vs 11.33 mm; p < 0.006), and the phosphoric acid and iodine-containing IodoFive had the smallest mean zone of inhibition (9.70 vs 11.33 mm; p = 0.0001). CONCLUSIONS: The alcohol-containing disinfectants had the largest zones of inhibition against resistant P. aeruginosa. These compounds may be more effective than glutaraldehyde for endoscopic equipment reprocessing.

Enhancing magnetic resonance images using water bags.

Fascial planes between tissues are separated by connective tissue, fat, and blood vessels. Magnetic resonance imaging displays surface anatomy and soft tissues. Our team has been successful in demonstrating brachial plexus nerves as a model of magnetic resonance anatomy. Radiologists have devised methods to increase the resolution of images by suppressing noise and increasing the sharpness of the image. We added water bags to a 0.3 tesla permanent magnet suppressing the noise and increasing the signal to image our patients. The images proved to be sharper.

DNA sequences which influence the selection and efficient utilisation of transcriptional start sites of a eukaryotic gene by RNA polymerase II in vitro and in vivo.

Experiments are described which probe the relationship between three sequence elements which make up the eukaryotic RNA polymerase II promoter. A cloned eukaryotic gene, from which the TATA-box and 400 base pairs of 5'-flanking sequence has been deleted, is still transcriptionally active in vivo (following its transfection into cultured mammalian cells) and in vitro. Deletion has appropriately positioned a cluster of five TATA box-like sequences upstream from multiple potential cap sites. Which cap sites are actually used can be predicted from the DNA sequence of TATA box-like sequences and their spatial relationship with respect to possible transcriptional start sites, although there appears to be some difference in cap site utilisation in vitro and in vivo. Data suggest that deletion has also removed "upstream" sequences which affect promoter function.

The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo.

We have studied how small changes in the distance between the TATA-box and cap site affect transcription of a eukaryotic gene in vitro and in vivo. The trout protamine gene TPG-3 [Gregory et al. (1982) Nucl. Acids Res. 10, 7581-7592] is a good model for such a study as it has (i) a consensus TATA-box 32 base pairs (bp) upstream from an A-residue which is the natural cap site (designated +1) (ii) two further A-residues at -5 and +5, providing alternative transcriptional start sites which are in significantly different sequence environments and (iii) a unique AvaII restriction site immediately downstream from the TATA-box which is ideal for the insertion or deletion of up to 3bp. Transcripts of the wild type and mutant genes were generated in vitro using a HeLa whole cell extract or 'in vivo' by transient expression following their transfection into HeLa cells. These 'spacer' mutations did not affect the efficiency of transcription of the gene in vitro but they did affect the selection of transcriptional start site both in vitro and 'in vivo'. Analysis of 5'-ends by S1-mapping and primer extension showed that the A-residue(s) selected are those which, by insertion or deletion, come to lie on the same face of the DNA double helix as the TATA-box, although the DNA sequence in the immediate vicinity of the potential start sites influences their utilisation. Comparison of the TPG-3 wild type transcripts in these experimental systems with natural mRNA suggests that cap site selection is more stringent in the developing trout testis.

The generation of a single nick per plasmid molecule using restriction endonucleases with multiple recognition sites.

Some restriction endonucleases generate a single-stranded nick at their recognition sequences in the presence of ethidium bromide (EtBr). This nick can then be extended to a single-stranded gap in which mutations can be introduced by a variety of techniques. To date, the templates used in these studies have largely contained a single recognition site for a given enzyme. Therefore, we have extended these studies to twelve enzymes for which multiple recognition sites exist in the template and show that, under appropriate conditions, one single-stranded nick is introduced per plasmid molecule.