Quantitative evaluation of prion inactivation comparing steam sterilization and chemical sterilants: proposed method for test standardization.

Prions are notoriously resistant to inactivation. To prevent accidental transmission of variant Creutzfeldt-Jakob disease (vCJD), various decontamination procedures have been adopted for re-usable medical devices by the authorities of countries at risk. As the vCJD agent in humans has a wide tissue distribution, practical methods of prion decontamination urgently need to be standardized, as do other sterilization and disinfection procedures (European Committee for Standardization). This article proposes a method using a quantitative murine model, combining observations of the decrease in the infection rate, the increase in the incubation period and a simultaneously performed chemical protein fixation control. In terms of practical application, autoclaving at 134 degrees C for 18 min or 121 degrees C for 30 min, and 1N sodium hydroxide for 15 min reduced the transmission of infectivity by a factor of at least 10(6). Partial efficacy can also be identified by the methodology, particularly for liquid cold sterilants such as glutaraldehyde and peracetic acid solutions.

[Hospital practice and prion risks]. / Pratiques hospitalières et risque Prion.

Procedures applicable in France for the prevention of prion diseases were first implemented in 1995, resulting from the threat of an epidemic extension of Creutzfeldt-Jakob Disease (CJD) following contamination resulting from the use of extracted growth hormone. It was found later that the bovine disease could infect humans via foodstuffs, and the human variant of the disease (v-CJD) transmissible through lymphoid formations was described in 1996. This led to generalizing precautions to a larger number of medical interventions, taking into account the risk for a population more broadly exposed to contamination. The principles for managing these new risks are described, as well for the use of medical devices or in patient as pathology laboratories.

Detection of Mycobacterium tuberculosis complex by PCR in hospital air samples.

Tuberculosis remains a health problem in many developed countries. After the development of a sedimentation method, a semi-quantitative approach for bioaerosol monitoring of Mycobacterium tuberculosis based on polymerase chain reaction (PCR) was adapted for direct detection in air. Bovine serum albumin was added to override airborne environmental particle PCR inhibitors. The method gave positive results in hospital rooms where tubercular pneumonia patients were hospitalized during the first days after their diagnosis.

Importance of cleaning for reprocessing endoscopes and thermolabile sterile medical devices: French use and regulations.

In France, endoscope maintenance regulations present some particularities in terms of the definitions and texts referring to the disinfection and sterilization of medical devices, with respect to the prion risk. The main measures specified are a double cleaning prior to disinfection, with some stipulations concerning the procedure itself, such as time limits, duration and rinsing. The use of aldehydes in cleaning products is prohibited and it is recommended that peracetic acid, or any chlorinated product, be used at the disinfection phase. For machines, the recycling of detergents or disinfectants is prohibited, and traceability procedures are mandatory. The French Agency for Safety of Health Products (AFSSAPS) is committed to providing standards that prevent any undesirable consequences for the patient, the operator or the equipment. All these measures will be described in a 'user's guide' intended for medical care units, to be released by the National Technical Committee on Nosocomial Infections (CTIN).

Inactivation of prions in daily medical practice.

The danger of transmission of non conventional agents such as prions constitutes a risk that is difficult to evaluate as the actual understanding of the infectious mechanism is minimal. The efficacy of each procedure is evaluated via submitting infectious particles mixed with cellular products to inactivating treatments, and then by inoculating animals with fractions of the preparation. With these protocols, some ineffective treatments can be identified (for example, soaking in aldehyde), and three reference treatments are recommended by the WHO: soaking in 1 N sodium hydroxide (1 h, 20 degrees C), soaking in 12.5% bleach (1 h, 20 degrees C) and steam sterilization in autoclave (134 degrees C-138 degrees C, 18 min). Universal precautions of asepsis and hygiene must be applied, especially in anatomo-pathological laboratories and for the re-use of medical devices in neurosurgery or in ophthalmology.

Flourescence-assay on traces of protein on re-usable medical devices: cleaning efficiency.

The cleaning of re-usable medical devices before disinfection or sterilization is recognized as being an essential phase. Detection of residual proteins can be used to validate the process, provided a sufficiently sensitive method is employed. A fluorescent method is presented, using orthophtalaldehyde (OPA) bound to N,N dimethyl-2-mercaptoethylammonium, to demonstrate the presence of amino acids on a medical device following cleaning. The sensitivity of this method (10-5 g/l) was assessed and the applicability of this detection technique is verified, using three types of carriers (steel blades, glass tubes or ceramic penicylinders), three types of contaminants (yeast extract, bovine albumin with native sheep's blood and formaldehyde fixed fibrin). In this context, studies involving formaldehyde-fixed fibrin are more sensitive and are to be recommended.

Investigation of an outbreak of wound infections due to Alcaligenes xylosoxidans transmitted by chlorhexidine in a burns unit.

Alcaligenes xylosoxidans, an environmental gram-negative bacillus, was isolated within a 1-month period from six patients in a pediatric burns unit. Twelve isolates were studied, one from each of the six patients (five from wound cultures and one from a blood culture) and one from each of six contaminated atomizers containing chlorhexidine diluted to 600 mg/l. The biochemical and susceptibility patterns of all the isolates were similar, and their DNA enzyme restriction patterns were identical. The epidemic strain of Alcaligenes xylosoxidans was probably introduced into the atomizers during handling of the diluted solution, which failed to eliminate it.

[Disinfection and sterilization of reusable devices]. / Désinfection et stérilisation du matériel réutilisable.

Official of quality assurance requirements for the disinfection and sterilization of reusable medical devices in health care facilities has been reminded recently. These requirements concern good manufacturing practices, essential to avoid adverse events. Identification of non critical and invasive devices, protocols for pre-disinfection, cleaning, high level disinfection and sterilization for thermosensitive materials must be evaluated. Monitoring and process control of these operations are defined by French regulations and European standards, and every health professional has to become aware of these limits.

Evidence of bacterial biofilm in tubing from hydraulic pathway of hemodialysis system.

Biofilms consist of microorganisms immobilized at a substratum surface embedded in an organic polymer matrix of bacterial origin. Tubing drawn from the fluid pathways within dialysis machines of various models were investigated for biofilm. Scanning electron microscopy (SEM), performed on approximately 2 cm2 samples of the tubing inner surfaces revealed that the inner surfaces of the tubing were covered with biofilms consisting of numerous deposits and glycocalix at different stages of formation with components containing bacteria and algae. Evaluations of biomass were performed from tubing sections of various lengths and inner diameters put in tubes containing water for injection and immersed in an ultrasound washtub for 1 h to ensure sloughing of the biofilm. Living bacteria were identified by plating on nutrient agar media and incubation for 48 h at 37 degrees C. Epifluorescent stains were used for the total bacteria count. Lipopolysaccharide levels were determined by the endotoxin activity measurements. Polyoside contents were determined by the colometric method, and the chemical oxygen demand was measured to evaluate the amount of organic substance. Biofilms detached from tubing samples drawn from the water path, bicarbonate path, and fresh dialysate path within dialysis machines contained approximately 1.10(3)-1.10(6) total bacteria/cm2, yet only some living bacteria were found. Endotoxin levels ranged from 1 to 12 EU/cm2. In contrast in the dialysate fluid, no bacteria were found, and the endotoxin content was under the detection level of the method. The polyoside content and chemical oxygen demand of the biomass ranged from 11 to 83 microg/cm2 and from 53 to 234 mg/cm2, respectively. It is concluded that a germ- and endotoxin-free dialysate does not exclude the risks and hazards of bacteria and endotoxin discharge from biofilm developed on the fluid pathway tubing, acting as a reservoir for continuous contamination, and efforts in the optimization of cleaning and disinfection procedures used for hemodialysis systems should aim to detach and neutralize biofilm when necessary.

Study protocol: stability of morphine injected without preservative, delivered with a disposable infusion device.

BACKGROUND AND OBJECTIVE: Morphine hydrochloride, a major analgesic drug, is being increasingly administered using portable disposable infusion devices. The objective of this study was to investigate the stability of morphine in such a system at two concentrations (2.50 and 5.00 mg/ ml) over a 30-day period. METHOD: High-performance liquid chromatography of stored morphine solutions. RESULTS: The best stability was observed with disposable infusion devices filled with a morphine solution containing sodium metabisulphite as a preservative. No breakdown products were detected after 1 month of storage at room temperature, in light or darkness. On the other hand, 2.50 and 5.00 mg/ml morphine solutions without sodium metabisulphite, stored in the infusion device led to the formation of 0.205% and 0.235% of pseudomorphine, respectively, after 6 days of storage in the light, and 1.50% and 0.94% after 30 days storage. CONCLUSION: Morphine hydrochloride solutions stored in disposable infusion devices degraded very slowly, particularly when preserved with sodium metabisulphite. The solutions are stable over 5 days, the maximum period of storage normally required when using disposable infusers.

Planetary protection program for Mars 94/96 mission.

Mars surface in-situ exploration started in 1975 with the American VIKING mission. Two probes landed on the northern hemisphere and provided, for the first time, detailed information on the martian terrain, atmosphere and meteorology. The current goal is to undertake larger surface investigations and many projects are being planned by the major Space Agencies with this objective. Among these projects, the Mars 94/96 mission will make a major contributor toward generating significant information about the martian surface on a large scale. Since the beginning of the Solar System exploration, planets where life could exist have been subject to planetary protection requirements. Those requirements accord with the COSPAR Policy and have two main goals: the protection of the planetary environment from influence or contamination by terrestrial microorganisms, the protection of life science, and particularly of life detection experiments searching extra-terrestrial life, and not life carried by probes and spacecrafts. As the conditions for life and survival for terrestrial microorganisms in the Mars environment became known, COSPAR recommendations were updated. This paper will describe the decontamination requirements which will be applied for the MARS 94/96 mission, the techniques and the procedures which are and will be used to realize and control the decontamination of probes and spacecrafts.

Bactericidal activity of beta-lactams and amikacin against Haemophilus influenzae: effect on endotoxin release.

Ampicillin or cefotaxime, alone or in combination with amikacin, were tested at levels achievable in CSF for bactericidal activity against eight clinical isolates of Haemophilus influenzae serotype b. Endotoxin release was determined by the limulus amoebocyte lysate test and by macrophage tumour necrosis factor production for each beta-lactam antibiotic, alone and in combination with amikacin. Accelerated killing was observed when amikacin was added to ampicillin or cefotaxime; however, the additional antibiotic-induced bacterial lysis observed after the addition of amikacin to beta-lactam antibiotics was not associated with an increase in endotoxin release.

A bacterial biofilm in a hemodialysis system. Assessment of disinfection and crossing of endotoxin.

Bacterial contamination may cause several adverse reactions, including pyrogenic consequences during hemodialysis. Some disorders, such as skin flush, pruritus, and other anaphylactoid reactions, are still unexplained. Bacterial bioproducts, i.e., endotoxins, are not removed by the highly porous membranes. Most studies have been done on the liquid phase of the dialysate. In this study, a biofilm has been made in a hemodialysis system, using an additional pump that continuously supplies several bioproducts: gram (+) or gram (-) bacteria, or extracted endotoxin. The attachment of bacteria and the role of the biofilm in different circumstances were determined: 1) A contaminated dialysate with 1,000 bacteria/ml is necessary to set up a biofilm in the device. AAMI, Canadian, and French standards (200 bacteria/ml) are validated. 2) In the presence of non-endotoxic bacteria (Staphylococcus conhii, delta hemolytic), and when solutions contaminated by endotoxin are used, the biofilm produces micellar forms of endotoxin that are not removed by the filter. 3) The efficiency of disinfectants differs. For example, the activities of sodium hypochlorite, formaldehyde, and hydrogen peroxide are lower in the biofilm than in static studies.

l-Methionine, a Precursor of Trace Methane in Some Proteolytic Clostridia.

The in vivo formation of methane and of several S-methyl volatile compounds from the terminal S-methyl group of l-methionine is reported for growing cultures of four Clostridium strains (C. hastiforme, C. histolyticum, C. subterminale, and Clostridium sp. strain DSM 1786). After growth in 5 ml of unamended medium, C. hastiforme formed the highest amount of methane (408 nmol per tube in the headspace). When the culture medium was amended with 100 mM l-[S-methyl-H(3)]methionine, the four strains formed [H(3)]methane (proportion in the methane peak, >85%) as well as methanethiol, dimethyl disulfide, dimethyl trisulfide, and S-methyl thioacetate labeled on the methyl moiety. Methanethiol is also a precursor of methane for Clostridium sp. strain DSM 1786. The trace methane formation observed for these four proteolytic, nonglucidolytic Clostridium strains can be of ecological interest, particularly in aquatic sediments and in the gastrointestinal tract of humans and animals. It can explain in part the trace methane formation which cannot be ascribed to methanogens sensu stricto.

[Comparative study of 3 methods of evaluation of antiseptic products and disinfectants in quality control]. / Etude comparative de trois méthodes d'évaluation des produits antiseptiques et désinfectants en contrôle de qualité.

For choosing a protocol of determination of bactericidal activity of antiseptic and disinfectant products, we have compared a method using an MS2 Abbott system and the classic methods: membrane filtration and dilution-method by transfer loops. The bactericidal-activity of 12 antiseptic solutions are determined. The results shown that there was no significative difference between the automated system and AFNOR specifications. The dilution-method by transfer loops gave higher bactericidal concentrations than the two other methods.

Development of a standard, freeze-dried serum containing the drugs assayed most often in French hospitals.

A quality control program for the therapeutic drug monitoring methodology used to determine serum levels of the fourteen drugs assayed most frequently by French hospital laboratories is described. The program has been adopted by the Societé Française de Biologie Clinique (SFBC), which provides participating laboratories with a statistical analysis of the performance data. The control samples developed for the program are prepared by spiking calf serum with, in one case, phenobarbital, phenytoin, carbamazepine, sodium valproate, amikacin, lithium carbonate, digitoxin and hydroquinidine, and, in the other case, caffeine, quinidine, digoxin, isoniazid and gentamycin. After freeze-drying the spiked serum, the drugs were shown to be stable for at least a year when stored at -20 degrees, +4 degrees and +22 degrees; at the end of this time, the samples were pathogen free. For checking therapeutic drug monitoring methods, the freeze-dried serum is diluted with distilled water (10 ml); these solutions are chemically stable, and remain pathogen-free, when stored in stoppered glass tubes at -20 degrees or +4 degrees for 4 weeks.

[Use of the MS2 automated system in the determination of the activity of 5 antiseptic drugs: quality control]. / Application de l'automate MS2 à la détermination de l'activité de cinq médicaments antiseptiques en contrôle de qualité.

The study of growth curves of Klebsiella pneumoniae and Staphylococcus aureus in presence of five antiseptics, established using a MS2 Abbott system is presented. From our results, the advantages of automation after the adaptation of the method for the determination of bactericidal properties are examined. This technique may be proposed for the quality control of such drugs.