Biocleaning in operating theatres: validation of cleaning techniques by revealing residual traces of blood.

BACKGROUND: In the operating theatre the biocleaning process is essential after each passage to guarantee the non-transmission of potentially pathogenic microbial agents from patient to patient. AIM: To evaluate the quality of this biocleaning, the Operational Hygiene Team used a very sensitive method to detect residual traces of blood: luminol (3-aminophthalhydrazide) on the basis of methods used by the police. METHODS: Luminol was used after conventional one-step biocleaning with the usual detergent/disinfectant, after bleach disinfection before biocleaning, and after biocleaning with a steam cleaner. FINDINGS: Lunimol revealed extended traces of blood corresponding to the passage of the strip on the floor, in the corners of the room and on certain pieces of furniture which are difficult to clean. However, no luminescence was detected on the surfaces cleaned by a single passage of the steam cleaner. CONCLUSIONS: In all cases, the rooms appeared visually clean and traces of blood only became visible when revealed by luminol. We also showed that usual detergents or disinfectants do not remove blood and instead actually spread it over surfaces that may seem visually clean. These results led us to modify our procedure and also confirmed our wish to generalize the use of the steam cleaning technique for immediate cleaning. Furthermore, our tests show the relevance of luminol as a validation tool for the quality and method of biocleaning.

[Effectiveness and limits of the cleaners steam in hospitals]. / Efficacité et limites des nettoyeurs vapeurs en milieu hospitalier.

We assessed bactericidal activity of the cleaners steam used for the bio-cleaning of the hospital surfaces. We performed of samples (Rodac) before and after use of cleaner steam and compared with bactericidal effect of disinfecting detergent used in hospital for surfaces. We studied this effectiveness for different time of steam contact. Finally, we wanted to prove, by air sampling, that aero-bio-contamination was possible generated by using cleaners steam. We show that bactericidal effect of the cleaner steam is superior of some tested disinfecting detergent, for the treatment of one square meter till 2 min. This effectiveness diminishes to be just identical in that some disinfecting detergent when use of the cleaner steam is up to two or four square meters surfaces till 2 min. On the other hand, the cleaner steam is less efficient in terms of bacterial destruction when the time of contact steam-soil is superior in 2 min for six square meter surface. The air bacterial pollution, generated by the use of the cleaner steam, is restricted and not significantly augmented if measured in 44 cm above the soil in the course of cleaning. The cleaner steam is indeed a very good equipment for the cleaning of surfaces but it is necessary to respect a time of minimal contact of 2 min for four square meters surfaces treaties to acquire an antibacterial effect at least so important as that acquired with used disinfecting detergent. The disinfection of surfaces is then user-dependent and the time of requested contact is can be not compatible with hospital obligations.

[Experimental studies of long-term effect of a detergent disinfecting for surface on a strain of Escherichia coli]. / Etudes expérimentales de l'effet rémanent d'un détergent désinfectant pour surfaces sur une souche d'Escherichia coli.

We offer three complementary, original methods and reproducibles to study the antibacterial and long-term effect of a detergent disinfecting for surfaces commercialized lately in France. Long-term activity noticed-effect is compared with that of other products. We first study the curves of growth of a strain of Escherichia coli put in presence with the surface of the wells of a microplate beforehand and for several days (from D-10 to D0), coated with disinfecting detergents. Another method consisted on surfaces firstly treated from D-10 to D0 by the one or the other product to be tested, which are artificially contaminated in a standardized manner by a velvet footprint with a suspension of E. coli. The surviving microbes are counted after transfer on a Rodac plate. Finally, doorhandles are cleaned and disinfected with the product. The natural bacterial recolonization doorhandles is studied by daily Rodac plate within a week. These studies allow to prove that Bacoban introduces a bactericidal activity on E. coli with an long-term effect for at least 10 days. The most competitive products have a bacteriostatic effect during nine to 10 days, but bactericidal effect only during two days. This bactericidal long-term effect may be particularly interesting in hospital hygiene for the biocleaning of the most manipulated surfaces and should restrict the role of bacterial reservoir of certain surfaces participating in care or near of the patients.

[Determination of the detergent efficiency of the detergents disinfecting for surfaces]. / Mesure de l'efficacité détersive des détergents désinfectants pour surfaces.

Since the disinfecting activity of disinfectants is evaluated by standards, the intrinsic detergent activity is not easily quantifiable and no standard have been suggested yet. Beyond the physicochemical parameters like wettability or foaming presented by the manufacturers, it appears necessary to us to objectively measure the real effect of the detergent agent. The objective of our work is to propose a simple, fast and reproducible method to evaluate detersive activity of the disinfecting detergents. We measured three factors (total amount of extracted bacteria, extraction efficiency and slope of extracting curve) by using Rodac prints technique on two different supports (PVC, stainless steel) that have been contaminated by either E. coli or S. aureus. An increasing mark from 1 to 6 is given to each of these factors in case of statistically differences. The three factors allowed us to calculate a "Specific Index of detersion" (SI) for each germ/support couple (3 to 18). Addition of the marks given to each couple for each disinfecting detergent allowed to calculate a "Globally Index of detersion" (GI) (9 to 72). We tested 4 commercialised disinfecting detergents: Surfanios, Aniosurf, Major C100 and Ecodiol. All detergents may be classified according to their effectiveness on a bacterium/support couple (value of the SI). This enlights a specific spectrum for each disinfecting detergents. As a result, Ecodiol seems to be the most effective deterging agent on 3 of the 4 germ/support couples (S. aureus/PVC, E. coli/PVC and E. coli/stainless steel), whereas Aniosurf is most effective on the S. aureus/stainless steel couple. The GI is very useful to choose the best compromise between activities for all situations. GI rankings of the tested agents were as follows: water < Aniosurf < Surfanios < neutralizing < Major C100 < Ecodiol. This experimental model will be used to test and compare the intrinsic detergent activities of other commercialised products which are usually used for the biocleaning of the medical devices (i.e. endoscopes or reusable dialysis device).

[Methods for microbiological control of textiles]. / Evaluation des méthodes de prélèvement pour le contrôle microbiologique des textiles.

Our laboratory is requested more and more by the establishments of health and the industrial laundries for the microbiological realization of control on the textile articles after completion. We checked the effectiveness of the techniques of the bacteriol prints or rodac and the bacterial extraction after maceration for the realization of these controls. The output of extraction of the bacteriol prints applied to woven material samples sterilized and then artificially contaminated by a titrated Staphylococcal suspension is lower than 1%. The technique recommended and largely used for the study in particular of the contamination of the blouses of doctor in the establishments of health thus does not appear to be relevant. We propose for the quality control of the linen a technique of maceration then extraction by the ultra sounds whose output is evaluated to 62% which requires to sacrifice the controled textile article. The choice of the bacteriological techniques as controls ultimate of a procedure must be carried out carefully to meet the needs for quality. Conclusions, when with the effectiveness of a process, deduced from results obtained by a bad method could be wrongfully reassuring.

[Factors associated with early invasive strategy in patients with acute coronary syndrome. A multicenter study]. / Facteurs associés a la réalisation d'une coronarographie précoce en présence d'un syndrome coronarien aigu sans sus-décalage du segment ST. Résultats d'une etude multicentrique.

OBJECTIVES: To identify the factors associated with early cardiac catheterization in patients with a non ST-segment elevation acute coronary syndrome. METHODS: We analyzed data collected by retrospective chart review for 208 patients presenting at seven French hospitals with an acute coronary syndrome (chest pain at rest within 24 h prior to presentation with positive cardiac markers and/or electrocardiographic changes) between January and March 2005. RESULTS: Eighty-seven patients (42%) were first admitted to hospitals with cardiac catheterization facilities. One hundred ten patients (53%, 95% confidence interval [95% CI], 46-60) underwent early cardiac catheterization less than 48 h following presentation. In addition to presentation at hospitals with catheterization facilities, factors independently associated with early catheterization included positive cardiac markers in patients first admitted to hospitals without catheterization facilities (adjusted odds ratio [aOR] 34.5, 95% CI, 4.4-268.0) and diabetes mellitus (aOR, 0.4, 95%CI, 0.2-0.9). With the exception of positive cardiac markers, no risk factors comprising the TIMI risk score were associated with increased odds of early cardiac catheterization. During the index hospital stay, six patients (3%) died, seven patients (3%) had pulmonary edema, three patients (1%) had major or minor bleeding, and none had ST segment elevation myocardial infarction. CONCLUSION: Despite the dissemination of international guidelines, the use of early cardiac catheterization remains related to initial presentation at hospitals with catheterization facilities rather than risk assessment in patients with a non ST-segment elevation acute coronary syndrome.

Pore formation by a two-component leukocidin from Staphylococcus aureus within the membrane of human polymorphonuclear leukocytes.

The effects of the Staphylococcus aureus leukocidin (PVL), a two-component non-hemolytic toxin, were investigated on the membrane permeability of human polymorphonuclear leukocytes (PMNs). In the absence of extracellular Ca2+, the fluorescence of ethidium bromide added to the extracellular medium increased after PVL application in a concentration-dependent manner and no variations in the free intracellular [Ca2+] of Fura2-loaded PMNs were detected. In the presence of extracellular Ca2+, the fluorescence of ethidium was not modified but the free intracellular [Ca2+] of PMNs increased after application of PVL in a concentration-dependent manner. The time lag observed before an increase in the ethidium fluorescence was longer than the time lag observed before a Fura2 fluorescence increase. Simultaneous recordings of the two probes fluorescence variations have shown the protective effect of Ca2+ and Zn2+ and the closing of the pore by 50 mM Ca2+ or 2 mM Zn2+. Moreover, the effect of Ca2+ could be reversed by the addition of EGTA. In the presence of 1 mM extracellular Ca2+ or 0.8 mM extracellular Zn2+, the pore induced by PVL had an ionic size allowing Ca2+, Mn2+, Zn2+ and Mg2+ fluxes. The addition of antibodies against either component of PVL inhibits the permeabilization provoked by the toxin even after it was initiated. It is concluded that leukocidin from S. aureus is a pore-forming toxin which, under physiological conditions ([Ca2+] = 1 to 1.5 mM), provokes the formation of an ion-sized pore inducing an increase in the free intracellular Ca2+ which may activate PMN functions.

A predicted beta-sheet from class S components of staphylococcal gamma-hemolysin is essential for the secondary interaction of the class F component.

Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal y-hemolysin locus, which originate two pore-forming toxins (HlgA + HlgB, HlgC + HlgB). As related proteins, HlgA and HlgC were found to bind first to cell membranes. Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved beta-sheet of the Chou and Fasman secondary structure prediction. The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells. The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited. In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least one of the functional domains involved.

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus.

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.

[Bacteriological sampling of surfaces: importance of the step of enrichment and the choice of the culture media]. / Prélèvements bactériologiques des surfaces: importance de l'étape d'enrichissement et du choix des milieux de culture.

This study tested the efficiency of four different procedures for isolating bacteria found on hospital surfaces. The techniques studied use both rich and poor media with or without enrichment in nutritive broth. The sampling of surfaces in hospital care departments was carried out using a dampened sterile flue brush. Bacteria samples were then placed on TCSA agar plates (method 1) and blood agar plates (GS) (method 2) before immersion in a nutritive broth for enrichment. The following day, the broth was used to produce two new media: TCSA (method 3) and GS (method 4). For each sample, we established the global amount of different bacterial species isolated by all 4 methods combined. These values were then used as a reference to evaluate the efficiency of each technique. 360 smears were carried out, and a total of 718 bacterial strains were isolated. Methods 1 and 2 (without enrichment) permitted the isolation of 10.86 and 13.37% respectively of the total number of strains. Methods 3 and 4, with preliminary enrichment, made it possible to isolate 69.08% of bacterial strains on TCSA medium and 90.53% on GS medium. The combination of the enrichment stage and an enriched culture medium lead to an excellent output that highlights and identifies bacteria isolated from samples taken from hospital surfaces.

Effect of peracetic acid and aldehyde disinfectants on biofilm.

The effects of two aldehyde (Cidex, Endosporine) and four peracetic acid (PAA) (Nu Cidex, Anioxyde 1000, Hydraseptic, Peralkan) disinfectants on an Escherichia coli biofilm model were studied. The biofilm was prepared in glass tubes, and evaluated indirectly using a colourimetric method. The ability of the disinfectants to fix or remove the biofilm from tubes was determined by their detergent activity (DA). The two aldehyde derivatives and two of the PAA (Nu Cidex, Anioxyde 1000) agents fixed the biofilm. However, the effects of Hydraseptic and Peralkan were equivalent to the control (sterile water). Regardless of their disinfectant activity, PAA agents display different DAs that could be used to select the weakest biofilm-fixing agents. Users should be concerned about the efficiency of the cleaning stage of medical devices, and when choosing a PAA product, non-fixing ability should be considered in addition to antimicrobial activity.

Effectiveness of detergent-disinfecting agents on Escherichia coli 54127 biofilm.

Detergent-disinfecting agents (dD) are used daily for cleaning reused medical devices. We have devised a simple method to test dD detergent activity (DA) using an E. coli 54127 biofilm prepared in haemolysis glass tubes, which are cleaned with test dD, according to supplier's recommendations. Crystal violet 0.05% is used to colour the residual biofilm after dD (or tap water control) application. The biofilm quantification was made indirectly by measuring the absorbance of crystal violet at 585 nm. A measure of the detergent effectiveness called DA was calculated as the percentage reduction of colour from a tap water control. Fifteen products including enzymatic and non-enzymatic dDs were evaluated. Most enzymatic dDs gave a high DA, as did some non-enzymatic products. Thus, the view that enzymatic dDs are more effective than non-enzymatic dDs, put forward by some manufacturers, should be regarded with caution. The DA determination should help infection control teams choose, within the wide range of products available on the market, the most effective dD based on both its detergent and disinfecting activity.

Antral and fundic D-cell numbers in Helicobacter pylori infection.

OBJECTIVES: Helicobacter pylori infection is associated with an exaggeration of gastrin release following meals or bombesin stimulation attributed to a defect of somatostatin secretion of antral D-cells. Nevertheless, these modifications of gastric physiology do not explain the increase of gastric acid secretion which is only observed in duodenal ulcer patients. The inhibitory effect of somatostatin secretion of fundic D-cells on parietal cells is well known. The aim of our prospective study was to compare the number of fundic D-cells and likewise the number of antral G-cells and D-cells between patients with duodenal ulcer and healthy subjects with and without H. pylori infection. METHODS: The numbers of D-cells and G-cells were compared between 19 infected patients with duodenal ulcer and 20 healthy subjects, 10 with and 10 without H. pylori infection. Fundic mucosal biopsy specimens were examined using immunohistochemical techniques specific for the presence of somatostatin, antral mucosal biopsy specimens for the presence of gastrin and somatostatin. RESULTS: The number of G-cells was significantly lower (P = 0.0012) in duodenal ulcer patients by comparison with infected subjects and controls. The number of antral D-cells was significantly less (P < 0.0001) in duodenal ulcer patients (mean of 10 random fields = 0.45 +/- 0.04) than in either asymptomatic infected patients (0.65 +/- 0.07) or uninfected controls (0.88 +/- 0.10). The number of fundic D-cells was significantly lower (P < 0.0001) in duodenal ulcer patients (mean = 0.20 +/- 0.03) than in either asymptomatic infected subjects (0.29 +/- 0.05) or controls (0.73 +/- 0.09); here the difference between the two groups of infected subjects was not significant. Multivariate analysis showed that the presence of H. pylori infection of the fundic mucosa did not influence the number of fundic D-cells. CONCLUSION: Changes in the number of fundic and antral D-cells induced by H. pylori infection did not explain abnormalities of gastric acid secretion usually observed in duodenal ulcer patients; it is suggested that pre-existing abnormalities in the regulation of parietal cell or increase of parietal cell mass are involved.

[What are the indications in France for vaccination against Japanese encephalitis?]. / Comment poser en France l'indication de la vaccination contre l'encéphalite japonaise?

Japanese encephalitis is a mosquito-borne viral disease occurring in rural and rice-growing areas of Asia, where mosquitoes proliferate, transmitting the Flavivirus from viremic animals, mostly pigs, to humans. Japanese encephalitis has recently spread to previously non-affected regions, leading to serious outbreaks among non-immune populations. Although it has a high proportion of unsymptomatic infection, clinical encephalitis is usually severe, resulting in a very high mortality rate, and neurologic sequellae are common among survivors. Vaccines are used in several Asian countries. One of these vaccines is now available to French travellers, but only in international vaccination centres with an authorization from the French drug agency (Agence française de sécurité sanitaire des produits de santé). The aim of this paper is to clarify the recommendations for immunisation in each country of the affected regions. The area can be divided into three epidemiological zones, with tropical, subtropical and temperate characteristics. For the first two, vaccination is recommended before a long stay in a rural area, especially during the rainy season; in temperate climates, outbreaks occur in summer and autumn. However, local variations such as intensive rice-growing or development of pig breeding may interfere with these patterns. Long-term visitors should consult a local physician and prevention of mosquito bites is always recommended.

[Role of hygiene and bacteriological laboratories in the management of an epidemic of Enterobacter aerogenes multiresistant to antibiotics]. / Rôles des laboratoires d'hygiène et de bactériologie dans la prise en charge d'une épidémie à Enterobacter aerogenes multirésistant aux antibiotiques.

We describe a multiresistant Enterobacter aerogenes outbreak in an intensive care-unit. An epidemiology study based on phenotypic characters (species diagnosis and antibiotype) was completed by a genotypic study (pulsed field electrophoresis) to confirm bacterial clonality. The hygiene laboratory proposed numerous preventive measures to limit bacterial dispersion. We describe the role of bacteriologists, hygienists and medical staff to stop the bacterial dispersion.

[Susceptibility of the hospital environmental bacterial strains to a detergent and disinfectant product for surfaces]. / Etude de l'efficacité d'un produit détergent désinfectant pour sols et surfaces sur les souches bactériennes isolées à partir de l'environnement hospitalier.

We studied the sensitivity of the hospital environmental bacterial strains to Surfanios, which is a detergent and disinfectant product for surfaces. This product is used in our establishment since nearly 10 years. This work which relates to 425 bacterial strains proposes to study the possible evolution of the resistance of the bacteria under the pressure of a product biocide used since many years without the change of active ingredient as recommended in pharmaceutical and agroalimentary industry. We developed a micromethod to study the sensitivity of many bacterial strains simultaneously into a 96 wells microplaque. All the Staphylococcus aureus strains (N = 20) and Staphylococcus with coagulase negative (N = 308) as 78 Acinetobacter sp. strains remain very sensitive to Surfanios according to our study. Target dilution, which is the last dilution not allowing the bacterial growth, is much lower than the manufacturer recommended use dilution. For the Pseudomonas aeruginosa strains tested, target dilution is equal or higher than the dilution of use. Rather than to propose the rotation or the alternation of the products, we recommend the use of Surfanios for the bionettoyage of dry surfaces which might be contaminated by Staphylococcus sp. or Acinetobacter sp. and the use of another product or Surfanios at a higher concentration, active on Pseudomonas sp. to disinfect wet surfaces which are possible reservoirs for the opportunist bacteria of the hydrous flora.

Application of flow cytometry in toxinology: pathophysiology of human polymorphonuclear leukocytes damaged by a pore-forming toxin from Staphylococcus aureus.

The pore-forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two-component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but allowed an influx of calcium as shown by the increase in fluorescence of Fluo-3 loaded in the cells. This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of their granule content measured by a decrease in side light scattering. Furthermore, ethidium fluorescence was used to discriminate the cells sensitive to PVL, and the analysis of differentiated HL-60 cells and cells obtained from a case of chronic myeloid leukemia led to the conclusion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage.