An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

The aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed.

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants.

AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

BACKGROUND: Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. OBJECTIVES: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. STUDY DESIGN: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. RESULTS: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. CONCLUSION: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.

A comparison of methods for counting viruses in aquatic systems.

In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1, 3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10(8) particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.

Role of infection control measures in limiting morbidity associated with multi-resistant organisms in critically ill patients.

A retrospective comparative study was performed to determine the impact of infection control measures (ICMs) on colonization and infections due to methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae (producing transferable extended-spectrum beta-lactamase, KPESBL), and multi-resistant Enterobacter aerogenes (MREA) in intensive care unit patients. Infection Control Measures included surveillance cultures, isolation procedures and mupirocin for MRSA nasal carriage. The numbers of patients infected and/or colonized by MRSA, KPESBL or MREA were compared during two consecutive one-year periods (Period 1 before ICMs, and Period 2 after ICMs). The antibiotic consumption during the two periods was analysed. In Period 1 and Period 2, respectively, the rate of patients infected or colonized by at least one of the three organisms was 15% and 6.8% (P=0.001); by MRSA 7.7% and 2.6% (P=0. 004); by KPESBL 1.7% and 0% (P=0.25); and by MREA 5.6% and 4.3% (P=0. 47). During Period 2, there was a clear-cut decrease in the percentage of patients infected by MRSA (P=0.018), a non-significant decrease in those infected by KPESBL (P=0.06), and no decrease in patients infected by MREA (P=0.22). When calculated per 1000 patient-days, for Period 1 and Period 2, respectively, the rate of patients infected or colonized by at least one of the three organisms was 11.9 and 8.8; for MRSA it was 4 and 2.2; for KPESBL it was 1 and 0; and for MREA it was 4 and 4. Antibiotic cost was pound98.7 in Period 1 and pound62.7 in Period 2. ICMs contributed to the control of infections and colonizations due to MRSA and KPESBL but not those due to MREA.

Comparison of in-vivo antibacterial activity of two skin disinfection procedures for insertion of peripheral catheters: povidone iodine versus chlorhexidine.

Skin disinfection is a key step in the prevention of nosocomial infections especially prior to invasive procedures such as the insertion of peripheral catheters. Alcohol-based antiseptics improve bactericidal activity and decrease the time needed for skin disinfection in emergencies. A randomized study was performed in two groups of 22 volunteers to compare the in vivo bactericidal effect of two rapid disinfection procedures using povidone iodine (PVP-I) in scrub formulation followed by alcoholic PVP-I, or chlorhexidine in scrub formulation followed by alcoholic chlorhexidine. Bacteria were recovered using the cylinder scrub method. Comparison of reductions in the aerobic and anaerobic flora from baseline levels to each of the three sampling times (30 sec, 3 min, 2 h) showed no significant difference between the two procedures Log(10)reduction after 30 seconds was around 1.5 for the aerobic flora and 1.1 for the anaerobic flora. After 3 minutes the corresponding values were 2.1 and 1.8, and after 2 hours 2.0 and 1.3. The products were well tolerated in both groups. The two procedures had comparable rapid bactericidal activity in vivo.

Dialysis and central venous catheter infections in critically ill patients: results of a prospective study.

OBJECTIVE: To determine the incidence of dialysis catheter (DC)-related infections in intensive care unit (ICU) patients, and to compare the frequency of DC and central venous catheter (CVC) infections in an ICU setting. DESIGN: Prospective, descriptive survey. SETTING: An adult, 10-bed medical/surgical ICU at a university hospital. PATIENTS: A total of 151 DCs and 230 CVCs placed in 170 patients were evaluated. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Catheter colonization was defined by a quantitative catheter tip culture yielding > or =10(3) colony-forming units/mL, catheter-related bacteremia was defined as catheter colonization and blood culture positive for the same organism, and site infection was defined as the presence of pus at the insertion site. The mean duration of catheterization was 6.8+/-6 days for DCs and 5.9+/-4.6 for CVCs (p = .52). There was no difference between DCs and CVCs in catheter colonization and catheter-related bacteremia incidence rates per 1000 days of catheter use (24.2 vs. 19.8 [p = .46] and 0.96 vs. 1.5 [p = .60], respectively). Site infection was observed in one patient (CVC placement). For DCs and CVCs the duration of catheterization was associated with catheter infection (p = .0007 and p = .04, respectively), but when the catheters were examined over 5-day intervals, the incidence of catheter infections did not increase with duration of catheter use (p = .23 and p = .10, respectively). CONCLUSIONS: DC-related infections are associated with DC longevity. As shown by the 5-day-interval analysis, the incidence of DC-related infections did not increase with DC duration, suggesting that the risk for DC-related infections remained unchanged with time. The characteristics of DC-related infections in ICU patients were comparable to those previously reported for CVC-related infections.

[Laryngoscope. Evaluation of a device for preventing blade contamination]. / Laryngoscope. Evaluation d'un dispositif de prévention de la contamination des lames.

OBJECTIVE: To evaluate the efficacy and the difficulty of use of a disposable sheath which prevents the contamination of blades. STUDY DESIGN: Prospective bacteriological, virological and clinical evaluation. MATERIAL: A translucid cover sheath, made of polyethylene enclosing the blade of the laryngoscope, and delivered in clean, non-sterile packaging (Prolam, Péters). METHOD: 1) A control of sterility performed by setting-up a culture derived from the solution used for rinsing the device before its use. 2) An in vitro study of the effectiveness of preventing contamination of the blades by a polio virus/RT-PCR technique. 3) Clinical evaluation: after 200 orotracheal intubations by 12 anaesthesiologists and 15 nurse anaesthetists, a questionnaire on the ease of use was completed. RESULTS: The bacteriological study of the sheats before use showed an acceptable level of contamination. The sheath was an effective barrier against poliovirus, even after 12 h of immersion. Clinically, the sheath was easily adapted over the blade of the laryngoscope in 98% of the cases. Insertion in the mouth was considered as easy in 94% of the patients. The visualization was good or excellent in 83% of the cases and in 16% of the patients, the users experienced difficulties to intubate. CONCLUSION: The laryngoscope blade sheath is simple and easy to use, efficient and not expensive.

Threonine and methionine are limiting amino acids for protein synthesis in patients with AIDS.

This study was conducted to identify the most rate-limiting amino acids for whole-body protein synthesis in acquired immunodeficiency syndrome (AIDS) patients. We postulated that an essential amino acid that would be rate limiting in AIDS should have a low basal plasma concentration and should remain at a low level during amino acid infusion. Seven male AIDS patients (median age 37 y, CD4 cell count: 76 mm-3) without any clinically active opportunistic infection during the month before the experiment were infused intravenously with a complete amino acid-glucose mixture for 2.5 h. Eight healthy volunteers were used as controls. Before the infusion, the concentrations of most free essential amino acids (methionine, threonine, histidine, isoleucine, leucine and tryptophan) were significantly lower (P < 0.05) in AIDS patients than in controls. Most plasma free essential amino acids increased significantly during infusion. However, the absolute increase above basal levels for threonine, valine, lysine, (P < 0.05) and methionine (P < 0.073) was smaller in AIDS patients than in control subjects. Thus, threonine and possibly methionine may be rate limiting for whole-body protein synthesis in AIDS patients, suggesting that there are selective amino acid requirements in patients with AIDS.

Reverse transcriptase PCR detection of astrovirus, hepatitis A virus, and poliovirus in experimentally contaminated mussels: comparison of several extraction and concentration methods.

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods. The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic flocculation. RT-PCR was performed with RNA extracts from crude shellfish extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and borate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extraction method. We assessed the efficiency of 20 combinations of extraction and concentration methods. The borate buffer-organic flocculation, borate buffer-PEG 6000, and glycine solution-PEG 6000 combinations gave RT-PCR-positive results for all 27 samples analyzed for the three viruses. Detoxification of the samples by Sephadex LH20 filtration significantly decreased the efficiency of RT-PCR virus detection.

[Current requirements of sterilization and disinfection of endoscopic materials]. / Les impératifs actuels de la stérilisation et de la désinfection du matériel endoscopique.

Bacterial and viral infectious risks related to gynecologic endoscopy and laparoscopy are a significant problem. Contamination between patients or by environmental bacteria can occur when sterilisation or disinfection procedures are inadequate. Endoscope steam sterilisation is the safest method and have to be employed whenever possible. For heat labile materials, disinfection procedure will comply with the recommendations stated in a recent ministerial decision, including a cleaning step with a non aldehydic detergent-disinfectant and a disinfection step with 2% glutaraldehyde. Rooms and equipment dedicated to disinfection have to be well adapted to this use and specific staff training must be achieved. Endoscope automatic washer disinfections are an alternative method to manual disinfection but they have to meet precise criteria to insure safety and efficacy.

Capsid protein composition of reference strains and wild isolates of human astroviruses.

Astroviruses are small RNA viruses that are frequently associated with gastroenteritis in humans and animals. Despite much work on the genetic analysis of astrovirus strains, little progress has been made in the characterization of the proteins composing mature virions. We have analyzed the capsid protein composition of the reference strains and several wild isolates of human astroviruses using high-resolution polyacrylamide gradient gels. For reference strains of the seven serotypes analyzed, a consistent pattern of three infection-specific proteins--designated P1, P2, and P3 -was generally observed. The strains could be divided into two groups, based upon the reactivity of these proteins in immune precipitation assays that used homologous rabbit serum. One group included reference types 1 4 for which all three proteins were precipitated by homologous rabbit sera; for the other group, types 5 7, only proteins P2 and P3 were precipitated. When wild isolates from around the world were compared to the reference strains, a correlation between genetic type and the pattern of protein sizes and immune reactivity was observed for strains of the common types (1-4). Strains of types 2 and 4 consistently exhibited P3 proteins larger than those of types 1 and 3. Unusual patterns of proteins or immune reactivity were detected in strains of types 5-7, indicating that there may be incomplete processing of the capsid precursor during growth in cell culture.

Outbreak of gastroenteritis in military recruits associated with serotype 3 astrovirus infection.

A serotype 3 astrovirus was identified in stool samples from an outbreak of acute gastroenteritis that occurred among military recruits in France. Sixteen stools samples and eight pairs of acute- and convalescent-phase serum were collected from affected individuals. Astrovirus was detected in two stool samples by electron microscopy and in four stool samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Seroconversion to the astrovirus present in one stool was detected in seven patients by using solid-phase immune electron microscopy (SPIEM) and dot blot. For three patients, the serological results were consistent with the PCR results, indicating that astrovirus was a cause of gastroenteritis in these young adults. This study describes the characterization of the serotype 3 astrovirus associated with this outbreak. The virus has a buoyant density in cesium chloride of 1.365 gm/ml and contains two proteins immuno-precipitated with rabbit serum. Only the larger protein was recognized by immunoblotting using a convalescent-phase human serum. The protein composition of this virus differs from that reported for serotype 1 astrovirus, indicating heterogeneity in the capsid composition among astrovirus serotypes.

[Bacterial contamination of eyedrops in clinical use]. / Etude de la contamination bactérienne de collyres en usage clinique.

PURPOSE: The aim of this study is to assess the importance of bacterial contamination of multidose eyedrops in a routine clinical setting. MATERIAL AND METHODS: 406 eyedrop vials were cultured about one week after clinical use: 204 collected from an Ophthalmic Department, and 202 from a Nursing Home. The microbiological analysis was performed on the tip and the residual eyedrop, counting the number of bacterial colonies. RESULTS: 66 (16.3%) from the 406 analyzed vials were contaminated, and 5.4% out of these were severely affected. There was no significant difference between the "Ophthalmic Department" and the "Nursing Home". Commensal germs were the most frequently encountered in both groups. 4 gram negative organisms were isolated from the "Nursing Home" group. CONCLUSION: These results are in agreement with the literature. Comparison between our two groups is difficult because the eyedrops and uses were different. However, we notice the presence of gram negative organisms in the "Nursing Home". These severe contaminations due to opportunistic pathogen organisms are rare (0.75%), probably underestimated, and represent a real infectious risk during instillation. The study of the contamination site shows that the eyedrop is more often contaminated than the tip. This can be in relation to germ desicsation and to an aspiration phenomenon of contaminated fluid at the tip level. At last, the role of preservatives is not sufficient to ensure the sterility of multidose eyedrops during their use, and this justifies safer (single dose or filtration system) eyedrop vials.

Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region.

Astroviruses are single-stranded, positive-sense RNA viruses that are associated with gastroenteritis in humans and animals. We describe a reverse transcription-polymerase chain reaction (RT-PCR) assay using primers targeted to a nonstructural protein coding region that allowed sensitive detection and genetic typing of representative strains of seven astrovirus serotypes. Phylogenetic analysis of the nucleotide sequences of PCR products from the reference strains and several wild isolates indicated two distinct genogroups of sequences in open reading frame 1a (ORF 1a). These genogroups correlated with serotype: genogroup A included strains of types 1 to 5, while genogroup B included strains of types 6 and 7. This phylogenetic arrangement differs from the nearly equidistant clustering of serotypes seen when comparing nucleotide sequences from either ORF 1b or ORF 2. It is possible that recombination was responsible for the observed difference in genetic relationships.

An in-vitro evaluation of the activity of povidone-iodine against nosocomial bacterial strains.

Two povidone-iodine (PVP-I) preparations, one, an antiseptic handwash and one, a skin disinfectant, were tested against 504 bacterial strains isolated from nosocomial infections in 12 French hospitals. In vitro bactericidal activity was determined by a micromethod, using specific interfering substances over a range of dilutions, after 1, 3 and 5 min exposure times. A 5 log10 reduction of the challenge inoculum was considered as the criterion of efficacy. Any resistant strains were tested with the French Standard (T72300). When the micromethod was carried out at 20 degrees C, 10.7% (54/504) of the strains were resistant to the PVP-I skin disinfectant (dilution 1:10) and 1.6% (8/504) were resistant to the handwashing formulations (dilution 1:3) after 1 min exposure. By increasing the temperature to 32 degrees C, the resistance rate to the skin disinfectant fell to 1.9% (10/504). All of the 18 strains found resistant with the micromethod were sensitive using the French standard.

Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103. Attempts to use this phage for therapy.

A bacteriophage for Escherichia coli 0103 was isolated during a study on E. coli diarrhoea in intensive breeding units of rabbits. The phage had an isometric head and a short tail and resembled coliphage N4 (Podoviridae). It had a very narrow host range and seemed to be specific for serogroup 0103, suggesting that it might be used for preliminary identification of E. coli strains of this serogroup instead of the usual slide agglutination. In view of its possible use as a therapeutic phage, we investigated its dissemination in rabbit organs after oral administration. The phage persisted in the spleen for at least 12 days. However, in vivo studies showed that this phage and a mixture of more virulent phages for E. coli 0103 were ineffective in preventing disease in rabbits inoculated with an enteropathogenic strain of E. coli 0103.

Activity of glutaraldehyde at low concentrations (less than 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures.

The activity of glutaraldehyde (GTA) at low concentrations (less than 2%) against poliovirus was assessed by a suspension procedure. The inactivation kinetics showed that concentrations of less than or equal to 0.10% were effective against purified poliovirus at pH 7.2; a 1 log10 reduction was obtained in 70 min with 0.02% GTA, and a 3 log10 reduction was obtained in 30 min with 0.10% GTA. GTA activity at low concentrations was greatly enhanced at alkaline pH, but was completely abolished at acid pH. In contrast, the inactivation assays on poliovirus RNA showed that it was highly resistant to GTA at concentrations up to 1.0% at pH 7.2. At pH 8.3 a low inactivation was noticed with 1.0% GTA. Our results are of relevance to hospital practice in digestive endoscopy investigations because there has been an increasing tendency to use low concentrations of GTA and very short contact times in disinfection procedures.

Sequential rotavirus infections: characterization of serotypes and electrophoretypes.

Few studies have characterized the rotaviruses observed in repeated infections in the same patient. Rotavirus serotypes and electrophoretypes were determined from faecal samples of 24 children hospitalized between 1984 and 1989 in a Clermont-Ferrand hospital and who had had repeated rotavirus shedding. The patients were aged between 3 days and 4 years. Fifty infections were recorded. Of the 48 serotypes obtained, 18 were serotype 1 (37.5%), 10 serotype 2 (20.8%) and 20 serotype 4 (41.7%). Serotype 3 was not found. Seven serotype 2 infections were characterized during rehospitalization. Six patients had two subsequent infections with the same serotype.