[Hygiene and security management in medical biology laboratory]. / Hygiène et sécurité au laboratoire de biologie médicale.

Risk management in Medical Biology Laboratory (MBL) which includes hygiene and waste management, is an integrated process to the whole MBL organisation. It is composed of three stages: risks factors identification, grading and prioritization, and their evaluation in the system. From the legislation and NF EN ISO 15189 standard's requirements viewpoint, prevention and protection actions to implement are described, at premises level, but also at work station environment's one (human resources and equipments) towards biological, chemical, linked to gas, to ionizing or non ionizing radiations and fire riks, in order not to compromise patients safety, employees safety, and quality results. Then, although NF EN 15189 standard only enacts requirements in terms of prevention, curative actions after established blood or chemical exposure accident are defined.

[Recommendations for waste management]. / Recommandations pour la gestion des déchets.

Laboratory waste management must ensure the safety of patients and staff, limiting the environmental impacts and control waste disposal budget. Sorting of waste must be carried out at the source. The packaging must be adapted, allowing easy identification of specific disposal routes. With regard to wastes for human or animal health care and/or related research (DASRI), packages must comply with the regulations, standards and ADR if necessary. Storage provisions differ according to the amount of DASRI produced. Waste collection is carried out directly on the place of activity by a certified service provider. Non pre-treated DASRI is incinerated in specific approved plants for a T ° > 1,200 °C. Special provisions also exist for chemical waste and radioactive waste, the latter being regulated by ANDRA.

Multicentre study of the molecular epidemiology, serotypes and antimicrobial susceptibility patterns of invasive Streptococcus pneumoniae invasive isolated from children in the Ille de France area.

Between January 1997 and April 2002, 73 consecutive invasive strains of Streptococcus pneumoniae were isolated from children under 16 years of age in four hospitals in suburban Paris. Their genetic diversity was investigated by serotyping and analysis of pulsed-field gel electrophoresis restriction patterns. Antibiotic susceptibility patterns were analysed by disk susceptibility testing and determination of minimal inhibitory concentrations. The genetic basis of macrolide resistance was investigated by polymerase chain reaction. Studies of penicillin and vancomycin tolerance were performed for each strain. Despite the high prevalence (45.2%) of penicillin-nonsusceptible Streptococcus pneumoniae, resistance to amoxicillin (1.4%) was rare, and no strain was resistant to cefotaxime. Overall, 4.1% of pneumococcal strains were resistant to penicillin. Penicillin or vancomycin tolerance was not detected in any of the 73 strains studied. Of the erythromycin-resistant strains (48%), all but one carried the ermB gene. No strains showing a decreased susceptibility to ciprofloxacin (MIC, >4 mg/l) or overexpressing an efflux pump inhibited by reserpine were isolated. The serotypes found, in order of frequency, were as follows: 18C, 14, 6B, 19F, 19A, 9V, 23F, 1, 7F, 9A, 38. Strains of penicillin-nonsusceptible Streptococcus pneumoniae belonged predominantly to serotypes 14, 6B, 9V, 9A, 23F, 19F and 19A. The seven-valent conjugated vaccine covered 85.5% of the serogroups isolated in children under 2 years of age and 65.6% of the serogroups identified in children over 2 years of age. The genetic analysis showed a high identity for some serotypes, such as 14/9V, 6B and 23F. The use of the seven-valent conjugated vaccine is a critical measure to prevent invasive pneumococci infections in children in the Ille de France area.

[Pediatric imported malaria. Experience of the hospital center of Versailles (1997-2001)]. / Paludisme d'importation de l'enfant. Expérience du centre hospitalier de Versailles (1997-2001).

UNLABELLED: Few data are available about pediatric imported malaria, whereas the number of cases seems in constant increase. PATIENTS AND METHODS: all pediatric malaria cases diagnosed by a positive thin or thick blood film at the Versailles Hospital, from January 1997 to December 2001, were studied retrospectively. RESULTS: sixty cases of pediatric imported malaria were studied. They were 58 cases of uncomplicated malaria and 2 cases of severe malaria; 85% of the children had travelled to sub-Saharan Africa and 15% to Oceania; 90% of the children were of African origin. Plasmodium falciparum was found alone in 84% of the cases. The anti-malarial chemoprophylaxis was inappropriate in 92% of the cases. No child had profited from preventive measures against mosquitos. Fever > 37,5 degrees C was observed in 100% of the cases. The other clinical signs were present in less than 50% of the cases. The median of haemoglobin and platelet was 10.5 g/dL and 141,000/mm(3), respectively. After treatment, the evolution was good in all the cases, without relapse or any consequences. DISCUSSION/CONCLUSION: our study, in agreement with the national data, confirms the increase in the number of case of pediatric imported malaria, and underlines the mediocrity of the prevention, in particular in term of anti-malarial chemo-prophylaxis. These data, in a context of regular increase of international travels to endemic areas, suggest the necessity to improve the information of the general public, and the urgency of a better staff training of health care workers concerning malaria, in order to improve the prevention and the treatment of this potentially fatal disease.

[Risk factors and screening strategy for group B streptococcal colonization in pregnant women: results of a prospective study]. / Facteurs de risque et stratégie de dépistage de la colonisation par le streptocoque du groupe B chez la femme enceinte: résultats d'une étude prospective.

OBJECTIVES: Group B Streptococcus (GBS) continues to be the most important bacterial cause of sepsis, meningitidis in newborns. American consensus guidelines have been published. They recommended the use of either risk-based strategy or screening-based approach for GBS colonization in pregnant women to identify candidates for intrapartum prophylaxis. Screening consists of obtaining vaginal and anorectal specimens for culture at 35 to 37 weeks' gestation. The aim of this prospective study was to assess the usefulness of systematic and concomitant GBS screening by rectal and vaginal swab in pregnant women. A questionnaire designed to determine the risk factors for colonization by GBS was completed. MATERIALS AND METHODS: We have screened 370 pregnant women with rectal and vaginal swab. RESULTS: Fifty seven (15.4%) women had positive GBS cultures. Of those women, the rectum and the vagina were the only site of colonization in 16 (4.3%) and 8 (2.2%) women respectively. None of the factors studied was significantly associated with GBS colonization. CONCLUSION: Detection of GBS is enhanced by 40% by using vaginal and anorectal swabs compared to a vaginal swab alone. No studied factor appeared to predict GBS colonization, which incited us to screen all pregnant women.

E-test antibiotics susceptibility of strict anaerobic bacteria.

The E-test is convenient for testing susceptibility of anaerobes. From September 1998 to September 1999, 194 strains (105 Gram-positive bacteria, 89 Gram-negative bacteria) of clinically relevant samples were tested against five antibiotics benzylpenicillin, amoxicillin-clavulanic acid, clindamycin, metronidazole and imipenem on blood agar plates. Resistance to benzyl penicillin is widespread and Gram-negative bacteria and resistance to amoxicillin-clavulanic acid is exceptional. Metronidazole is very effective against anaerobes except non-spore-forming aerotolerant Gram-positive rods and Peptostreptococcus micros.

[Routine use of gene amplification for pertussis diagnosis in children]. / Utilisation en routine de l'amplification génique pour le diagnostic de coqueluche chez l'enfant.

OBJECTIVES: The resurgence of whooping cough observed in France convinced us to develop a specific PCR assay to detect B. pertussis in nasopharyngeal secretions in parallel of the culture. The aim of our study was to show the value of the PCR in routine diagnosis. MATERIAL AND METHODS: From November 1996 to August 2000, in two hospitals located in the Yvelines (France), the children consulting for a cough compatible with the diagnosis of whooping cough were included in this study. A questionnaire including clinical, biological and radiological items was completed for each one of these patients. A culture of Bordetella and a detection by PCR of B. pertussis were carried out on each nasopharyngeal aspirate. The diagnosis of whooping cough was retained if the detection was positive in PCR and/or culture. RESULTS: Among the 215 investigated children with suspected cases of whooping cough, the diagnosis was positive for 45 (20.9%), of which 39 were less than one year old (median: three months). Sixteen (35.5,%) were positive at the same time for both the PCR and the culture, 26 (57.8%) for only PCR and three (6.7%) for only culture. The PCR was positive in 93.3% of the cases. The results were obtained with an average time of 48 hours. The culture was positive in 61.2% of the cases with an average time of six days. The monthly distribution of the cases of whooping cough was very inhomogeneous and of epidemic appearance. The majority of the cases was located between two periods: 42% between November 1996 and September 1997 and 40% between November 1999 and August 2000. Among the infected children, 15 were less than two months old and were not yet vaccinated; among the 24 others infants, a delay in the vaccine calendar was noted in 50% of the cases. Four children between six and 14 years old were correctly vaccinated. The evolution was favourable in all the children. CONCLUSION: The PCR due to its sensitivity, its specificity and its rapidity offers to the clinician a powerful tool for the diagnosis of whooping cough. Nevertheless, the culture must be associated with the PCR, in order to follow the epidemiology and the sensitivity to antibiotics of B. pertussis.

[Detection of circulating Candida albicans mannan and antimannan antibodies: useful for diagnosis of deep seated candidiasis]. / Intérêt de l'antigénémie mannane, du dosage des anticorps totaux par Elisa Platelia dans le diagnostic des infections profondes à Candida.

In deep seated candidiasis, only 40% of blood cultures are positive. The aim of the study was to investigate circulating Candida albicans mannan and anti-mannan antibodies as a possible help for the diagnosis of deep seated candidiasis. We have compared the results to the detection of IgM by Elisa and antibodies by immunoflourescence. The best tests, in accord to their sensitivity and specificity, are the mannan antigenemia (43% and 100%) and IgM (86% and 100%) and have to be used together.

Susceptibility of Enterobacteriaceae to beta-lactam agents and fluoroquinolones: a 3-year survey in France.

OBJECTIVE: To assess trends in the susceptibility to beta-lactam agents and to fluoroquinolones of clinically relevant Enterobacteriaceae isolated over a 3-year period in 14 French hospital laboratories. METHODS: During the second quarter of 1996, 1997 and 1998, 180 consecutive non-duplicate isolates of Enterobacteriaceae were collected in each center. Sixteen beta-lactams and four quinolones were tested by the disk diffusion method. In addition, the double-disk synergy test was used to screen for the production of extended-spectrum beta-lactamase (ESBL). RESULTS: Totals of 2507, 2312 and 2506 clinical isolates were obtained in each period, respectively. The distribution of Enterobacteriaceae species according to clinical specimens and wards was similar in each study period. No significant variation in the susceptibility rates to beta-lactams and fluoroquinolones was observed, except in Klebsiella pneumoniae and Enterobacter aerogenes. The prevalence of ESBL-producing isolates decreased from 18% to 9% in the former, while it increased from 32% to 54% in the latter. At the same time, the susceptibility to ofloxacin and pefloxacin increased for K. pneumoniae (P < 0.003) and cephalosporinase-producing species (P < 0.05), except Enterobacter spp. CONCLUSION: Over the 3-year study period beta-lactams and fluoroquinolones remained highly active against Enterobacteriaceae clinical isolates, with the exception of E. aerogenes, probably as a result of the dissemination of multiresistant clones in French hospitals.

[Evaluation of Hemofast MRSA for identification of Staphylococcus aureus and detection of methicillin-resistance staphylococci, directly in blood culture broths]. / Evaluation d'Hémofast MRSA pour l'identification de Staphylococcus aureus et la détection des staphylocoques résistants à la méticilline, directement à partir des bouillons d'hémocultures.

The newly developed Hemofast MRSA system for Staphylococcus aureus identification and methicillin-resistant staphylococci (MRS) detection in blood culture broths was evaluated in 106 Bactec broths containing grapelike clusters of Gram-positive cocci. All 26 S. aureus positive broths were correctly identified by Hemofast MRSA within two hours, and 81% were identified within one hour. Sensitivity and specificity were both 100%. Accuracy of MRS detection was 100%, i.e., no discrepancies versus the agar diffusion method were found. When the 28 broths containing coagulase negative staphylococci (CNS) were tested using an inoculum ten fold larger than for S. aureus testing, a number of discrepancy were recorded. For 49 other broths containing CNS, accuracy was 98.5% when the test was interpreted based on the growth rate of the strain, according to the manufacturer's instructions. One broth containing S. epidermidis strain susceptible yielded a false result with Hemofast MRSA, indicating that it probably contained a contaminant. The other discrepancies occurred with specimens containing mixed populations with CNS or a Micrococcus strain. Most (85%) results were available within 4 hrs 30 min, irrespective of the S. aureus or CNS strains. Avoiding the isolation step on agar, Hemofast MRSA saves 24 to 48 hours, thus allowing earlier antistaphylococcal treatment.

[Identification of Staphylococcus aureus within 2 hours in blood culture broths]. / Identification en 2 heures de Staphylococcus aureus dans les bouillons d'hémocultures.

Staphylococcus aureus identification is one of the priorities of the microbiological diagnosis of the staphylococcal infections. Current identification methods are carried out after a first step of colony isolation on agar media. We describe a fluorogenic method for S. aureus identification, which is directly applied to blood culture broths. This method uses a gel tube which allows an optimized microculture of the bacteria. 129 clinical samples of blood cultures (HEC) containing gram positive cocci in grapelike clusters (35 Vital bottles, 94 Bactec bottles), and 77 inoculated blood culture (HE) with collection strains of S. aureus are included in the study. Bacteria are concentrated and separated from other components of sample in the gel tube. Staphylococci are revealed during a microculture in the gel phase, by using a colorimetric substrate of their dehydrogenases. Then, staphylococci are recovered in an adapted culture medium containing human prothrombin and a fluorogenic substrate, which is specific for the staphylocoagulase. After 1 to 2 h incubation at 37 degrees C, a blue fluorescence shows the presence of S. aureus. Among the 40 HEC containing S. aureus the test is positive for 37 samples. For 3 cases, the test is not interpretable, due to non lysis red blood cells in the gel phase of the tube. No false positive result is observed for the HEC containing coagulase-negative staphylococci. Moreover, our method is positive with the 77 HE. 94.7% of tested samples (HEC and HE) show a fluorescence after only one hour and half. Sensibility and specificity are both 100%. We propose a rapid method for S. aureus identification directly applied to blood culture broths. This method saves 24 h, avoiding the isolation step on agar. Therefore, the treatment of staphylococcal infections and possible isolation measures could earlier set up.

[A chromogenic method for rapid identification of Staphylococcus aureus]. / Méthode chromogénique d'identification rapide de Staphylococcus aureus.

Staphylococcus aureus is responsible for septicaemia and serious nosocomial infections. A rapid and specific identification of this species is of great importance in clinical microbiology. Current methods for S. aureus identification require a 18 to 24 h-incubation. We describe a two hour-identification method based on the detection of the staphylocoagulase, using human prothrombin and a chromogenic substrate. 242 staphylococcal strains (160 S. aureus, 82 coagulase-negative staphylococci (CNS)) were collected from 4 French hospitals. They have been identified by the following methods: (i) clotting of citrated rabbit plasma, which is considered as reference method; (ii) biochemical tests (Rapidec Staph and Api Staph or ID 32 Staph); (iii) and agglutination test (Pastorex Staph or Pastorex Staph-plus). A strain of S. intermedius was provided by the Collection of the Pasteur Institute (Paris). An adapted culture medium is inoculated with staphylococci and adjusted to 2 Mac Farland unities. This medium is then mixed to an equal volume with a human prothrombin solution and the chromogenic substrate. After 1 to 2 hours incubation at 37 degrees C, the strength of the yellow colour of the mixture is observed to the naked eye, or measured at 405 nm with a spectrophotometer. Fifteen chromogenic tripeptides having a thrombin-like affinity and paranitroanilin as leaving group were compared. With the substrate which has the higher hydrolysis velocity and enzymatic affinity (SQ149), all S. aureus strains gave a positive result: 94.7% of the methicillin-susceptible S. aureus were detected after 1 hour incubation, but only 52.3% of the methicillin-resistant S. aureus. 98.4% of the methicillin-resistant S. aureus were detected after 2 hours. No false positive result was observed for the 82 CNS strains. The chromogenic method shows good within-run and day-to-day precision tests. It doesn't need any complementary test. The sensitivity and the specificity are 99.4% and 100% respectively.

[An Acinetobacter baumanii outbreak at the Versailles Hospital Center]. / Investigation d'une épidémie à Acinetobacter baumannii au Centre Hospitalier de Versailles.

A. baumannii is a multiresistant bacteria which is recognised as responsible for nosocomial infections and hospital outbreaks. The control of these outbreaks depends on the strain's typing and on the fight's policy against nosocomial infections. An outbreak of A. baumannii is occurred to patients who were hospitalized in Centre Hospitalier de Versailles. To investigate this outbreak, we have determined the biotype (Bouvet's method), the succeptibility pattern (disk diffusion and agar dilution results were analysed with the hierarchical classification and main component analysis) and the total DNA macrorestriction pattern (Pulse Field Gel Electrophoresis using SmaI restriction enzyme). A risk factors for A. baumannii acquisition were delineated in case-control study. During 2 years, 38 patients have been infected or colonized to A. baumannii. Thirty two patients were hospitalized in ICU. We studied 38 non repetitive clinical isolates and 9 strains of the patient's rooms. Four biotypes were defined by the Bouvet's typing method. Fourteen groups were obtained when succeptibility results were analysed with the hierarchical classification and 6 with the main composant analysis. The molecular typing permit us to define 4 epidemic and 6 sporadic strains. All the epidemic strains were isolated on ICU hospitalized patients. Our study has shown wide contamination in patient's rooms (Water tap, dry surfaces, patient's mattresses...). Environmental objects have been a major risk factor for A. baumannii acquisition. The control of this outbreak has been possible by application of hygienic measures (hands washing, isolment, meticulous cleaning of the ICU and environmental controls). No new case is occurred in the last year. Typing methods and case-control study are necessary to investigate cross-infections and take efficient measures against these outbreaks.