Evaluation of the MRSA/SA ELITe MGB Assay for the Detection of Staphylococcus aureus in Bone and Joint Infections.

Bone and joint infections represent a potentially devastating complication of prosthetic orthopedic joint replacement, thus requiring both rapid and appropriate antibiotic treatment. Staphylococcus aureus is one of the most common pathogens involved in this pathology. Being able to assert its presence is the first step of efficient patient management. This monocenter study evaluated the MRSA/SA ELITe MGB assay for the molecular detection of S. aureus and methicillin-resistant S. aureus (MRSA) in bone and joint biopsy specimens and synovial fluids. This test, together with conventional techniques, including standard cultures and the 16S rRNA amplification assay, was performed on 208 successive perioperative samples collected prospectively for 1 year obtained from 129 patients. Using conventional techniques, we detected a microbial pathogen in 76 samples from 58 patients, 40 of which were identified as S. aureus. The limit of detection (LOD) of the MRSA/SA ELITe MGB assay was experimentally determined for bone and joint biopsy specimens and synovial fluids using negative samples spiked with S. aureus ATCC 43300. The sensitivities of S. aureus detection with the MRSA/SA ELITe MGB assay were 82.5% (33/40 samples) and 97.5% (39/40 samples) using the manufacturer's LOD and an experimentally determined LOD, respectively. Interestingly, using the osteoarticular specific LOD, 15 additional samples were determined to be positive for S. aureus DNA with the MRSA/SA ELITe MGB assay; in all cases, these samples were obtained from patients considered to be infected with S. aureus according to their clinical and microbiological records. The results were available within 24 h, which could help to expedite therapeutic decisions.

Intracellular activity of antimicrobial compounds used for Staphylococcus aureus nasal decolonization.

Background: Staphylococcus aureus is able to invade mammalian cells during infection and was recently observed inside nasal mucosa of healthy carriers. Objectives: To determine the intracellular activity of antimicrobial compounds used for decolonization procedures using a cell model mimicking S. aureus nasal epithelium invasion. Patients and methods: HaCaT cells and human nasal epithelial cells (HNECs) recovered from nasal swabs of S. aureus carriers were visualized by confocal laser scanning microscopy to detect intracellular S. aureus cells. An HaCaT cell model, mimicking S. aureus internalization observed ex vivo in HNECs, was used to assess the intracellular activity against S. aureus of 21 antimicrobial compounds used for nasal decolonization, including mupirocin and chlorhexidine. Results: HaCaT cells and HNECs were found to internalize S. aureus with the same focal pattern. Most antimicrobial compounds tested on HaCaT cells were shown to have weak activity against intracellular S. aureus. Some systemic antimicrobials, including fusidic acid, clindamycin, linezolid, minocycline, ciprofloxacin, moxifloxacin, rifampicin and levofloxacin, reduced S. aureus intracellular loads by 0.43-1.66 log cfu/106 cells compared with the control (P < 0.001). By contrast, mupirocin and chlorhexidine reduced the S. aureus intracellular load by 0.19 and 0.23 log cfu/106 cells, respectively. Conclusions: These data indicate that most of the antimicrobial compounds used for nasal decolonization, including mupirocin and chlorhexidine, exhibit weak activity against intracellular S. aureus using the HaCaT cell model. This work emphasizes the need to better understand the role of the S. aureus intracellular reservoir during nasal colonization in order to improve decolonization procedures.

Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples.

AIMS: Legionella isolation from environmental samples is often difficult because of the presence of heterotrophic-associated bacteria that frequently overgrow when using standard culture (ISO 11731, 1998; NF T90-431, 2003) methods. To improve Legionella pneumophila recovery from complex water samples (water from cooling towers, biofilms), we evaluated an immunomagnetic separation (IMS) assay using a monoclonal antibody raised against the lipopolysaccharide of Leg. pneumophila sg1 in combination with culture. METHODS AND RESULTS: This study was conducted on 51 environmental specimens. The comparison between IMS-culture and standard culture (ISO 11731, 1998; NF T90-431, 2003) methods was made using ISO 17994, 2004 criteria for establishing equivalence between microbiological methods based on the upper and lower (XH and XL) values of the relative difference (95% confidence limit) and D as maximum acceptable deviation (value of the confidence limit). CONCLUSIONS: We found that the average performance of IMS culture was higher than the reference method.

Is nasal carriage of Staphylococcus aureus the main acquisition pathway for surgical-site infection in orthopaedic surgery?

The endogenous or exogenous origin of Staphylococcus aureus, responsible for orthopaedic surgical-site infections (SSI), remains debated. We conducted a multicentre prospective cohort study to analyse the respective part of exogenous contamination and endogenous self-inoculation by S. aureus during elective orthopaedic surgery. The nose of each consecutive patient was sampled before surgery. Strains of S. aureus isolated from the nose and the wound, in the case of SSI, were compared by antibiotypes or pulsed-field gel electrophoresis (PFGE). A total of 3,908 consecutive patients undergoing orthopaedic surgery were included. Seventy-seven patients developed an SSI (2%), including 22 related to S. aureus (0.6%). S. aureus was isolated from the nose of 790 patients (20.2%) at the time of surgery. In the multivariate analysis, S. aureus nasal carriage was found to be a risk factor for S. aureus SSI in orthopaedic surgery. However, only nine subjects exhibiting S. aureus SSI had been found to be carriers before surgery: when compared, three pairs of strains were considered to be different and six similar. In most cases of S. aureus SSI, either an endogenous origin could not be demonstrated or pre-operative nasal colonisation retrieved a strain that was different from the one recovered from the surgical site.

Hospital-acquired infections documented by repeated annual prevalence surveys over 15 years.

OBJECTIVE: To estimate the benefits of iterative prevalence surveys in detecting trends of hospital-acquired infections (HAIs). METHODS: On the basis of the French protocol for national prevalence studies, HAI data of 15 consecutive annual surveys performed at the same period by the same group of investigators was gathered in a single database to describe the trend of HAIs in a University Hospital over a 15-year period. RESULTS: A total of 20,401 patients were included. Overall, the prevalence of patients presenting with at least one HAI acquired in our University Hospital was 5.1% [95% CI, 4.8-5.4%]. The prevalence of HAIs and antimicrobial drug use significantly decreased over time (P<0.01). CONCLUSION: Despite limitations, repeated prevalence surveys can be a useful tool for promoting control measures to better prevent HAIs.

Decolonization of Staphylococcus aureus carriage.

Staphylococcus aureus nasal colonization is a well-known independent risk factor for infection caused by this bacterium. Screening and decolonization of carriers have been proven effective in reducing S. aureus infections in some populations. However, a gap remains between what has been proven effective and what is currently done. We aimed to summarize recommendations and current knowledge of S. aureus decolonization to answer the following questions: Why? For whom? How? When? And what are the perspectives?

Carbapenemase-producing Acinetobacter baumannii: An outbreak report with special highlights on economic burden.

OBJECTIVE: We aimed to describe the management of a carbapenemase-producing Acinetobacter baumannii (CP-AB) outbreak using the Outbreak Reports and Intervention Studies of Nosocomial Infection (ORION) statement. We also aimed to evaluate the cost of the outbreak and simulate costs if a dedicated unit to manage such outbreak had been set-up. METHODS: We performed a prospective epidemiological study. Multiple interventions were implemented including cohorting measures and limitation of admissions. Cost estimation was performed using administrative local data. RESULTS: Five patients were colonized with CP-AB and hospitalized in the neurosurgery ward. The index case was a patient who had been previously hospitalized in Portugal. Four secondary colonized patients were further observed within the unit. The strains of A. baumannii were shown to belong to the same clone and all of them produced an OXA-23 carbapenemase. The closure of the ward associated with the discharge of the five patients in a cohorting area of the Infectious Diseases Unit with dedicated staff put a stop to the outbreak. The estimated cost of this 17-week outbreak was $474,474. If patients had been managed in a dedicated unit - including specific area for cohorting of patients and dedicated staff - at the beginning of the outbreak, the estimated cost would have been $189,046. CONCLUSION: Controlling hospital outbreaks involving multidrug-resistant bacteria requires a rapid cohorting of patients. Using simulation, we highlighted cost gain when using a dedicated cohorting unit strategy for such an outbreak.

Molecular dynamics of Staphylococcus aureus nasal carriage in Hajj pilgrims.

During the 2012 Hajj season, the risk of acquisition of Staphylococcus aureus nasal carriage in a cohort of French pilgrims was 22.8%, and was statistically associated with the acquisition of viral respiratory pathogens (p 0.03). The carriage of S. aureus belonging to the emerging clonal complex 398 significantly increased following the pilgrimage (p < 0.05).

Epidemiologic and molecular investigations of genital mycoplasmas from women and neonates at delivery.

The rates of colonization by Ureaplasma urealyticum and Mycoplasma hominis were evaluated in 208 women at delivery and in their neonates. Mycoplasmas were isolated from the cervicovaginal specimens of 100 mothers (48.1%) and from the gastric secretions of 40 neonates (19.2%). The prevalences of U. urealyticum and M. hominis were 47.6% and 11.0% in women and 19.2% and 1.0% in neonates, respectively. Premature rupture of membranes was significantly associated with colonization of women by U. urealyticum (P = 0.031), and colonization of their neonates by U. urealyticum (P = 0.002) and/or M. hominis (P = 0.023). Forty-four selected strains of mycoplasmas were further characterized by arbitrarily primed polymerase chain reaction. All strains of U. urealyticum belonged to the parvo biovar of the species. Arbitrarily primed polymerase chain reaction demonstrated the similarity of strains isolated from mother-neonate pairs, confirming the importance of vertical transmission of mycoplasmas at delivery.

Characterization of nosocomial strains of Enterobacter aerogenes by arbitrarily primed-PCR analysis and ribotyping.

OBJECTIVE: To study the spread of strains of Enterobacter aerogenes in our hospital in 1992 and 1993 by using two genotypic markers, and to evaluate these methods for the epidemiological investigation of this species. DESIGN: Ribotyping (using two endonucleases) and arbitrarily primed (AP)-PCR (using two different 10-mer primers) were applied to the epidemiological typing of clinical strains of E aerogenes isolated from hospitalized patients. SETTING AND PATIENTS: The intensive care unit (ICU; 5 patients, 13 isolates), nephrology units (3 patients, 5 isolates), and surgery units (2 patients, 2 isolates) of the university hospital of Saint-Etienne (France). RESULTS: Eight epidemiologically unrelated isolates, chosen as controls, exhibited distinct profiles, both by AP-PCR and ribotyping. Two clones of E aerogenes circulated in the ICU; both were isolated successively from samples of a single patient who stayed in the unit for almost 1 year. A third clone was recovered from patients of surgery units. A fourth clone was shown to have infected patients of nephrology units. CONCLUSIONS: Ribotyping and AP-PCR appear to be reliable methods for typing E aerogenes strains implicated in nosocomial infection. The spread of independent clones of E aerogenes in different units of our hospital in 1992 and 1993 was demonstrated by both methods. This study emphasizes the need to choose the endonucleases or primers with care to obtain high discriminatory results in genotypic investigations.

Investigation of a nosocomial outbreak due to Serratia marcescens in a maternity hospital.

OBJECTIVES: To investigate an outbreak of Serratia marcescens in a maternity hospital (November 1994 to May 1995). DESIGN: Retrospective analysis of epidemiological data and prospective study of systematic bacteriological samples from patients and environment, with genotyping of strains by arbitrarily primed polymerase chain reaction. SETTING: A private maternity hospital, Saint-Etienne, France. RESULTS: In the neonatal unit, 1 newborn developed a bacteremia, and 36 were colonized in stools with S marcescens. As the colonization of some newborns was shown to occur only a few hours after delivery, the inquiry was extended to other maternity wards, where 8 babies and 4 mothers were found to be colonized. Environmental sampling led to the isolation of S marcescens from a bottle of enteral feed additive in the neonatal unit and from the transducers of two internal tocographs in the delivery rooms. The genotyping of 27 strains showed two different profiles: a major epidemic profile shared by 22 strains (18 from babies of the neonatal unit, 2 from babies of other units, and 2 from breast milk) and another profile shared by 5 strains (2 from transducers of internal tocographs, 2 from babies, and 1 from a mother). The strain isolated from lipid enteral feeding was not available for typing. Although this source of contamination was removed soon from the neonatal unit, the outbreak stopped only when infection control measures were reinforced in the delivery rooms, including the nonreuse of internal tocographs. CONCLUSIONS: In delivery rooms, the quality of hygiene needs to be as high as in surgery rooms to prevent nosocomial colonization or infection of neonates at birth.

Nosocomial colonization of premature babies with Klebsiella oxytoca: probable role of enteral feeding procedure in transmission and control of the outbreak with the use of gloves.

OBJECTIVE: To investigate the persistence of colonization of premature babies by Klebsiella oxytoca, with special emphasis on the mode of transmission of the bacterium and evaluation of Standard Precautions to stop the epidemic. DESIGN: Retrospective analysis of cases and prospective study of systematic bacteriological samples (stools and throat) from babies, healthcare workers (HCWs), and environment, with genotyping of strains by arbitrarily primed polymerase chain reaction. SETTING: A premature baby unit (PBU) and a neonatal intensive care unit in the university hospital of Saint-Etienne, France. RESULTS: An outbreak of K oxytoca was suspected in two pediatric wards after the occurrence of a fatal bacteremia in a newborn hospitalized in the PBU and the colonization of other babies 2 months later. Retrospective analysis showed that 24 babies' digestive tract had been colonized. No environmental reservoir was recovered in the units nor in enteral feeding. No K oxytoca was isolated from HCW samples. Genotyping confirmed the presence of epidemic strains, although independent clones were responsible for infections or colonizations in each of the two units. The chronology and the site of babies' colonization (isolation of K oxytoca in stools before throat) were determined during a prospective study and suggested that enteral feeding procedures could be the source of contamination. Therefore, use of gloves during this practice by HCWs was recommended and, after readjustment of Standard Precautions, stopped the outbreak. CONCLUSION: To prevent cross-contamination among high-risk babies, careful attention must be paid to Standard Precautions. Bacteriological surveillance of the digestive tract of neonates could help to check compliance with these guidelines

Prospective study of nosocomial colonization and infection due to Pseudomonas aeruginosa in mechanically ventilated patients.

OBJECTIVE: To investigate the respective contribution of endogenous and exogenous transmission of Pseudomonas aeruginosa in the colonization of lungs in the mechanically ventilated patient, to estimate the role of P. aeruginosa colonization in the occurrence of severe infections, and to extrapolate appropriate control measures for the prevention of P. aeruginosa ventilator-associated pneumonia. DESIGN: Prospective study of the presence of P. aeruginosa (in stomach fluid, throat specimens, stool, and sputum) on admission, twice a week throughout the patient's stay, and in their environment. O-serotyping, pulsed-field gel electrophoresis, and arbitrarily-primed polymerase chain reaction were used to characterize the strains. SETTING: The two intensive care units (ICUs 1 and 2) of a university hospital. PATIENTS: During a 6-month period, 59 patients were included (21 in ICU 1 and 38 in ICU 2). RESULTS: P. aeruginosa was isolated in 26 patients, including ten pneumonia cases and seven colonizations on admission. The incidence of acquired colonization was statistically different between the two ICUs: 5.5 and 20.5 per 1000 days of mechanical ventilation, in ICUs 1 and 2, respectively. Endogenous acquisition was the main origin of P. aeruginosa colonization (21 of 26 patients) and the upper respiratory tract was the main bacterial reservoir in broncho-pulmonary colonization and infection. However, during the 6-month period of the study, a multidrug-resistant strain of P. aeruginosa O:11, isolated in the sink of the room of 12 patients, was found responsible for two colonizations (1 digestive, 1 throat/lungs) and one pneumonia. As a whole, from 26 cases of colonization/infection with P. aeruginosa, 5 were related to an exogenous contamination (environmental reservoir in 4 patients and cross-contamination in one patient). CONCLUSIONS: These results emphasize the need for applying various infection control measures to prevent colonization of patients with P. aeruginosa, including strategies to limit the potential of sinks from acting as a source or reservoir for this bacterium.

Differentiation of Pseudomonas aeruginosa strains by ribotyping: high discriminatory power by using a single restriction endonuclease.

The genotypic diversity of 40 presumably epidemiologically unrelated strains of Pseudomonas aeruginosa belonging to nine different O-serotypes was analysed according to ribosomal DNA fingerprints. Ribotyping was performed with a digoxigenin-labelled DNA probe and four restriction endonucleases. Characteristic banding patterns of three to 12 bands were obtained with the different endonucleases. Among the 40 strains, eight, nine, 10 and 29 different ribotypes were differentiated with EcoRI, the combination EcoRI+HindIII, BamHI and PvuII, respectively. Poor correlations were noted between the results of serotyping and those of ribotyping. With the latter method, indices of discrimination were calculated for each enzyme from the data of the 40 unrelated strains: the values ranged from 0.678 for EcoRI to 0.979 for PvuII. Epidemiologically related samples were also tested; this enabled assessment of whether the method was able to cluster strains from a common origin with each of the enzymes tested. Ribotyping with PvuII endonuclease is proposed for screening large numbers of P. aeruginosa strains in epidemiological studies. Additional enzymes could be used to further increase the discrimination between isolates found to be indistinguishable with PvuII enzyme.

Competition binding studies with biotinylated echovirus 11 in cytofluorimetry analysis.

Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.

Evaluation of a prototype HCV NS5b assay for typing strains of hepatitis C virus isolated from Tunisian haemodialysis patients.

Hepatitis C virus (HCV) strains isolated from 68 haemodialysis Tunisian patients exhibiting chronic infection were genotyped targeting the NS5b region of the HCV genome using a prototype assay developed by Bayer HealthCare-Diagnostics (TRUGENE NS5b HCV). The overall results were compared to those obtained with another assay of the same company based on sequencing of the 5' non-coding region (TRUGENE HCV 5'NC genotyping kit). All strains could be typed by the 5'NC typing kit, but only 62 (91; 2%) by the NS5b prototype assay. All the 62 strains typed by both methods exhibited the same pattern at the type level: 57 were type 1, 3 were type 2, and 2 were type 4. At the subtype level, eight strains that gave undetermined results by the 5'NC kit were successfully typed by the NS5b kit; eight additional strains exhibited discrepant results. The overall agreement between the two assays was 74.2% at the subtype level. In conclusion, the NS5b region appears to be much more accurate than the 5'NC region to subtype HCV strains, especially in those isolated from patients attending haemodialysis centres where the subtype distribution suggests frequent nosocomial transmissions.

Detection of GB virus C/hepatitis G virus in semen and saliva of HIV type-1 infected men.

OBJECTIVE: To investigate the presence of the genome of GB virus C/hepatitis G virus (GBV-C/HGV) in semen and saliva from HIV-1-infected men. METHODS: Samples of blood from 33 men seropositive for HIV-1 were tested for the presence of GBV-C/HGV markers of infection, RNA by RT-PCR, and anti-E2 antibodies by ELISA, respectively. The cell-free fractions of seminal fluid and saliva samples of the patients with positive blood samples for GBV-C/HGV RNA or anti-E2 antibodies were then analyzed for the presence of the RNA of this virus. In addition, six semen samples and 11 saliva samples from GBV-C/HGV-negative men were tested. RESULTS: The GBV-C/HGV RNA tested by RT-PCR was recovered from blood in 11 patients of 33 (33.3%), and the antibodies to E2 envelope protein were detected in six patients (18.2%). Since no patient was positive for both markers, the overall prevalence of GBV-C/HGV infection was 51.5% in the studied population. Four-all belonging to the homosexual risk group-of the 17 men with markers to GBV-C/HGV in blood were found to be positive for GBV-C/HGV RNA in mucosal samples: two of them exhibited genomic RNA in both semen and saliva, and two others were positive for semen only. The absence of inhibitors of the PCR technique was confirmed in all mucosal fractions found negative for GBV-C/HGV RNA, except for one saliva sample and one seminal fluid sample. CONCLUSION: These results confirm the high prevalence of GBV-C/HGV infection in patients infected with HIV-1 by sexual exposure and the presence of GBV-C/HGV RNA in seminal fluid and saliva of men with markers of this virus in the blood, suggesting that mucosal fluids could be a potential source for the spread of the GBV-C/HGV infection.

Ventilator temperature sensors: an unusual source of Pseudomonas cepacia in nosocomial infection.

A prospective study was undertaken to determine the source of Pseudomonas cepacia colonization and infection that had affected ventilated patients in an Intensive Care Unit (ICU) for three years. Thirty-eight patients undergoing mechanical ventilation were enrolled during a six-week period. Samples were taken from patients, ventilator circuits and the environment for culture. P. cepacia was isolated from the condensate formed in the ventilator circuit and the source of the contamination was shown to be the temperature sensor. Ribotyping of the representative strains of P. cepacia performed with two endonucleases, EcoRI and PvuII, confirmed the homogeneity of the isolates from patients and ventilator circuits. A modification of the procedure for disinfection of the temperature sensors resulted in the eradication of P. cepacia from the ICU.

[Tolerance of influenza vaccination in the aged and the nursing staff in a geriatric hospital]. / Tolérance de la vaccination grippale chez les sujets âgés et le personnel soignant d'un hôpital gériatrique.

The tolerance of influenza vaccination (Vaxigrip, Pasteur-Mérieux) was evaluated during four consecutive years (1989-1992) in the geriatric hospital of Saint-Etienne from questionnaires concerning 327 vaccinations in the aged (group 1) and 88 vaccinations in members of the nursing staff (group 2). Minor local symptoms were the more common incidents, respectively for each group: blotch (9.8 and 21.6%), pain (9.2 and 47.7%), nodule (2.4 and 13.6%). Fatigue (4.0 vs 12.5%) and fever (6.1 vs 4.5%) were the more frequent among general symptoms, respectively in each group. No major vaccinal accident was recorded. These results underline that influenza vaccination is well tolerated, much more in aged people than in members of the nursing staff.

Acute encephalitis associated to a respiratory infection due to Chlamydophila pneumoniae.

OBJECTIVE: Chlamydophila pneumoniae is a common agent of respiratory infections. Severe acute neurological infections are very infrequently linked to this bacterium. We report such a case and give a rapid overview of published cases of acute encephalitis occurring after a respiratory infection due to C. pneumoniae. PATIENT AND METHODS: A 12-year-old child without any prior medical history was hospitalized for encephalitis associated to respiratory symptoms. RESULTS: C. pneumoniae DNA was identified by multiplex PCR assay in respiratory secretions and C. pneumoniae IgM and IgG antibodies were assessed in the serum. This bacterium was not detected in CSF, nor was any other pathogen. A macrolide treatment was prescribed for two weeks. The outcome was good without any sequels. CONCLUSIONS: This observation correlates to the few similar cases reported in the medical literature. C. pneumoniae must be suggested in the etiological diagnosis of acute encephalitis, notably in a context of respiratory infection, when no more common cause can be identified.