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.

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.

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.

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?

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).

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.

Nosocomial transmission of NDM-1-producing Escherichia coli within a non-endemic area in France.

Two patients with no travel history and sharing the same room were colonized by the same strain of New Delhi metallo-ß-lactamase 1 (NDM-1)-producing Escherichia coli within a geographical area not endemic for this highly multidrug-resistant bacterium. It was documented an absence of an epidemiological and bacteriological link with a third patient returning from India after surgery and found to be infected by an NDM-1-producing Citrobacter strain during the same period. Despite extensive investigation, the source of contamination of the two former patients was not elucidated. This case report illustrates the need of investigating rapidly the emergence of highly multidrug-resistant Enterobacteriaceae, to stop their dissemination in a nosocomial setting.

An algorithm based on one or two nasal samples is accurate to identify persistent nasal carriers of Staphylococcus aureus.

Persistent Staphylococcus aureus nasal carriers are at high risk of S. aureus infection. The present study delineates a simple strategy aimed at identifying rapidly and accurately this subset of subjects for clinical or epidemiological purposes. Ninety healthy volunteers were each identified as persistent, intermittent or non-nasal carriers of S. aureus by using seven specimens sampled over a 5-week period. By reference to this so-called reference standard, six other strategies aimed at simplifying and speeding the identification of persistent carriers and based on the qualitative or quantitative detection of S. aureus in one to three nasal samples were evaluated by the measure of the area under the curve of receiver operating characteristic diagrams. Among strategies using qualitative results, there was no statistical difference between protocols using seven and three samples. A threshold of 10(3) CFU of S. aureus per swab was found capable of defining persistent nasal carriage with a sensitivity of 83.1% and a specificity of 95.6%. These figures reached 95.5% and 94.9%, respectively, by using an algorithm including one or two nasal specimens according to the threshold of 10(3) CFU of S. aureus in the first swab. The latter two strategies were shown to be costly equivalents. The proposed algorithm-based strategy proved to be relevant to identify properly and consistently persistent nasal carriers of S. aureus. However, as it was built from data of healthy volunteers, it needs to be confirmed prospectively on patients potentially at risk for S. aureus infection.

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.

Evaluation of a total core antigen assay for the diagnosis of hepatitis C virus infection in hemodialysis patients.

Hemodialysis patients are recognized as a group at high risk of infection with hepatitis C virus (HCV). Therefore, such a population should be screened routinely for the presence of HCV viremia. Since nucleic acid techniques remain expensive and largely unavailable in many laboratories in the developing world, the present study assesses the clinical usefulness of the HCV core antigen enzyme immunoassay for the diagnosis of HCV infection in dialysis patients. One hundred seventy-five dialysis patients were screened for the presence of anti-HCV antibodies and HCV RNA in the serum. One hundred twenty-eight serum samples were collected from the 76 patients who were anti-HCV antibody- and/or HCV RNA-positive. These were evaluated for total HCV core antigen. Of these samples, 55 had sufficient volume to be further tested to quantify HCV RNA by reverse transcription polymerase chain reaction (RT-PCR). Genotyping of the HCV strains showed that the majority belonged to genotype 1b (77%). The HCV core antigen assay showed a sensitivity and specificity of 84% and 89%, respectively. The use of core antigen assay has enabled the early detection of three patients who developed an acute hepatitis C infection during the period of study. A correlation study was undertaken between the quantitative values of viral load, expressed as pg/ml of HCV core antigen in serum, and viral RNA in UI/ml. A significant correlation was observed (Pearson's correlation coefficient: 0.552; P<0.001). In conclusion, detection of HCV core antigen in serum is an inexpensive, reliable, and highly specific assay that can be useful in most laboratory settings to diagnose HCV infection, and especially in laboratories where nucleic acid technologies are not yet available.

[Epidemiology of nosocomial infections due to Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia]. / Epidémiologie des infections nosocomiales à Pseudomonas aeruginosa, Burkholderia cepacia et Stenotrophomonas maltophilia.

Non-fermentative Gram negative rods are opportunistic pathogens responsible for nosocomial infections. Using phenotypic markers (serotypes for Pseudomonas aeruginosa and antibiotic susceptibility) allows a preliminary screening of epidemiologically-related strains. However, genotypic markers are necessary to better characterize nosocomial strains for the investigation of outbreaks or cross-transmissions in the hospital setting. Infections due to P. aeruginosa, Burkholderia. cepacia or Stenotrophomonas. maltophilia are usually hospital-acquired and responsible for a high mortality rate as illustrated by the lethality of nosocomial pneumonia due to P. aeruginosa. The severity of these infections is due to the virulence factors of the bacteria and to their occurrence in debilitated patients in whom invasives devices are used. The hospital environment can act as a reservoir with a rate of exogeneous transmission of these bacteria as high as 50% in some studies. To better prevent nosocomial infections related to Gram negative non fermentative rods, the control of the aqueous hospital environment, the strict application of hand disinfection and the investigation of potential cross-transmission in the hospital setting are needed.

Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR.

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 x 10(-3) IFU, 5 x 10(-2) color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study.

This cross-sectional study aimed to investigate, during a short period between 2000 and 2001, in a large population of patients with chronic hepatitis C, the epidemiological characteristics of hepatitis C virus (HCV) genotypes in France. Data from 26 referral centres, corresponding to 1769 patients with chronic hepatitis C were collected consecutively during a 6-month period. HCV genotyping in the 5'-non-coding region (NCR) was performed in each center using the line probe assay (LiPA, in 63% of cases), sequencing (25%) or primer-specific polymerase chain reaction (PCR) (12%). HCV genotypes 1a, 1b, 2, 3, 4, 5, non-subtyped 1 and mixed infection were found in 18, 27, 9, 21, 9, 3, 11 and 1% of our population, respectively. HCV genotype distribution was associated with gender, age, source and duration of infection, alanine aminotransferase (ALT) levels, cirrhosis, alcohol consumption, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection. In multivariate analysis, only the source of infection was the independent factor significantly associated with genotype (P = 0.0001). In conclusion, this study shows a changing pattern of HCV genotypes in France, with i.v. drug abuse as the major risk factor, an increase of genotype 4, and to a lesser extent 1a and 5, and a decrease of genotypes 1b and 2. The modification of the HCV genotype pattern in France in the next 10 years may require new therapeutic strategies, and further survey studies.

Bacterial risk and sperm cryopreservation.

Prior to sperm cryopreservation, French guidelines only recommend viral screening for serological status towards human immunodeficiency virus, hepatitis B and C viruses and Treponema palidum. The probability of semen infection by other bacterial pathogens is not taken into consideration by the current recommendations. The objective of the present study was to evaluate this risk and a strategy to reduce it prospectively. Ninety-six patients consulting for sperm cryopreservation underwent a semen culture simultaneously to cryopreservation. The patients were classified into three groups following semen culture results: negative culture (group 1, 77/96, 80.2%), positive culture with saprophytic agents (group 2, 9/96, 9.4%) and positive culture with pathogen agents (group 3, 10/96, 10.4%). For six patients of the latter group showing a genital infection with Ureaplasma urealyticum, a discontinuous gradient selection performed on the cryopreserved sample was efficient to discard bacteria. These data emphasize the usefulness to cultivate semen simultaneously to cryopreservation and demonstrate the ability to remove some microbial agents from semen before its use in assisted reproductive techniques.

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.

[Epidemic outbreaks control involving Staphylococcus aureus with reduced sensitivity to glycopeptides]. / Contrôle de phénomènes épidémiques dus à des souches de Staphylococcus aureus de sensibilité diminuée aux glycopeptides.

This study describes two epidemic outbreaks involving Staphylococcus aureus with reduced sensitivity to glycopeptides, one in 2000 involving eight patients and the other in 2001-2002 involving 16 patients. These strains were detected rapidly, thanks to routine screening for the offending organisms in the bacteriology laboratory of our hospital. The clonal character of these strains was confirmed by pulsed field electrophoresis. The management of these epidemic outbreaks confirmed (i) the need for systematic adoption of standard precautions, (ii) the importance of circulating information in combating multi-resistant bacteria, as well as the difficulties in transferring colonised patients to different hospital wards, and (iii) the intermittent nature of S. aureus carriage, resulting in a need for prolonged surveillance of colonised and/or infected patients. In addition, our study underlines the value of a multi-disciplinary approach to the management of diffusion of multi-resistant bacteria.

[Possible implication of student nurses in the transmission of methicillin-resistant Staphylococcus aureus during a nosocomial outbreak]. / Implication potentielle d'étudiants infirmiers dans la transmission de Staphylococcus aureus résistant à la méthicilline lors d'une épidémie nosocomiale.

We report an outbreak of infections due to methicillin-resistant Staphylococcus aureus (MRSA) in a medical unit and the possible implication of student nurses in the dissemination of the epidemic strain. A retrospective epidemiological study looking for hospitalised patients colonised or infected with MRSA from the 1st of June to the 30th of September 2001 in the unit was conducted. An audit of delivered cares and a nasal screening of health care workers (HCW) was performed. Six patients were colonised or infected with a MRSA strain, four of them exhibiting a bacteremia. Six HCW had a nasal carriage of MRSA. Typing of the MRSA strains by pulsed field gel electrophoresis demonstrated an epidemic clone isolated from five of six patients, two student nurses and one HCW not implicated in nursing cares. This report illustrates the risk of nosocomial outbreak linked to cares delivered by student nurses.