Aline Wenger

Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.

Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.

Extended-Spectrum β-Lactamases of the CTX-M Type Now in Switzerland

ABSTRACT The epidemiology of clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) was investigated among infection-associated enterobacterial isolates at the University Hospital in Lausanne, Switzerland, from January 2004 to June 2005. Out of 57 nonrepetitive ESBL producers (prevalence rate of 0.7%), 45 produced CTX-M-like ESBLs. CTX-M enzymes were mostly from clonally nonrelated Escherichia coli isolates, from urinary infections and community-acquired infections. Pediatric patients (20 out of 57) accounted for a large number of CTX-M producers. CTX-M-15 was the most frequent CTX-M-type enzyme. The plasmid-located blaCTX-M genes were associated with either ISEcp1 or ISCR1 insertion sequences. This study is the first published report of CTX-M-type β-lactamases in Switzerland.

Exogenous sources of pseudomonas aeruginosa in intensive care unit patients: implementation of infection control measures and follow-up with molecular typing.

BACKGROUND In 1998, a study in the intensive care unit (ICU) of our institution suggested possible transmission of Pseudomonas aeruginosa from faucet to patient and from patient to patient. Infection-control measures were implemented to reduce the degree of P. aeruginosa colonization in faucets, to reduce the use of faucet water in certain patient care procedures, and to reduce the rate of transmission from patient to patient. OBJECTIVE To evaluate the effect of the control measures instituted in 1999 to prevent P. aeruginosa infection and colonization in ICU patients. DESIGN Prospective, molecular, epidemiological investigation. SETTING A 870-bed, university-affiliated, tertiary care teaching hospital. METHODS The investigation was performed in a manner identical to the 1998 investigation. ICU patients with a clinical specimen positive for P. aeruginosa were identified prospectively. Swab specimens from the inner part of the ICU faucets were obtained for the culture on 9 occasions between September 1997 and December 2000. All patients and environmental isolates were typed by pulsed-field gel electrophoresis (PFGE). RESULTS Compared with the 1998 study, in 2000 we found that the annual incidence of ICU patients colonized or infected with P. aeruginosa had decreased by half (26.6 patients per 1,000 admissions in 2000 vs 59.0 patients per 1,000 admissions in 1998), although the populations of patients were comparable. This decrease was the result of the decreased incidence of cases in which an isolate had a PFGE pattern identical to that of an isolate from a faucet (7.0 cases per 1,000 admissions in 2000, vs 23.6 per 1,000 admissions in 1998) or from another patient (6.5 cases per 1,000 admissions in 2000 vs 16.5 cases per 1,000 admissions in 1998), whereas the incidence of cases in which the isolate had a unique PFGE pattern remained nearly unchanged (13.1 cases per 1,000 admissions in 2000 vs 15.6 cases per 1,000 admissions in 1998). CONCLUSIONS These results suggest that infection control measures were effective in decreasing the rate of P. aeruginosa colonization and infection in ICU patients, confirming that P. aeruginosa strains were of exogenous origin in a substantial proportion of patients during the preintervention period.

Evaluation of a Novel Medium for Screening Specimens from Hospitalized Patients To Detect Methicillin-Resistant Staphylococcus aureus

ABSTRACT A novel medium, Oxacillin Resistant Screening Agar (ORSA) medium, was evaluated for the screening of specimens for methicillin-resistant Staphylococcus aureus (MRSA) in the hospital setting. Screening swabs (swabs of the nose, throat, perineum, and infected sites) were inoculated onto the new ORSA medium and into an enrichment broth (Muller-Hinton broth supplemented with NaCl and oxacillin). After 24 h of incubation, the enrichment broth was subcultured onto one ORSA plate and one lipovitellin Chapman salt agar plate. The sensitivities for the detection of MRSA were calculated for each medium alone and for the media in combination. A low sensitivity (74%) was obtained when ORSA medium was used alone as a primary culture, whereas the sensitivity was 88% when a single selective enrichment broth was used. Among the 414 blue colonies observed on ORSA plates, only 47% were found to be MRSA, 40% were coagulase-negative staphylococci, 7% were Enterococcus species, and 2% were methicillin-sensitive S. aureus. The optimal incubation time for the ORSA plates was evaluated. On primary culture, 38% of the blue MRSA colonies were visible only after 48 h of incubation (no blue colonies were not seen after 24 h of incubation), whereas 94% of the colonies were already visible at 24 h when ORSA plates were used for subcultures. In conclusion, the advantage of the novel ORSA medium is the ease of recognition of mannitol-fermenting bacteria, but further identification tests are needed to confirm the identification of S. aureus. An enrichment broth is still needed to ensure a good sensitivity for the recovery of MRSA, and an incubation time of 48 h is required for primary culture on ORSA medium.

Comparison of automated strategies for surveillance of nosocomial bacteremia.

OBJECTIVE Surveillance of nosocomial bloodstream infection (BSI) is recommended, but time-consuming. We explored strategies for automated surveillance. METHODS Cohort study. We prospectively processed microbiological and administrative patient data with computerized algorithms to identify contaminated blood cultures, community-acquired BSI, and hospital-acquired BSI and used algorithms to classify the latter on the basis of whether it was a catheter-associated infection. We compared the automatic classification with an assessment (71% prospective) of clinical data. SETTING An 850-bed university hospital. PARTICIPANTS All adult patients admitted to general surgery, internal medicine, a medical intensive care unit, or a surgical intensive care unit over 3 years. RESULTS The results of the automated surveillance were 95% concordant with those of classical surveillance based on the assessment of clinical data in distinguishing contamination, community-acquired BSI, and hospital-acquired BSI in a random sample of 100 cases of bacteremia. The two methods were 74% concordant in classifying 351 consecutive episodes of nosocomial BSI with respect to whether the BSI was catheter-associated. Prolonged episodes of BSI, mostly fungemia, that were counted multiple times and incorrect classification of BSI clinically imputable to catheter infection accounted for 81% of the misclassifications in automated surveillance. By counting episodes of fungemia only once per hospital stay and by considering all cases of coagulase-negative staphylococcal BSI to be catheter-related, we improved concordance with clinical assessment to 82%. With these adjustments, automated surveillance for detection of catheter-related BSI had a sensitivity of 78% and a specificity of 93%; for detection of other types of nosocomial BSI, the sensitivity was 98% and the specificity was 69%. CONCLUSION Automated strategies are convenient alternatives to manual surveillance of nosocomial BSI.

SME-2-Producing Serratia marcescens Isolate from Switzerland

Carbapenemases in Serratia marcescens have been rarely reported, being related either to metallo-β-lactamases IMP-1, IMP-6, and VIM-2 (10) or to SME-type Ambler class A β-lactamases (6). SME-1 had been identified first from the carbapenem-resistant S. marcescens strain S6, isolated in London in 1982 (5), and then in carbapenem-resistant S. marcescens strains isolated in 1999 in the United States (1). It hydrolyzes penicillins, aztreonam, cephalosporins, and carbapenems and is inhibited by clavulanic acid (4). The blaSME-1 gene was chromosome encoded in isolate S6 (5). The SME-2 and SME-3 variants have been identified (being just point mutant analogues) from S. marcescens isolates recovered from the United States and United Kingdom (8, 9). Our study was initiated by the isolation in May 2006 at the University Hospital of Lausanne, Switzerland, of a carbapenem-resistant S. marcescens isolate. This isolate was from an exudate obtained after a parotidectomy of a 62-year-old patient who received a prophylaxis containing amoxicillin-clavulanate. Three days after surgery, the patient suffered from a pulmonary deficiency and received an imipenem-containing treatment. Two days later, culture of the exudate gave S. marcescens strain AW, which was resistant to penicillins and imipenem. The MICs of imipenem, meropenem, and ertapenem were 32, 8, and 4 μg/ml, respectively. It was also resistant to cefoxitin and aztreonam, whereas it remained susceptible in vitro to expanded-spectrum cephalosporins. The results for double-disk synergy testing, performed as described previously (2), were slightly positive with the clavulanate-imipenem combinations, indicating that S. marcescens AW produced a carbapenemase that was inhibited by clavulanic acid. PCR amplification using various primers, including blaSME-specific primers and whole-cell DNA of S. marcescens AW, followed by sequencing performed as described previously (7), identified a gene encoding SME-2. No other similar isolate was recovered from the same hospital during the same period of time, and no history of travel abroad or previous hospitalization was identified. The clonal relationship between isolate AW and isolate S6 from London was evaluated by pulsed-field gel electrophoresis as described previously (8), showing that isolate AW was not clonally related to isolate S6. By analyzing the sequences of the rpoB genes as reported previously (3), we found that the sequences obtained from isolates AW and S6 and also from two randomly selected carbapenem-susceptible and blaSME-negative S. marcescens isolates were identical. This observation likely rules out a possible identity for SME producers as part of a given subspecies. Conjugation, electrotransformation, and plasmid analysis, performed as described previously (5, 7), failed to identify a plasmid-borne location for the blaSME-2 gene, suggesting a likely chromosomal location for this gene. Further PCR mapping showed the presence of the LysR-type regulatory gene smeR upstream of blaSME-2, as previously identified (4). This was in accordance with the observation of a slight antagonism observed between the cefoxitin- and imipenem-containing disks. This report underlines the possible identification of SME-type S. marcescens producers worldwide. Along with other clavulanic acid-inhibited carbapenemases (NMC-A, IMI, KPC), SME-type enzymes may confer clinically significant resistance to carbapenems. Interestingly, SME producers may confer a lower level of resistance to ertapenem than to the other carbapenems.

Candida dubliniensis in Recurrent Polymicrobial Tricuspid Endocarditis

This report describes a successful operative case of tricuspid infective endocarditis in an IV drug user. Despite cessation of IV drug use, there were further recurrences. Six different microorganisms with multiple portals of entry were identified, including one episode of fungal endocarditis, To our knowledge, this is the first case of recurrent infective endocarditis involving Candida dubliniensis in an HIV‐negative patient.

Ampicillin-Sensitive, Imipenem-Resistant Strains of Enterococcus faecium

Weinstein has in an interesting paper (3) suggested that testing the susceptibilities of isolates of enterococci to penicillin or ampicillin accurately predicted the in vitro activity of imipenem, and also pointed out that there are no NCCLS guidelines for testing the susceptibility of enterococci to imipenem. However, we found three strains of Enterococcus faecium that were sensitive to ampicillin but resistant to imipenem in blood and an abdominal abscess from an elderly patient being treated with imipenem at an intensive care unit (ICU) in a Swedish hospital. MICs of ampicillin were from 0.25 to 1 μg/ml, and those of imipenem were 4 to 16 μg/ml. Resistance to imipenem in these strains was caused by increased production of PBP5 with decreased affinity to imipenem (1). Similar strains have been isolated in Switzerland from the blood of eight hospitalized patients (V. Brandt, A. Wenger, and J. Bille, 10th Eur. Congr. Clin. Microbiol. Infect. Dis., poster WeP14, 2000), and studies on their resistance mechanisms are in progress. MICs of ampicillin ranged from 0.5 to 6 μg/ml, while MICs of carbapenems (imipenem and meropenem) were ≥16 μg/ml. Interestingly, six of the strains were benzyl penicillin resistant by NCCLS standards, making benzyl penicillin a better, though not perfect, indicator of decreased susceptibility to carbapenems. In a recent investigation, hospitals (2), ampicillin-sensitive, imipenem-resistant strains were found in ICUs in Swedish hospitals (2), but since breakpoints for imipenem have not been defined by NCCLS, it is hard to estimate how frequently, if at all, these strains occur outside ICUs. In conclusion, we feel that sensitivity testing with carbapenem should be done whenever treatment of enterococcal infections with this type of drug is considered.