Detection of colonization by Streptococcus agalactiae: prospective study comparing real-time gene amplification with a new chromogenic medium Strepto B ID

We carried out a prospective study including 554 vaginal swabs simultaneously tested for antenatal screening of Group B Streptococcus (GBS or Streptococcus agalactiae) using culture on the chromogenic medium Strepto B ID (Biomérieux, Marcy l'Etoile, France) and real time gene amplification on LightCycler (Roche Applied Science). We centrifuge the swabs with "SETS" device and separate centrifugates in 2 parts: one for the culture and the other one for molecular biology. First half of the centrifugate is inoculated onto Todd-Hewitt broth enriched with antibiotics. This broth is incubated to 35 degrees C during 24 hours and then subcultured on a Strepto B ID medium. This last one is incubated during 24 hours to 35 degrees C in capnophilic conditions before interpretation. DNA extraction for molecular biology is simply obtained by heating the microtubes to 95 degrees C in a water bath. The cfb gene is amplified, allowing a specific gene amplification of GBS even within a polymorphic flora. The concordance between both methods is 94.8%. The sensitivity and negative predictive values obtained are respectively 88.0 and 97.4% for real time PCR and 83.0 and 96.4% for culture on Strepto B ID. Both methods are thus concordant, with equal sensitivity and valid for detection of GBS colonization in pregnant women. However real time gene amplification allows reducing turn around time since molecular biology process (extraction+amplification) does not exceed 1 hour.     

Detection of methicillin-resistant Staphylococcus aureus directly from nasal swab specimens by a real-time PCR assay

Screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories, including molecularly based techniques, require a culture step and the isolation of pure colonies that result in a minimum of 20 to 24 h until a result is known. We describe a qualitative in vitro diagnostic test for the rapid detection of MRSA directly from nasal swab specimens (IDI-MRSA; Infectio Diagnostic, Inc., Sainte-Foy, Quebec, Canada), based upon a real-time PCR and direct detection of MRSA via amplicon hybridization with a fluorogenic target-specific molecular beacon probe. Samples from 288 patients were analyzed for the presence of MRSA with the IDI-MRSA assay, compared to detection by either direct plating or enrichment broth selective culture methods. The diagnostic values for this MRSA screening method were 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods. The time from the start of processing of specimen to result was approximately 1.5 h. In our hands, the IDI-MRSA assay is a sensitive and specific test for detection of nasal colonization with MRSA and providing for same-day results, allowing more efficient and effective use of infection control resources to control MRSA in health care facilities.     

Staphylococcus aureus nasal colonization level and intracellular reservoir: a prospective cohort study

European Journal of Clinical Microbiology & Infectious Diseases - Staphylococcus aureus is a major pathogen in humans. The nasal vestibule is considered as the main reservoir of S. aureus....     

Comparison of culture screening methods for detection of nasal carriage of methicillin-resistant Staphylococcus aureus: a prospective study comparing 32 methods

Screening for carriage of methicillin-resistant Staphylococcus aureus (MRSA) is fundamental to modern-day nosocomial infection control, both for epidemiologic investigation and day-to-day decisions on barrier isolation. Numerous microbiologic techniques have been advocated for screening for nasal carriage of MRSA, including the use of charcoal rather than rayon swabs, preincubation of swabs in Stuart's medium, preincubation of swabs in salt-containing trypticase soy broth (TSB), use of mannitol-salt agar (MSA), use of MSA containing oxacillin (MSA(Ox)), use of Mueller-Hinton agar containing oxacillin (MHA(Ox)), and the use of MSA containing lipovitellin with an oxacillin disk (MSAL(Ox)). We report a prospective clinical trial undertaken to test all of these methods concurrently. Patients at high risk for MRSA carriage were screened with eight consecutive nasal swabs (four standard rayon, four charcoal-coated rayon), which were processed by primary plating on MSA, MSA(Ox), MHA(Ox), and MSAL(Ox); Stuart's preincubation for 72 h followed by plating on the solid media; overnight enrichment in salt-containing TSB followed by plating; and Stuart's preincubation for 72 h followed by overnight enrichment in TSB and plating. All of the above methods were repeated with charcoal swabs. Each patient was screened by 32 culture methods. Forty-three (42%) of 102 patients studied were positive for MRSA by one or more methods. Among the four media evaluated with direct plating, MSAL(Ox) was 11 to 25% more sensitive for detecting MRSA (MSAL(Ox) versus MSA(Ox) or MHA(Ox) or MSA, each P < 0.01). Preincubation in Stuart's medium for 72 h did not enhance recovery of MRSA. Enrichment in salt-containing TSB further increased yield 9%. MSAL(Ox) also showed the best specificity, 93%. Charcoal swabs showed no advantage over standard rayon swabs. Our results suggest that the highest yield will be achieved by using standard rayon swabs that are enriched overnight in TSB with inoculation onto MSAL(Ox) medium. Direct inoculation of swabs onto MSAL(Ox) allows detection of 90% of MRSA carriers.     

Comparison of three selective chromogenic media for Methicillin-Resistant Staphylococcus aureus detection

PURPOSE: Rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) is of major importance in hospital hygiene. In order to reduce the response time from screening laboratories, new selective chromogenic media have been developed and marketed by major microbiology companies. In this context, an evaluation of their performances was needed. MATERIALS AND METHODS: Media produced by Bio-Rad Laboratories (MRSASelect), Becton Dickinson (CHROMagar MRSA) and bioMérieux (chromID MRSA) were studied by 203screening samples, 110 of which were MRSA positive. Each Stahylococcus aureus was identified by catalase detection, Staphytect Plus Dryspot latex agglutination test and free coagulase detection, in addition to mannitol fermentation and Voges-Proskauer tests in the case of doubtful identification. Resistance was verified by checking the inhibition zone diameter of under 20 mm on 30 microg cefoxitin disks. RESULTS: Bio-Rad Laboratories, Becton Dickinson and bioMérieux media read at 24 or 24/48 hours have a respective sensitivity of 91, 71 and 85/92%. The specificity of these media is 99, 100 and 99/93%. CONCLUSION: These media proved to be new powerful and rapid tools used in screening for MRSA detection. MRSASelect is one of the most effective media showing high sensitivity and specificity and the easiest interpretation. chromID MRSA exhibits similar performance but needs more time to be as effective as Bio-Rad media while CHROMagar MRSA isn't enough efficient with its slightly lack of sensitivity to perform a reliable screening.     

Staphylococcus aureus colonization during military service: a prospective cohort study

ObjectivesStaphylococcus aureus colonization leading to skin and soft-tissue infections (SSTI) are known challenges in crowded settings such as the military. The aim of the study was to establish and compare the prevalence of S. aureus colonization in recruits at enrolment and discharge after the first year of military service, and to investigate the prevalence of S. aureus SSTI.MethodsAll recruits entering first year of military service in January 2013 to be stationed at three garrisons in the northern part of Norway were invited to join this prospective cohort study. Swabs were taken from nose, throat and perineum. Staphylococcus aureus was identified using standard culturing methods. Methicillin resistance was determined by a cefoxitin disc diffusion test.ResultsOf the 923 eligible recruits, 512 were included at enrolment; 265/512 (52%) were also screened at discharge. Staphylococcus aureus colonization was high, and increased significantly during military service (166/265 versus 224/265, p < 0.001) mainly caused by increase in throat colonization alone or in combination with nasal colonization. All S. aureus isolates were susceptible to methicillin. SSTI was self-reported in 7/265 (3%) recruits, of which only one was confirmed by a military physician.ConclusionStaphylococcus aureus colonization increased during military service, but there were few confirmed reports of SSTI. Inclusion of throat swab provides important information as ∼20% of the recruits were only positive in their throat. Further analyses need to be performed to investigate if the increase in colonization is caused by specific S. aureus stains.     

Rapid screening of methicillin-resistant Staphylococcus aureus using PCR and chromogenic agar: a prospective study to evaluate costs and effects

Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is considered essential for controlling the spread of MRSA, but noncolonized patients will be isolated unnecessarily as a result of a delay in diagnosis of 3–5 days with conventional cultures. We determined costs per isolation day avoided, and incremental costs of rapid MRSA screening tests when added to conventional screening, but with decisions on isolation measures based on PCR results. A prospective multicentre study evaluating BD GeneOhm MRSA PCR (‘IDI’) (BD Diagnostics, San Diego, CA, USA), Xpert MRSA (‘GeneXpert’) (Cepheid, Sunnyvale, CA, USA) and chromogenic agar (MRSA-ID) (bioMérieux, Marcy-l’Etoile, France) was performed in 14 Dutch hospitals. Among 1764 patients at risk, MRSA prevalence was 3.3% (n = 59). Duration of isolation was 19.7 and 16.1 h with IDI and GeneXpert, respectively, and would have been 30.0 and 76.2 h when based on chromogenic agar and conventional cultures, respectively. Negative predictive values (at a patient level) were 99.5%, 99.1% and 99.5% for IDI, GeneXpert and chromogenic agar, respectively. Numbers of isolation days were reduced by 60% and 47% with PCR-based and chromogenic agar-based screening, respectively. The cost per test was €56.22 for IDI, €69.62 for GeneXpert and €2.08 for chromogenic agar, and additional costs per extra isolation day were €26.34. Costs per isolation day avoided were €95.77 (IDI) and €125.43 (GeneXpert). PCR-based decision-making added €153.64 (IDI) and €193.84 (GeneXpert) per patient to overall costs and chromogenic testing would have saved €30.79 per patient. Rapid diagnostic testing safely reduces the number of unnecessary isolation days, but only chromogenic screening, and not PCR-based screening, can be considered as cost saving.     

Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar

We evaluated the use of a chromogenic selective medium (MRSA ID) as a useful tool for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patient samples. Fifty-four MRSA isolates were detected by MRSA ID, while only 24/54 (44%) (odds ratio [OR], 2.79; 95% confidence interval [CI], 1.63 to 4.76) were detected by conventional methods. A chromogenic selective medium for MRSA detection may improve its surveillance in CF patients.     

Dynamics of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus carriage in pig farmers: a prospective cohort study

Our purpose was to determine the dynamics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage and its determinants in persons working at pig farms, in order to identify targets for interventions. This prospective cohort study surveyed 49 pig farms in the Netherlands on six sampling dates in 1 year (2010-11). Nasal and oropharyngeal swabs were collected, as well as environmental surface samples from stables and house. Of 110 pig farmers, 38% were persistent MRSA nasal carriers. The average cross-sectional MRSA prevalence was 63%. Methicillin-susceptible S. aureus (MSSA) nasal carriage was associated with fewer MRSA acquisitions (prevalence rate (PR) = 0.47, p 0.02). In multivariate analysis, an age of 40-49 years (PR = 2.13, p 0.01), a working week of ≥40 h (PR=1.89, p 0.01), giving birth assistance to sows (PR=2.26, p 0.03), removing manure of finisher pigs (PR=0.48, p 0.02), and wearing a facemask (PR = 0.13, p 0.02) were significantly related with persistent MRSA nasal carriage. A higher MRSA exposure in stables was associated with MRSA in pig farmers (p <0.0001). This study describes a very high prevalence of LA-MRSA carriage in pig farmers, reflecting extensive exposure during work. We identified the possible protective effects of MSSA carriage and of continuously wearing a facemask during work.     

Prevalence of methicillin-resistant Staphylococcus aureus nasal colonization among Taiwanese children in 2005 and 2006

From July 2005 to October 2006, a total of 3,046 children, of ages between 2 months and 5 years, presented for a well-child health care visit to one of three medical centers, which are located in the northern, central, and southern parts of Taiwan, and were surveyed for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA). The overall prevalences of S. aureus and MRSA nasal carriage among the children were 23% and 7.3%, respectively (18% and 4.8% in the central region, 25% and 6.7% in the southern region, and 27% and 9.5% in the northern region). Of the 212 MRSA isolates (96%) available for analysis, a total of 10 pulsed-field gel electrophoresis (PFGE) patterns with two major patterns (C [61%] and D [28%]) were identified. One hundred forty-nine isolates (70%) contained type IV staphylococcal cassette chromosome mec (SCCmec) DNA, and 55 isolates (26%) contained SCCmec V(T). The presence of Panton-Valentine Leukocidin (PVL) genes was detected in 60 isolates (28%). Most MRSA isolates belonged to one of two major clones, characterized as sequence type 59 (ST59)/PFGE C/SCCmec IV/absence of PVL genes (59%) and ST59/PFGE D/SCCmec V(T)/presence of PVL genes (25%). We concluded that between 2005 and 2006, 7.3% of healthy Taiwanese children were colonized by MRSA in nares. MRSA harbored in healthy children indicates an accelerated spread in the community.     

Molecular epidemiologic analysis of methicillin-resistant Staphylococcus aureus isolates from bacteremia and nasal colonization at 10 intensive care units: multicenter prospective study in Korea

We investigated molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated at 10 intensive care units (ICUs) in Korea. MRSA isolates from bacteremia and nasal colonization were collected prospectively from October 2008 through May 2009 at 10 University-affiliated hospital ICUs. A total of 83 and 175 MRSA strains were isolated from bacteremia and nasal colonization, respectively. Acquired group accounted for 69.9% (n = 58) of bacteremia and 73.1% (n = 128) of nasal colonization. Pulsed-field gel electrophoresis (PFGE) type B (SCCmec type II/ST5) was dominant in the acquired group followed by PFGE type D (SCCmec type IVA/ST72; a community genotype). Seven of 58 (12.1%) acquired bacteremia and 15 of 128 (11.8%) acquired nasal colonizations had SCCmec type IVA/ST72 genotype, which indicated that the community genotype had already emerged as a cause of ICU acquired MRSA infection or colonization. Antibiotic resistance rates to ciprofloxacin, tetracycline, clindamycin and trimethoprim/ sulfamethoxazole were 84.4%, 67.1%, 78.1%, and 12.0%, respectively. Susceptibility to ciprofloxacin best predicted a community genotype (sensitivity 96.5%; specificity 96.9%; odds ratio 861; 95% confidence interval 169-4,390, P < 0.001) and the positive predictive value was 90.2%. Among 23 nasal re-colonized strains, 7 MRSA strains (30.4%) were different from the originally colonized strains on the basis of PFGE types.     

Factors associated with nasal colonization of methicillin-resistant Staphylococcus aureus among healthy children in Taiwan

Methicillin-resistant Staphylococcus aureus (MRSA) has been identified as a major cause of community-associated (CA) S. aureus infections in the past decade. The main reservoir in the community for MRSA and the factors contributing to its worldwide spread remain poorly defined. Between July 2005 and June 2008, a total of 6,057 healthy children 2 to 60 months of age were screened for carriage of S. aureus and Streptococcus pneumoniae in Taiwan. The prevalence and epidemiological factors influencing MRSA carriage were determined. MRSA strains were tested for antimicrobial susceptibility and underwent molecular characterization. The overall prevalences of MRSA and S. aureus carriage were 7.8% and 23.2%, respectively. A majority (88%) of MRSA isolates belonged to a common Asian-Pacific CA-MRSA lineage, multilocus sequence type 59, and were resistant to multiple non-beta-lactam antibiotics. The carriage rate of MRSA was higher among subjects 2 to 6 months old (P < 0.0001), residing in northern Taiwan (P = 0.0003), and enrolled later in the study (P < 0.0001). MRSA colonization was associated with the number of children in the family (adjusted odds ratio [aOR], 1.114; 95% confidence interval [CI], 1.002 to 1.240; P = 0.0463) and day care attendance (aOR, 1.530; 95% CI, 1.201 to 1.949; P = 0.0006). Breast feeding (P < 0.0001) and colonization with S. pneumoniae (P = 0.0170) were protective against MRSA colonization. We concluded that epidemic CA-MRSA strains increasingly colonized Taiwanese children between 2005 and 2008. The carriage rate varied significantly across different demographical features. Crowding was an independent environmental risk factor that might accelerate CA-MRSA transmission in the community.     

Performance of the BD GeneOhm MRSA achromopeptidase assay for real-time PCR detection of methicillin-resistant Staphylococcus aureus in nasal specimens

We evaluated the BD GeneOhm MRSA achromopeptidase (ACP) assay, which incorporates a new specimen preparation approach. A total of 1,216 leftover nasal samples were tested; using culture as the gold standard, the sensitivity and specificity were 92% and 94.6%, respectively. The new lysis method provides good sensitivity and simplifies specimen preparation.     

Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus

We describe here the development and evaluation of MRSA ID, a new chromogenic agar medium for the specific isolation and identification of methicillin-resistant Staphylococcus aureus (MRSA). We used S. aureus ID (bioMérieux, La Balme Les Grottes, France) and supplemented it with various antimicrobials, including cefoxitin, ciprofloxacin, oxacillin, and methicillin. Cefoxitin proved to be superior to the other antimicrobials for the selection of MRSA from other strains of S. aureus. MRSA ID (consisting of S. aureus ID supplemented with 4 mg of cefoxitin/liter) was evaluated by the use of 747 swabs from various clinical sites. All specimens were also cultured on CHROMagar MRSA and oxacillin resistance screening agar base (ORSAB) and in selective mannitol broth (SMB). A total of 85 MRSA strains were isolated by a combination of all methods. After 22 to 24 h of incubation, 80% of the MRSA strains were isolated as green colonies on MRSA ID, compared with 59 and 62% of the strains that were isolated as colored colonies on CHROMagar MRSA and ORSAB, respectively. After 48 h of incubation, 89, 72, and 78% of the MRSA strains were isolated on MRSA ID, CHROMagar MRSA, and ORSAB, respectively. Sixty-five percent of the strains were isolated by growth in SMB. The specificities of MRSA ID, CHROMagar MRSA, ORSAB, and SMB were 99.5, 99.3, 97.9, and 92.8%, respectively, after 22 to 24 h of incubation. We conclude that MRSA ID is a sensitive and specific medium for the isolation and identification of MRSA.     

Lack of evidence for persistent nasal colonization with community-acquired methicillin-resistant Staphylococcus aureus in a central European cohort

One hundred and three patients who had previously tested positive for community-acquired methicillin-resistant Staphylococcus aureus (cMRSA) were followed up for a mean time of 32.6 months. Eighty patients had a history of skin or soft tissue infection, and the remainder were mostly asymptomatic carriers. Of 103 patients, only two reported ongoing symptoms with abscess formation. Of 81 nasal swabs available, 30.9% were positive for S. aureus but only four yielded Panton–Valentine leukocidin-positive methicillin-resistant S. aureus. In summary, we were unable to find persistent health issues or nasal colonization with cMRSA in a cohort of previously cMRSA-infected/colonized patients.     

Evaluation of different chromogenic media for the detection of methicillin-resistant Staphylococcus aureus CC398 in broilers

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged in a wide variety of animal species, including poultry. The objective of this study was to evaluate three different chromogenic media for MRSA clonal complex (CC) 398 detection in broilers. On three Belgian poultry farms, 50 broiler chickens were sampled per farm from both nose shell and cloaca. All swab specimens were enriched and inoculated the following day on three chromogenic media: chromID MRSA (bioMérieux), Brilliance MRSA 2 Agar (Oxoid) and MRSASelect (Bio-Rad). ChromID had the highest isolation rates, yet, Brilliance MRSA 2 Agar demonstrated the highest relative sensitivity, while MRSASelect and Brilliance MRSA 2 Agar showed the highest relative specificity. A subset of MRSA isolates was confirmed to be CC398 by the polymerase chain reaction (PCR) targeting sau1-hsdS1. In conclusion, Brilliance MRSA 2 Agar outperformed MRSASelect and chromID MRSA for the detection of MRSA in broilers.     

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.     

Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes

We describe a real-time PCR assay for the detection of methicillin-resistant Staphylococcus aureus and genes encoding toxic shock syndrome toxin 1 and Panton-Valentine leukocidin. Rapid screening and detection of toxins is a useful tool for surveillance studies and outbreak investigations involving large numbers of isolates.