International multicenter evaluation of latex agglutination tests for identification of Staphylococcus aureus

A newly marketed rapid agglutination kit for the identification of Staphylococcus aureus, Slidex Staph Plus (bioMérieux), was compared to Staphaurex Plus (Murex Diagnostics) and Pastorex Staph-Plus (Sanofi Diagnostics Pasteur). The study took place in three clinical microbiology laboratories in three different European countries. A total of 892 staphylococcal isolates, including 278 methicillin-sensitive S. aureus (MSSA) isolates, 171 methicillin-resistant S. aureus (MRSA) isolates, and 443 coagulase-negative staphylococcal isolates, were analyzed. The sensitivities (MSSA/MRSA) and specificities, respectively, were 98. 2% (98.9%/97.1%) and 98.9% for Slidex Staph Plus, 98.2% (98.2%/98. 2%) and 96.2% for Staphaurex Plus, and 98.7% (98.6%/98.8%) and 95.7% for Pastorex Staph Plus. The specificity of the Slidex Staph Plus kit was statistically significantly higher than the specificities of Staphaurex Plus and Pastorex Staph-Plus. The Slidex Staph Plus is a very reliable test for the identification of S. aureus.     

Sensitivity and Specificity of an Improved Rapid Latex Agglutination Test for Identification of Methicillin-Sensitive and -Resistant Staphylococcus aureus Isolates

The performance of a second-generation rapid agglutination kit, Slidex Staph Plus (SSP; bioMérieux), was compared to those of the Slidex Staph (SS; bioMérieux), Staphaurex (SRX; Murex Diagnostics), and BBL Staphyloslide (BBL; Becton Dickinson) kits by using 508 clinical isolates composed of 150 methicillin-sensitive Staphylococcus aureus (MSSA) organisms, 154 methicillin-resistant S. aureus (MRSA) organisms, and 204 non-S. aureus Staphylococcus spp. Of the 508 isolates tested, 75% were fresh clinical isolates, with the remainder taken from five different freezer collections. All four agglutination tests had comparable sensitivities for MSSA and MRSA. However, the SS kit was significantly less specific (93.1%) than the three other tests (P > 0.05, McNemar test). These results demonstrate that the new rapid latex agglutination kit, SSP, was more specific for the identification of S. aureus than the previous version and performed comparably to the SRX and BBL kits.     

False-negative test results in the Slidex Staph Plus (bioMérieux) agglutination test are mainly caused by spa-type t001 and t001-related strains

The relative sensitivity of commercial agglutination kits for fast identification of S. aureus is usually given to be about 98%. This reported sensitivity has sometimes been questioned. In this study, three collections of molecularly defined, single-copy strains of S. aureus were used to compare the sensitivities of agglutination-based identification and the MALDI-TOF mass spectrometry-based identification using the Biotyper 2.0 database to a molecularly defined reference method. Clinical isolates (n = 363) of methicillin-susceptible S. aureus (MSSA) and 240 clinical isolates of methicillin-resistant S. aureus (MRSA) were included. In order to rule out a predominance of local MRSA-strains, a collection of 104 pulsed-field-gel electrophoresis divergent MRSA strains were also tested. MALDI-TOF MS using Biotyper database (Bruker) identified all isolates, whereas the Slidex Staph Plus (bioMérieux) detected only 98.0% of the MSSA, 94.5% of the MRSA and only 70.1% of the MRSA of the molecularly divergent strain collection. Interestingly, strains with a false-negative test result in the agglutination methods were mostly spa-type t001 and t001 related. The MALDI-TOF MS based identification can thus be used as an alternative identification method for suspected false-negative results from the agglutination tests, especially if the local prevalence of t001 and t001 related strains is high.     

Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus.

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.     

Comparison of five tests for identification of Staphylococcus aureus from clinical samples.

Five different laboratory tests for the identification of Staphylococcus aureus were compared. Analyses of 271 presumptive S. aureus strains, supplemented with 59 well-defined methicillin-resistant S. aureus (MRSA) isolates, were performed. Only the Staphaurex Plus (Murex Diagnostics, Dartford, United Kingdom) and the Pastorex Staphplus (Sanofi, Marnes-La-Coquette, France) tests displayed 100% sensitivity. The observed difference with the free-coagulase test (Bacto coagulase plasma; Difco, Detroit, Mich.), a bound-coagulase (clumping factor) test, and the former Staphaurex test (Murex Diagnostics) was caused mainly by the inability of these three tests to identify some MRSA strains correctly. Among Polish MRSA isolates included in the analysis, a group of free-coagulase-negative S. aureus strains was detected. Genetic typing by random amplification of polymorphic DNA revealed that the strains showing aberrant behavior when the different test results were compared belonged to limited number of S. aureus clones.     

Comparison of five agglutination tests for identification of Staphylococcus aureus.

Various commercially produced agglutination kits are widely used for the identification of Staphylococcus aureus. These kits detect the presence of protein A and/or clumping factor on S. aureus. The literature has shown that methicillin-resistant S. aureus (MRSA) isolates which are deficient in both clumping factor and protein A may be misidentified. Two products, Slidex and Staphaurex Plus, utilize specific anti-S. aureus antibodies, potentially giving them greater sensitivity compared to products without these antibodies. We report a prospective study designed to compare the performance characteristics of Fastaph, Slidex, Staphaurex, Staphaurex Plus, Staphyloslide, and the tube coagulase test for the identification of staphylococcal isolates. All discrepant isolates were tested with the Gen-Probe AccuProbe S. aureus test and were identified to the species level with conventional reference biochemicals. A total of 1,193 isolates were tested, including 33 MRSA and 423 methicillin-sensitive S. aureus isolates. The sensitivities and specificities of the tests, respectively, were as follows: Fastaph, 99.1 and 98.9%; Slidex, 99.6 and 96.4%; Staphaurex, 98.9 and 99.9%; Staphaurex Plus, 99.6 and 93.9%; Staphyloslide, 99.1 and 98.9%; and tube coagulase, 99.3 and 100%. Sensitivity was excellent for all of the products tested. The specificities of Fastaph, Staphaurex, and Staphyloslide were excellent, while Staphaurex Plus and Slidex demonstrated less optimal results.     

Evaluation of a new Staphylococcus aureus latex agglutination kit, Prolex Staph Xtra, against other third-generation kits

Staphylococcus aureus, including methicillin-resistant strains, continues to be a common cause of infection/colonisation, which necessitates accurate and prompt diagnosis in the laboratory. Several rapid agglutination tests that aid this function are available, and some have been modified to improve their performance. One such kit, Prolex Staph Xtra, has been released recently. This study aims to compare this kit with other improved kits (i.e., Pastorex Staph-Plus, Staphaurex Plus and Staphytect Plus) and investigate their ability to confirm the identity of 100 strains of S. aureus. Results showed that 50 were resistant to methicillin. Specificity was checked against 30 strains of coagulase-negative staphylococci and 20 Enterococcus species isolates. Of the four kits tested, Prolex Staph Xtra and Pastorex Staph-Plus proved superior in terms of sensitivity and speed.     

Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures

The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2' (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.     

Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

Fifty methicillin-resistant Staphylococcus aureus (MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitive Staphylococcus aureus (MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen for S. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps. S. aureus strains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins of S. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negative S. aureus strains as methicillin resistant even prior to antimicrobial susceptibility testing.     

Use of commercial particle agglutination systems for the rapid identification of methicillin-susceptible and methicillin-resistantStaphylococcus aureus

The performance of a recently introducedStaphylococcus aureus identification system (Slidex Staph-Kit) was compared with that of currently available systems (Immuno Scan Staphlatex, Staphyloslide, Staphaurex and SeroSTAT II) for the identification of methicillin-resistantStaphylococcus aureus (MRSA) and methicillin-susceptibleStaphylococcus aureus. The new system, which detects a capsular antigen common in MRSA, performed with equal or greater sensitivity than the other systems. None of the commercial systems was adversely affected by the methicillin susceptibility of the staphylococci when isolates were recovered from non-selective media. The greatest advantage of the anti-capsular monoclonal reagent was its improved performance on isolates recovered from selective media.     

Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones

Staphylococcus aureus infections continue to pose a global public health problem. Frequently, this epidemic is driven by the successful spread of single S. aureus clones within a geographic region, but international travel has been recognized as a potential risk factor for S. aureus infections. To study the molecular epidemiology of S. aureus infections in the Caribbean, a major international tourist destination, we collected methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from community-onset infections in the Dominican Republic (n = 112) and Martinique (n = 143). Isolates were characterized by a combination of pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) typing. In Martinique, MRSA infections (n = 56) were mainly caused by t304-ST8 strains (n = 44), whereas MSSA isolates were derived from genetically diverse backgrounds. Among MRSA strains (n = 22) from the Dominican Republic, ST5, ST30, and ST72 predominated, while ST30 t665-PVL+ (30/90) accounted for a substantial number of MSSA infections. Despite epidemiological differences in sample collections from both countries, a considerable number of MSSA infections (~10%) were caused by ST5 and ST398 isolates at each site. Further phylogenetic analysis suggests the presence of lineages shared by the two countries, followed by recent genetic diversification unique to each site. Our findings also imply the frequent import and exchange of international S. aureus strains in the Caribbean.     

Positive predictive value of the Xpert MRSA assay diagnostic for universal patient screening at hospital admission: influence of the local ecology

The purpose of this study was to assess the accuracy of the Xpert MRSA assay (XP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission. Nasal swabs were prospectively collected for MRSA screening from 1,891 patients admitted to a teaching hospital. XP results were compared to chromogenic agar culture results. MRSA was cultured in 61 specimens (3%). Compared with culture, XP had a sensitivity, specificity, positive, and negative predictive value of 60.7, 97.3, 37.8, and 98.9%, respectively. The median turnaround time (TAT) for the results was 3 h. Of 24 MRSA isolated from XP-negative samples, three harbored composite SCCmec. Among 61 samples with culture-negative but XP-positive results, 15 methicillin-susceptible S. aureus (MSSA) isolates tested positive by XP on pure colony lysates. These MSSA included: (i) strains with SCCmec deletion encompassing mecA and (ii) multilocus sequence typing (MLST) clonal complex (CC) 1 strains harboring a chromosomal sequence homologous to one of the orfX–SCCmec junction sequences targeted by XP. On account of the low sensitivity and positive predictive value in a hospital patient population with moderate prevalence of MRSA, culture still appears to be necessary in order to confirm polymerase chain reaction (PCR) results. The emergence of new SCCmec variants and the presence of MSSA harboring cross-reactive SCCmec-like elements may challenge the successful implementation of such detection systems.     

Comparative evaluation of five agglutination techniques and a new miniaturized system for rapid identification of methicillin-resistant strains of Staphylococcus aureus.

The speciation of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant diagnostic problem when rapid identification methods such as slide agglutination tests, are used, because of the high proportion of false-negative reactions. 150 perfectly identified MRSA strains were tested on 5 commonly used agglutination reagents ("Bacto staph latex test", "Monostaph", "Pastorex staph", "Staphaurex", and "Staphyslide test") in comparison with a new micromethod ("RAPIDEC staph") which detects a type of staphylocoagulase within 2 hours by a fluorescence test. The "RAPIDEC staph" reagent enabled identification of all the MRSA while the agglutination tests gave poorer results: "Monostaph" correctly identified 64.6% of strains, "Staphyslide", 59.3%, "Bacto staph latex test", 44.6%, "Pastorex staph", 38.6% and "Staphaurex", 28.6%. These results show that agglutination slide tests are not reliable enough for the identification of MRSA which are more and more encountered in hospital wards. The authors recommend not to use slide agglutination methods. They suggest the tube test for coagulase which is the reference technique, although it is time-consuming and not well standardized. The results of this evaluation encourage the use of the "RAPIDEC staph" reagent since it is an easy-to-use, reliable technique for the rapid identification of Staphylococcus aureus.     

Comparison of seven coagulase tests for identification ofStaphylococcus aureus

Four coagglutination tests for the identification of Staphylococcus aureus were compared with the ordinary slide and tube coagulation tests using two groups of staphylococcal strains (one isolated in the authors' laboratory and the other identified by a reference laboratory). The correct identification rate in the two groups was respectively for slide test 96.9 % and 92.7 %, tube coagulation (citrate plasma) 99.0 % and 94.8%, tube coagulation (EDTA plasma) 99.0% and 91.7%, API Staphase III 100% and 91.7%, Staph Rapid 97.9% and 91.3%, Staphyslide 99.0% and 94.6 %, Staphaurex 96.9 % and 92.7 %. The sensitivity of Staph Rapid, Staphyslide and Staphaurex was slightly higher than that of the other tests. These three tests and the slide test were considerably more rapid as regards identification than the other tests.     

High frequency of Panton-Valentine leukocidin genes in invasive methicillin-susceptible <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains and the relationship with methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Córdoba,Argentina

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Córdoba, Argentina, during 1999–2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Córdoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.     

External quality assessment of the molecular diagnostics and genotyping of meticillin-resistant Staphylococcus aureus

Two multicentre external quality assessments (EQA) for the molecular detection and genotyping of meticillin-resistant Staphylococcus aureus (MRSA) were arranged. Firstly, 11 samples containing various amounts of inactivated MRSA strains, meticillin-susceptible S. aureus (MSSA), meticillin-resistant coagulase-negative staphylococci (MRCoNS) or Escherichia coli were distributed to 82 laboratories. Samples containing 10² or 10³ MRSA cells were correctly scored in only 16 and 46% of the datasets returned, respectively. Two of the used MSSA strains contained an SCCmec cassette lacking the mecA gene. There was a marked difference in the percentage of correct results for these two MSSA strains (37 and 39%) compared to the MSSA strain lacking the SCCmec cassette (88%). Secondly, a panel for MRSA genotyping, consisting of ten samples (two identical, three genetically related and five unique strains) was distributed to 19 laboratories. Seventy-three percent of the datasets recorded all samples correctly. Most pulsed-field gel electrophoresis (PFGE) protocols proved to be suboptimal, resulting in inferior resolution in the higher or lower fragment regions. The performance of molecular diagnostics for MRSA shows no significant changes since our first EQA in 2006. The first molecular typing results are encouraging. Both assessments indicate that programme expansion is required and that major performance discrepancies continue to exist.     

Genotypic and phenotypic characterization of methicillin-susceptible Staphylococcus aureus isolates misidentified as methicillin-resistant Staphylococcus aureus by the BD GeneOhm MRSA assay

Twenty-three nasal swab samples that tested positive for methicillin-resistant Staphylococcus aureus (MRSA) on initial testing by the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA) were culture positive only for methicillin-susceptible S. aureus (MSSA) from an enrichment broth. The 23 recovered isolates were confirmed as MSSA by a variety of phenotypic methods, including the BD Phoenix automated microbiology system (BD Diagnostics, Sparks, MD), oxacillin screening agar (BD Diagnostics), BBL CHROMagar MRSA (BD Diagnostics), and a PBP2' assay (Denka Seiken Co., Tokyo, Japan); susceptibilities were determined by using Mueller-Hinton agar with oxacillin. All were positive by nuc PCR, specific for S. aureus, but negative for mecA with one exception. Isolates were characterized by using multiplex PCR methodology to determine structural types and variants (SCCmec typing); additional PCRs were performed for the detection of the ccr and mec complexes, the junkyard regions as well as the Panton-Valentine leukocidin. Pulsed-field gel electrophoresis was used to determine clonality. One phenotypic MSSA isolate contained an intact SCCmec. Twelve MSSA isolates tested positive for MRSA by the BD-MRSA PCR because of amplification of the mec priming site flanking the SCC insertion point, although these isolates lacked mecA. The 10 remaining isolates were not MRSA and tested as MSSA by phenotypic and genotypic assays. In our patient population, diagnostic and surveillance testing and subsequent infection control practices may be impacted by the frequency of these excision events when using the BD-MRSA PCR for MRSA detection.     

Comparative evaluation of a latex test for the identification ofStaphylococcus aureus

A rapid latex agglutination test, Staphaurex, was tested for its ability to identifyStaphylococcus aureus using 72 reference strains and 785 clinical isolates of the familyMicrococcaceae. All reference strains ofStaphylococcus aureus were Staphaurex-positive. Non-Staphylococcus aureus reference strains were negative. Using clinical strains, the results of the Staphaurex test were compared with the results of other tests commonly used to identifyStaphylococcus aureus. A total of 393 clinical isolates were classified asStaphylococcus aureus. The Staphaurex, slide coagulase, tube coagulase/human plasma and tube coagulase/rabbit plasma tests correctly identified 98%, 93.6%, 93.6% and 97.5% of theStaphylococcus aureus strains, respectively. The performance of the Staphaurex test, in terms of sensitivity and specificity, was significantly better than the slide coagulase test. It was as sensitive and almost as specific as the tube coagulase rabbit test and more sensitive than the tube coagulase human test.