Evaluation of laboratory methods for the classification of oxacillin-resistant and oxacillin-susceptible Staphylococcus aureus

The purpose of this study was to examine the efficacy of the oxacillin disk diffusion test and the methicillin and oxacillin agar screen tests as predictors of oxacillin resistance as defined by the reference broth microdilution method. A total of 444 clinical isolates of Staphylococcus aureus collected from individual patients over a four-year period were tested by (1) the oxacillin disk diffusion test, with particular attention to the presence of fine growth of a resistant subpopulation within the zone of inhibition (Ox Grow interpretive criteria); (2) the agar screen method using agar plates with 4% NaCl and either 6 mg/L oxacillin or 10 mg/L methicillin; and (3) the oxacillin and methicillin broth microdilution test methods with 2% NaCl supplementation. Overall, 62 (14%) isolates were resistant and 382 (86%) isolates were susceptible to oxacillin with the use of the reference broth microdilution system. The results indicate that the disk diffusion test with the use of the Ox Grow criteria had a high sensitivity (94%) and negative predictive value (98%) but a low specificity (67%) and positive predictive value (32%) when compared with the reference broth dilution test. Similarly, the agar screen tests had a high sensitivity (95-97%) and negative predictive values (99%) but low specificity (64-74%) and positive predictive values (30-37%). These data indicate that the agar screen tests and the oxacillin disk test with the use of the Ox Grow interpretive criteria may be useful as screening tests for detecting resistance to the penicillinase-resistant penicillins but that all resistant isolates should be confirmed by the reference broth dilution method because of the large number of false-resistant screening test results.     

Evaluation of a commercial latex agglutination test for identification of Staphylococcus aureus.

A new, commercially available latex agglutination test (SeroSTAT Staph; Scott Laboratories, Inc., Fiskeville, R.I.) was compared with the tube coagulase and slide coagulase tests as means for identifying Staphylococcus aureus. Of 160 clinical isolates of S. aureus, 159 (99.4%) yielded positive results with the latex agglutination test. Negative latex agglutination test results were obtained with 266 of 267 clinical isolates of Micrococcus spp. and staphylococcal species other than S. aureus (99.6%). The latex agglutination test was found to be a rapid, technically nondemanding method for identifying S. aureus. It was as accurate as the tube coagulase test and more accurate than the slide coagulase test.     

Evaluation of the latex slide agglutination test for identification of Staphylococcus aureus.

This study evaluated the reliability of the latex slide agglutination test for identifying Staphylococcus aureus. A total of 806 clinical isolates of staphylococci were tested for latex agglutination, clumping factor, and free coagulase. Positive latex tests occurred in 98.3% of coagulase-positive strains, whereas 99.6% of coagulase-negative strains gave negative latex tests. It is concluded that in most instances, the latex slide agglutination test is a reliable method for identifying S. aureus in the clinical laboratory.     

New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.     

Failure of rapid agglutination methods to detect oxacillin-resistant Staphylococcus aureus.

Although a latex agglutination test (StaphAurex) and a hemagglutination test (Staphyloslide) correctly identified all strains of Staphylococcus aureus that were susceptible or had intermediate susceptibility to oxacillin, 17 of 73 (23%) and 18 of 73 (25%) strains of oxacillin-resistant S. aureus were not identified by StaphAurex and Staphyloslide, respectively. All strains not detected were resistant to trimethoprim-sulfamethoxazole and rifampin.     

Evaluation of three commercial agglutination tests for the identification of Staphylococcus aureus.

Three commercially available rapid slide agglutination tests for the identification of Staphylococcus aureus were evaluated with 354 recent clinical isolates (165 strains of S. aureus). The test results of two latex agglutination products, SeroSTAT Staph (Scott Laboratories, Inc.) and Staphylatex (American Micro Scan), and one hemagglutination product, Staphyloslide (BBL Microbiology Systems), were compared with the results of the tube coagulase test, which was read at 4 h (4-h tube coagulase test) and, if negative, again after overnight incubation at room temperature (24-h tube coagulase test). Discrepancies between agglutination and tube coagulase identifications were resolved by use of the thermonuclease, mannitol fermentation, and slide coagulase tests. All sensitivities, specificities, predictive values of a positive result, and predictive values of a negative result for the three agglutination tests were at least 98.8% and comparable with the 4-h tube coagulase test. Best results were obtained with the 24-h tube coagulase test, which yielded one false-negative and no false-positive tests. Agglutination identifications may be performed on organisms taken directly from a primary plate when sufficient growth is present. Kit agglutination procedures yield rapid and reliable identifications and are easy to perform. This study also demonstrates the usefulness of the 24-h tube coagulase test.     

Evaluation of various rapid agglutination methods for the identification of Staphylococcus aureus

A latex agglutination test (SeroSTAT Staph; Scott Laboratories, Fiskeville, R.I.) and two hemagglutination tests (Staphyloslide; BBL Microbiology Systems, Cockeysville, Md.; and Hemastaph; Remel, Lenexa, Kans.) were compared with the slide coagulase (SC) and tube coagulase (TC) tests at room temperature (22 to 25 degrees C) and at 37 degrees C for the rapid identification of Staphylococcus aureus. A total of 380 clinical strains of staphylococci were tested. The TC test performed at room temperature yielded the largest number of TC-positive results (n = 239), and based on this observation 239 organisms were classified as S. aureus and 141 were classified as non-S. aureus. The SC, TC (37 degrees C), SeroSTAT Staph, Staphyloslide, and Hemastaph tests correctly identified 210 (87.9%), 221 (92.5%), 238 (99.6%), 239 (100%), and 236 (98.7%) of the S. aureus isolates, respectively. Of the S. aureus isolates that were TC positive at room temperature 68% required 24 h of incubation before coagulase production was detected. There was one false-negative SeroSTAT Staph result and one false-negative Hemastaph result. The Staphyloslide test yielded two noninterpretable results (both organisms were later confirmed as non-S. aureus), whereas there were six noninterpretable results recorded with the Hemastaph test (four organisms were classified as non-S. aureus, and two were classified as S. aureus). The SeroSTAT Staph, Staphyloslide, and Hemastaph tests were all more sensitive than the conventional SC and TC (37 degrees C) tests and were considerably more rapid than the TC test at either temperature.     

Rapid and reliable identification of Staphylococcus aureus by a latex agglutination test

A latex slide agglutination test detecting clumping factor and protein A simultaneously is recommended for rapid and reliable routine identification of Staphylococcus aureus. Strains (836) of staphylococci isolated from clinical specimens were examined, all S. aureus strains identified by conventional methods were correctly differentiated by the latex test, and no false-positive results occurred with other staphylococci. The reagent is easy to prepare since plasma is the coating material.     

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.     

Direct identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test.

A commercial latex agglutination slide test (SeroSTAT Staph, Scott Laboratories, Inc., Fiskeville, R.I.) accurately identified Staphylococcus aureus when applied directly to blood culture fluid containing staphylococci. This latex agglutination test exhibited 100% accuracy when 30 seeded aerobic and anaerobic radiometric blood cultures (15 strains of S. aureus, 15 strains of other staphylococcal species) were tested blindly. In 36 actual clinical specimens yielding 16 isolates of S. aureus and 20 isolates of Staphylococcus epidermidis, 94.4% accuracy was achieved. The latex agglutination test provided positive test results before objective criteria of blood culture positivity such as radiometric growth indices and Gram stains became positive.     

Evaluation of three test procedures for identification of Staphylococcus aureus from clinical sources.

A total of 520 clinical and environmental isolates of the family Micrococcaceae that fermented glucose anaerobically were tested for their ability to produce coagulase, thermostable nuclease, and deoxyribonuclease. Of these, 450 isolates coagulated rabbit plasma, produced thermostable nuclease, and were identified as Staphylococcus aureus, 447 of which produced a 3+ to 4+ clot. The remaining three isolates produced a 2+ clot, deoxyribonuclease, and thermostable nuclease. It was found that three of the S. aureus isolates failed to produce deoxyribonuclease. A total of 70 isolates which did not coagulate rabbit plasma and which were thermostable nuclease negative were identified as S. epidermidis. Three of them produced deoxyribonuclease. It is suggested that the thermostable nuclease test be performed on all isolates producing a 2+ (or 1+) clot in the coagulase test before identifying them as S. aureus.     

Reliability of latex agglutination tests for identification of Staphylococcus aureus resistant to oxacillin.

Commercial latex agglutination tests (LATs) for the simultaneous detection of clumping factor and protein A are gaining increased acceptance as a means of identifying Staphylococcus aureus. We evaluated two LATs (Accu-Staph; Carr-Scarborough, Decatur, Ga.; Staphaurex; Wellcome, Dartford, England) with particular emphasis on their ability to correctly identify oxacillin-resistant S. aureus. We tested 59 oxacillin-resistant S. aureus, 136 oxacillin-susceptible S. aureus, and 92 coagulase-negative staphylococcal strains with the two LATs and with thermonuclease, slide clumping factor, tube coagulase, and protein A hemagglutination tests. Clumping factor and protein A were present in 96.9 and 82.1% of our S. aureus strains, respectively. Accu-Staph correctly identified 92.8% and Staphaurex correctly identified 91.3% of S. aureus strains. No significant difference in LAT positivity rates, presence of clumping factor, or presence of protein A was found between oxacillin-resistant and -susceptible S. aureus. Overall, there were 31 false-negative LATs for 20 S. aureus strains, 14 with Accu-Staph and 17 with Staphaurex. Ninety-five percent of these strains possessed either clumping factor or protein A or both when these factors were determined independently. There were five false-positive LATs for four strains of coagulase-negative staphylococci (three Staphylococcus epidermidis and one Staphylococcus warneri), four with Accu-Staph and one with Staphaurex. Clumping factor was present in one S. warneri strain. Thus, the specificities of Accu-Staph, Staphaurex, and the clumping factor test were 95.6, 98.9, and 98.9%, respectively. Our results indicated that LATs identify oxacillin-resistant and -susceptible S. aureus equally well; however, they offer no greater sensitivity or specificity than the clumping factor test for identification of S. aureus.     

Problems with rapid agglutination methods for identification of Staphylococcus aureus when Staphylococcus saprophyticus is being tested.

Six rapid agglutination tests for identification of Staphylococcus aureus were evaluated by using 62 strains of S. aureus, 63 strains of S. saprophyticus, and 67 strains of other coagulase-negative staphylococci. S. saprophyticus was responsible for 19 of 26 false-positive results and 20 uninterpretable reactions. Thus, urinary staphylococcal isolates that are positive by rapid agglutination tests may require other confirmatory tests for the identification of possible S. saprophyticus.     

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.     

Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in U.S. hospitals: epidemiological significance.

For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, greater than 4 micrograms/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7/47/53/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 micrograms/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-beta-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-beta-lactamase producing S. aureus are distinct epidemic strains.     

Efficacies of rapid agglutination tests for identification of methicillin-resistant staphylococcal strains as Staphylococcus aureus.

Four commercially available rapid agglutination tests for the identification of Staphylococcus aureus were compared with the tube coagulase test for the identification of 300 methicillin-resistant isolates of staphylococci. Isolates tested included 207 methicillin-resistant S. aureus and 93 coagulase-negative staphylococci, collected from five medical centers. Strain variability was documented by phage typing and antimicrobial susceptibility patterns. Results of rapid identification tests ranged between 82 and 86% sensitivity, significantly poorer than the 98% sensitivity which the tube coagulase test provided.     

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.     

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.     

Predominance of capsular polysaccharide type 5 among oxacillin-resistant Staphylococcus aureus.

The relationship between capsular polysaccharide types 5 and 8 and resistance of Staphylococcus aureus to oxacillin was studied with a collection of 406 clinical isolates from six French hospitals. Of 175 type 5 isolates, 84 (48%) were resistant to oxacillin. In contrast, only 8 of 160 type 8 isolates (5%) and 5 of 71 nontypeable isolates (7%) were resistant to oxacillin. Therefore, capsular typing of clinical isolates of S. aureus may facilitate the choice of first-line antibiotic therapy.     

Thermonuclease test for same-day identification of Staphylococcus aureus in blood cultures.

We used a thermonuclease test to presumptively identify Staphylococcus aureus directly in blood cultures on the same day that a smear of the broth showed gram-positive cocci in clusters. There were no discrepancies between the identification of S. aureus directly from 250 blood cultures and identification by the tube coagulase test which was performed on the 18- to 24-h subculture isolates. These rapid results were clinically relevant and enabled physicians to make more timely and cost-effective decisions about antibiotic therapy.