Performance of four slide agglutination methods for identification of Staphylococcus aureus when testing methicillin-resistant staphylococci.

Four commercially available slide agglutination systems for identifying Staphylococcus aureus were compared with the conventional slide (clumping factor) and tube coagulase tests. The systems evaluated included Bacto Staph Latex (Difco Laboratories, Detroit, Mich.), Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.), Mini ID Accu-Staph (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.), and Staphaurex (Wellcome Diagnostics, Research Triangle Park, N.C.). A total of 338 clinical isolates, including methicillin-resistant S. aureus (n = 149), methicillin-susceptible S. aureus (n = 78), methicillin-resistant, coagulase-negative staphylococci (n = 45), and methicillin-susceptible, coagulase-negative staphylococci (n = 66), were tested by each method. The slide test for clumping factor, the 4-h tube coagulase test, Bacto Staph Latex, Staphyloslide, Mini ID Accu-Staph, and Staphaurex detected 212 (93.4%), 218 (96%), 223 (98.2%), 223 (98.2%), 221 (97.4%), and 224 (98.7%) of the S. aureus (44% methicillin-resistant) isolates, respectively. There were no false-positive results with any of the methods when the 111 strains of coagulase-negative staphylococci were tested. The results of this evaluation suggest that the four slide identification methods tested can provide rapid and accurate identification of methicillin-resistant S. aureus strains.     

Comparison of rapid identification assays for Staphylococcus aureus.

A total of 137 strains of Staphylococcus species were blindly tested by four rapid serological assays, and the results were compared with those of the tube coagulase assay. For the S. aureus isolates, the Sero-STAT Staph assay gave six false-negative results, four of which were for methicillin-resistant strains. The Accu -Staph, Staphylatex , and Staphyloslide assays identified all the coagulase-positive strains as Staphylococcus aureus. Among the coagulase-negative staphylococci, false-positive results were seen with strains of S. capitis. S. saprophyticus, and S. cohnii. The overall accuracy of the kits compared with the tube coagulase test ranged from 95.1 to 100%.     

An evaluation of three rapid coagglutination tests: Sero-STAT, Accu-Staph, and Staphyloslide, for differentiating Staphylococcus aureus from other species of staphylococci

Three commercial coagglutination tests--Sero-STAT, Accu-Staph, and Staphyloslide--were performed in parallel with slide coagulase, tube coagulase, and thermostable nuclease tests on 100 methicillin-susceptible Staphylococcus aureus (MSS) strains, 100 methicillin-resistant S. aureus (MRS) strains, and 100 non-S. aureus staphylococcal strains (NSA). All three coagglutination tests showed sensitivities of 100% for MSS strains. For MRS strains, sensitivities were, respectively, 99%, 100%, and 99%. False-positive reactions were, respectively, 10%, 2%, and 2%. A marked difference in slide coagulase test sensitivity was found for MSS strains (79%) and MRS strains (14%). These findings suggest that the coagglutination tests may be less sensitive for detecting MRS strains than for detecting MSS strains and that these properties may be related to clumping factor reactivity. The high false-positive rate for Sero-STAT and even the 2% false-positive rate for Accu-Staph and Staphyloslide make clinical usefulness at this time somewhat problematic and debatable. In view of these findings, the authors prefer to retain the tube coagulase test and thermostable nuclease test for differentiation of S. aureus from non-S. aureus strains in their laboratory.     

Evaluation of RapiDEC Staph for identification of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.

RapiDEC Staph is a test for presumptive identification of the principal human staphylococcal species, Staphylococcus aureus, S. epidermidis, and S. saprophyticus. The test includes control and test cupules for fluorogenic detection of coagulase and chromogenic substrates for alkaline phosphatase and beta-galactosidase. These tests identify S. aureus, S. epidermidis, and S. saprophyticus, respectively. Positive results with both chromogenic substrates provide a presumptive identification of S. xylosus or S. intermedius (S. xylosus-S. intermedius). Test cupules are inoculated with an organism suspension, and reactions are read after a 2-h incubation. RapiDEC-Staph was evaluated with 303 clinical and stock staphylococcal strains. Identifications were compared with those obtained by the tube coagulase test, a latex slide coagulase test (StaphAUREX), another commercial identification system (Staph-TRAC), and additional conventional tests. RapiDEC-Staph correctly identified 100% of 130 S. aureus strains, 70.3% of 74 S. epidermidis strains, and 81.3% of 32 S. saprophyticus strains. Four of five S. xylosus isolates were called S. xylosus-S. intermedius. Unidentified S. epidermidis and S. saprophyticus strains were called "Staphylococcus spp." Among the 62 other coagulase-negative staphylococci, 4 were misidentified as S. epidermidis and 7 were misidentified as S. saprophyticus. While the sensitivity and specificity of the fluorogenic coagulase test for S. aureus were 100%, failure to detect alkaline phosphatase activity in several S. epidermidis isolates resulted in fewer correct identifications by the RapiDEC-Staph test for this species.     

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.     

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.     

Identification and susceptibility testing of Staphylococcus aureus by direct inoculation from positive BACTEC blood culture bottles

This study explored the possibility of combining direct inoculation of tube coagulase and DNase tests, and the VITEK2 system, from BACTEC blood culture bottles in order to achieve rapid identification and susceptibility testing of Staphylococcus aureus. All isolates were identified correctly as S. aureus or coagulase-negative staphylococci (CNS). Antimicrobial susceptibility testing with the VITEK2 system gave 99.6% correct category agreement, with 0.1% very major errors and 0.3% minor errors among S. aureus isolates, and 97.4% correct category agreement, with 0.9% very major errors and 1.7% minor errors among CNS isolates. The results suggested that direct identification and susceptibility testing is sufficiently accurate for immediate reporting.     

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.     

The combined oxacillin resistance and coagulase (CORC) test for rapid identification and prediction of oxacillin resistance in Staphylococcus species directly from blood culture

The combined oxacillin resistance and coagulase (CORC) protocol for rapid identification and determination of oxacillin-susceptibility in Staphylococcus spp from blood culture is described. It incorporates a modified direct tube coagulase test (TCT) and a novel 4-hour multiplication-induction step, which increases the expression of staphylococcal PBP2a if present, facilitating detection by a commercial PBP2a latex agglutination kit. The protocol shows excellent sensitivity and specificity for determination of coagulase-positivity in staphylococci from patient blood cultures (96.8% (95% CI 81.5 to 99.8) and 100% (95% CI 75.9 to 100), respectively, n = 47), and for prediction of oxacillin resistance in S aureus directly from patient blood cultures (100% (95% CI 59.8 to 100) and 100% (95% CI 82.2 to 100), respectively (100% accuracy), n = 31) within 5 hours of blood culture positivity.     

Differentiation of coagulase-positive and coagulase-negative staphylococci by lectins and plant agglutinins.

The screening of staphylococci with a panel of 14 lectins and extracts demonstrating lectin-like activity led to the development of a rapid agglutination slide test for the differentiation of certain coagulase-negative staphylococci and human strains of Staphylococcus aureus. The coagulase-negative staphylococci were agglutinated by agglutinins from Mangifera indica, Triticum vulgaris, and crude Limulus polyphemus. The test is rapid, requiring only 5 to 15 min to identify an unknown strain of staphylococci, as opposed to the 4 to 16 h required to perform the conventional tube coagulase test.     

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.     

Comparison of commercial slide agglutination kits with a tube coagulase test for the rapid identification of Staphylococcus aureus from blood culture.

Eighty clinical specimens of BACTEC 9240 blood culture vials, culture positive for staphylococci (38 Staphylococcus aureus and 42 coagulase negative staphylococci), were tested directly for the presence of clumping factor/protein A and free coagulase. Seven commercial slide agglutination kits were compared with a direct-tube coagulase (DTC) method. All tests were performed on blood culture pellets. Sensitivity, specificity, and negative and positive predictive values for the seven commercial kits were extremely variable, whereas a two-hour DTC test was highly predictive of S aureus. There was no significant difference between a two-, six- or 24-hour DTC test. Three (8%) S aureus isolates remained DTC negative even after 24 hours' incubation. Staphylococcal slide agglutination kits should not be used directly on blood culture broths. In contrast, a two-hour DTC test is a useful, rapid screening test for S aureus bacteraemia, provided isolates from DTC negative blood culture broths are re-tested using standard laboratory techniques.     

Pharmacoeconomic analysis of microbiologic techniques for differentiating staphylococci directly from blood culture bottles

Differentiating staphylococci in blood cultures is a critical issue, particularly when only one of two cultures is positive by Gram staining for staphylococci. New tests for the identification of Staphylococcus aureus allow faster results and definitive treatment compared to the tube coagulase test interpreted at 24 h (TCT24). These newer tests, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and real-time PCR (RT-PCR), offer improved sensitivity at higher cost. Data suggest that the tube coagulase test may be interpreted at 4 h (TCT4) with little loss of sensitivity. The impact of variability in turnaround time, sensitivity, specificity, and cost on comparative cost-effectiveness is unknown. Our aim was to establish the cost-effectiveness of TCT24, PNA-FISH, RT-PCR, and TCT4 for direct identification of staphylococci in blood cultures. Decision analysis comparing these strategies was done from the institutional perspective. Besides test variables, other variables included patient risk factors, empirical treatment, and follow-up cultures. Probability and cost estimates came from the literature and institutional data. Base case estimates were derived from institutional rates of 73% contamination when coagulase-negative staphylococci were identified, 67.6% prevalence of risk factors, and 12.4% prevalence of S. aureus when one of two cultures yielded staphylococci. Sensitivity analysis was done across a range of probabilities and costs. In the base case, TCT4 and TCT24 were more cost-effective than RT-PCR and PNA-FISH ($78 versus $120 versus $165 per patient, respectively). The advantage of TCT4 and TCT24 remained robust upon sensitivity analysis. TCT4 should be further evaluated as a rapid, cost-effective means for identification of S. aureus in blood cultures.     

Evaluation of rapid coagulase methods for the identification of Staphylococcus aureus.

Four rapid latex agglutination assays, StaphAurex (Wellcome Diagnostics, Research Triangle Park, N.C.), Bacto Staph (Difco Laboratories, Detroit, Mich.), SeroSTAT (Scott Laboratories, Inc., Fiskeville, R.I.), Veri-Staph (Zeus Technologies, Raritan, N.J.), and two hemagglutination tests, Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.) and Hemastaph (Remel, Lenexa, Kans.), were compared with the conventional slide coagulase, tube coagulase (TC), and thermonuclease (TNase) tests for the identification of Staphylococcus aureus. A total of 118 clinical isolates of S. aureus (52 methicillin resistant), 50 S. epidermidis, 5 S. capitis, 2 S. hominis, 3 S. simulans, 6 S. saprophyticus, and 2 S. warneri were tested. The slide coagulase, TC and TNase tests detected 115 (97.5%), 117 (99.2%), and 118 (100%) of the S. aureus isolates, respectively. All showed 100% specificity. The StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph assays correctly identified 117 (99.2%), 117 (99.2%), 116 (98.3%), 110 (93.2%), 108 (91.5%), and 107 (90.7%) of the S. aureus isolates, respectively. For methicillin-resistant S. aureus isolates, StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph showed 1 (2%), 1 (2%), 2 (4%), 7 (13.5%), 7 (13.5%), and 8 (15.4%) false-negative results, respectively. All the commercial agglutination assays demonstrated false-positive results with strains of S. capitis, S. saprophyticus and S. warneri. The overall accuracy of the commercial agglutination assays compared with TC and TNase ranged from 90.7 to 99.2%. We recommend that negative reactions with the rapid commercial test kits for methicillin-resistant Staphylococcus isolates be confirmed with the TC or TNase test.     

Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing.

The detection of thermonuclease activity in 86 blood culture samples containing gram-positive cocci showed 100% correlation with the subsequent identification of the isolate as Staphylococcus aureus by the coagulase test. No positive thermonuclease results were found with 66 samples containing coagulase-negative staphylococci and 56 samples containing other gram-positive organisms. The thermonuclease test provides a rapid, reliable method to identify S. aureus in blood cultures.     

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 a yellow latex reagent with other agglutination methods for the identification of Staphylococcus aureus.

A new commercial yellow latex agglutination reagent (Bacto-Staph) was compared with the slide and tube coagulase tests and three other commercial reagents for the identification of 283 Staphylococcus aureus and 54 non-S. aureus staphylococcal strains. Test sensitivities for the identification of S. aureus were as follows: tube coagulase, 99.6%; slide coagulase, 98.6%; Bacto-Staph, 99.6%; Staphylatex, 98.6%; Sero STAT Staph, 98.2%; and Staphyloslide, 97.5%. No false-positive reactions were observed with any of the commercial reagents.     

Comparative evaluation of identification systems for testing methicillin-resistant strains of Staphylococcus aureus.

Several commercial systems are available to distinguish between Staphylococcus aureus and the coagulase-negative species of the Micrococcaceae family. Four latex agglutination systems (Accu-Staph, SeroSTAT, Staphaurex, and Staphylatex) and two hemagglutination systems (Hemastaph and Staphyloslide) were compared for their performance in the rapid identification of 232 isolates of staphylococci, including 114 of methicillin-resistant S. aureus. Accu-Staph, Staphaurex, and Staphyloslide correctly identified 100% of the methicillin-resistant S. aureus isolates; Hemastaph and Staphylatex, 99.1%; and SeroSTAT, 94.7%. Most reactions were easy to interpret, although 15% of the SeroSTAT reactions were weak. Autoagglutination occurred only with isolates of coagulase-negative staphylococci. False-positive reactions were rare and occurred only with systems which did not detect autoagglutination. Five of these six systems appear to be adequate for the rapid identification of S. aureus, including methicillin-resistant isolates.     

Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.