Comparison of the Vitek Gram-Positive Susceptibility 106 Card and the MRSA-Screen Latex Agglutination Test for Determining Oxacillin Resistance in Clinical Bloodstream Isolates of Staphylococcus aureus

Abstract

The Vitek automated susceptibility testing system with a modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and a rapid slide latex agglutination test (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their ability to detect oxacillin resistance in Staphylococcus aureus. The oxacillin-salt agar screen (OS) test, the reference broth microdilution method, and the detection of the mecA gene by PCR were compared with the commercial products. A total of 200 contemporary (1999) bloodstream infection isolates were collected from the SENTRY Antimicrobial Surveillance Program, representing diverse geographic areas throughout the world. Among the 99 mecA-positive isolates, 3 isolates were found negative by the MRSA-Screen. Another two isolates did not grow on OS plates and had MICs of 0.5 and 2 μg/ml with the Vitek GPS card. All 101 mecA-negative isolates were also found negative by the MRSA-Screen and were categorized as susceptible by the GPS card. Overall, the MRSA-Screen, GPS card, and OS test had sensitivities of 96.9, 98.0, and 98.0% and specificities of 100.0, 100.0, and 98.0%, respectively. MRSA-Screen was a rapid (≤15 min) and simple test to perform, and the GPS card provided results in <8 h. Both methods were sensitive and specific for detecting staphylococcal oxacillin resistance in the clinical microbiology laboratory.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has steadily emerged over the last four decades to now cause both nosocomial and community-acquired infections (2). The optimal phenotypic method for detecting (methicillin) oxacillin resistance in S. aureus remains controversial. Moreover, errors in the detection of oxacillin resistance can lead to important adverse clinical consequences. False-susceptible results may contribute to treatment failure as well as to the unchecked spread of MRSA due to a failure to apply appropriate infection control measures. False-resistant results contribute to increased health care costs related to unnecessary isolation precautions, in addition to the consequences of overuse of glycopeptide antimicrobials agents, with associated resistance risk (8). The definitive genotypic test for oxacillin resistance in S. aureus (mecA gene detection by PCR) is not practical for routine performance in clinical laboratories. Therefore, there is an urgent need for a rapid, sensitive, and specific test for MRSA that can be easily performed in clinical microbiology laboratories.

This study was performed to compare two newly available tests: (i) the Vitek automated susceptibility testing system with the recently modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and (ii) a rapid slide latex agglutination test (16) for the detection of oxacillin resistance in recent (1999) blood culture isolates of S. aureus obtained from patients worldwide.

MATERIALS AND METHODS

Bacterial isolates.

A total of 200 bloodstream infection isolates were obtained from the SENTRY Antimicrobial Surveillance Program (19), representing diverse geographic areas (81 isolates from North America, 33 isolates from Latin America, 48 isolates from the Western Pacific, and 38 isolates from Europe). These isolates were selected on the basis of oxacillin MIC distribution using SENTRY-based results from reference broth microdilution testing as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The isolates used in this study had the following oxacillin MICs (number of isolates): ≤0.06 μg/ml (1), 0.12 μg/ml (13), 0.25 μg/ml (31), 0.5 μg/ml (19), 1 μg/ml (23), 2 μg/ml (14), 4 μg/ml (4), 8 μg/ml (2), and >8 μg/ml (93). All of the isolates had species level identification confirmed by conventional tests (9), specifically, slide and tube coagulase tests and an automated procedure (bioMerieux Vitek). All isolates were frozen at −70°C until processed. Prior to being tested, each isolate was subcultured at least twice on blood agar plates (Remel, Lenexa, Kans.) to ensure purity and optimal growth characteristics. All isolates were subjected to blind testing with the MRSA-Screen (Denka Seiken Co., Ltd., Tokyo, Japan) test, oxacillin-salt agar screen (OS) test, and determination of oxacillin MICs by use of the Vitek GPS 106 Card and the reference NCCLS broth microdilution test (17, 18). Control strains used for all assays included MRSA ATCC 43300 and a well-characterized MRSA clinical strain as well as the quality control strain S. aureus ATCC 29213 (oxacillin susceptible).

Susceptibility testing methods.

The MRSA-Screen test was performed according to the manufacturer's instructions. Briefly, a 1-μl loop of bacterial cells was taken from a fresh subculture, suspended in 4 drops of extraction reagent 1, and boiled for 3 min. After the suspension was allowed to cool to room temperature, 1 drop of extraction reagent 2 was added and vortexed thoroughly. The suspension was centrifuged at 1,500 × g for 5 min. A 50-μl aliquot of the supernatant was mixed with 1 drop of anti-PBP 2a monoclonal antibody-sensitized latex beads. A negative control consisted of 50 μl of supernatant mixed with 1 drop of negative control latex. The samples were placed on a shaker for 3 min prior to visual assessment of agglutination.

Five isolates of MRSA, including control strains, and five isolates of oxacillin-susceptible S. aureus (including ATCC 29213) were retested three times in triplicate to determine test reproducibility.

Automated susceptibility testing was performed using the Vitek GPS 106 Card (software configuration version R07.01) according to manufacturer recommendations. The current NCCLS breakpoint for oxacillin susceptibility of S. aureus was used (MIC for susceptible, ≤2 μg/ml; MIC for resistant, ≥4 μg/ml [17, 18]. Antimicrobial susceptibility testing of isolates using OS plates (Mueller-Hinton agar plates supplemented with 4% NaCl and 6 μg of oxacillin per ml [Remel]) and broth microdilution trays was performed in accordance with NCCLS guidelines (17, 18).

PCR was performed for the detection of mecA as previously described (3, 5). When S. aureus isolates yielded discrepant results among the tests used, the tests were repeated in triplicate with specimens from the same organism source.

RESULTS

Table 1 summarizes the results obtained with the MRSA-Screen test, the Vitek GPS 106 Card, the OS test, and the reference broth microdilution method. Of the 200 isolates tested, 99 isolates were found positive by mecA PCR and 101 were found negative. The MRSA-Screen test was able to accurately detect oxacillin resistance in almost all isolates (sensitivity, 96.9%). There were no false-positive results (specificity, 100.0%). However, oxacillin resistance was not detected in three isolates (Tables 1 and 2). Upon retesting of these isolates, two isolates produced visible agglutination with anti-PBP 2a-sensitized latex within the 3 min recommended by the manufacturer (sensitivity, 99.0%). The other isolate was found positive when the slide latex agglutination reaction was extended to 10 min.

TABLE 1.

Evaluation of MRSA-Screen test, Vitek GPS 106 Card, OS test, and broth microdilution method for detection of oxacillin resistance in 200 S. aureus isolates

PCR mecA detection	No. of isolates	No. of isolates with the indicated result a:
		MSRA-Screen test		OS test		Vitek MIC (μg/ml) of:		Microdilution MIC (μg/ml) of:
		Negative	Positive	No growth	Growth	≤2	≥4	≤2	≥4
Positive	99	3 (1)	96 (98)	2	97	2	97	1	98
Negative	101	101	0	99 (101)	2 (0)	101	0	100	1

^aPercent sensitivities and specificities for MRSA-Screen, OS, Vitek, and microdilution tests were 96.9 (99.0), 98.0, 98.0, and 99.0, respectively, and 100.0, 98.0 (100.0), 100.0, and 99.0, respectively. Numbers in parentheses indicate results after repeated tests. See the text for further explanations. 

TABLE 2.

Characteristics of the S. aureus isolates (coagulase positive) which had discrepant results among mecA PCR detection, MRSA-Screen test, Vitek GPS 106 Card test, OS test, and the broth microdilution method

Isolate	Resulta of the following test:
	mecA PCR	MRSA-Screen	Vitek GPS 106 Card	OS	Broth microdilution
013	+	+	0.5	−	2
058	+	+	2	−	4
011	+	−b	≥8	+	8
018	+	−b	≥8	+	>8
212	+	−c	≥8	+	>8
098	−	−	2	+d	2
123	−	−	2	+d	2
032	−	−	2	−	4

^a+, positive; −, negative; results for Vitek and broth microdilution tests are given as MICs in micrograms per milliliter. 

^bThese isolates were positive when retested. 

^cThis isolate was positive when the result was read at 10 min of rotation. 

^dThese isolates were negative when retested. 

The Vitek GPS 106 Card also had high sensitivity (98.0%) and specificity (100.0%). However, the GPS card falsely categorized two isolates as oxacillin susceptible, with MICs of 0.5 and 2 μg/ml (Table 2). One of these isolates also was categorized as oxacillin susceptible by the broth microdilution method (MIC, 2 μg/ml), and the other isolate was borderline oxacillin resistant (MIC, 4 μg/ml). Both isolates were found negative (susceptible) by the OS test. The OS test was also reliable for the detection of MRSA, with a sensitivity and a specificity of 98.0%. Two false-positive (susceptible) tests resolved when the test was repeated in triplicate (specificity, 100.0%).

Upon replicate testing of a panel of isolates, the MRSA-Screen was found to be 100% reproducible (data not shown).

DISCUSSION

This investigation showed that the detection of PBP 2a by the MRSA-Screen test and the Vitek automated susceptibility testing system with the modified GPS 106 Card were sensitive and specific for detecting staphylococcal oxacillin resistance in clinical bloodstream infection isolates. Since the isolates used in this study were selected from diverse geographic areas (epidemiologically unique) and their oxacillin MIC distribution approximated that of all S. aureus isolates in the SENTRY Program, this collection can be regarded as a reflection of S. aureus strains seen in clinical laboratories throughout the world. This study validated each rapid method tested (15 min to 8 h), as well as the NCCLS methods, in this high-priority clinical setting.

The MRSA-Screen test was very rapid and easy to perform, providing results within 15 min. This test has been evaluated by several investigators, and our results were similar (1, 14, 15, 21, 22). Previous studies were done with locally endemic clinical strains from western Europe, Canada, and Australia. When false-negative results were observed (three mecA-positive isolates), most (two) were positive when repeated. We assume that the false-negative results occurred secondary to a low inoculation volume. The recent study by Louie et al. (14) indicated that the use of a larger inoculum, of approximately 50 colonies, resulted in improved sensitivity without a loss of specificity. The remaining single discordant isolate showed no agglutination after 3 min of rotation (recommended by the manufacturer), but when the reaction time was extended to 10 min, visible agglutination occurred, confirming the observations of others (21). von Griethuysen et al. (21) reported five mecA-positive isolates that demonstrated only weak positive results after 3 min of rotation, but agglutination became stronger when rotation was extended to 6 min. The manufacturer has recently modified the package insert recommendations, indicating the need for extended time (10 min) for agglutination. On the other hand, some oxacillin-susceptible isolates may show false-positive reactions due to the prolonged reaction time. In fact, we found weak positive reactions for oxacillin-susceptible isolates, including the ATCC 25923 control strain, when incubation was prolonged for ≥15 min. Therefore, it is suggested that any strain showing agglutination after 10 min should undergo a mecA PCR as the final and conclusive confirmatory assay.

The Vitek system with the GPS 106 Card was easy to use and could provide results within 8 h. The GPS card was revised to a total of three oxacillin-containing wells, and new software (R07.01) was developed to analyze this pattern of concentrations. Although the GPS card was also modified to meet the new NCCLS oxacillin breakpoint for coagulase-negative staphylococci (17, 18), an assessment of the ability of the GPS card to detect methicillin-resistant coagulase-negative staphylococci is pending.

Prior studies using earlier versions of Vitek system cards have raised persisting concerns over their accuracy for detecting oxacillin resistance (6, 7, 12, 20), but interim modifications of the system have led to improved detection of this resistance (4, 10, 11). As an example of earlier problems, Skulnick et al. (20) compared results obtained by reference methods, including broth microdilution and mecA gene probe, with those obtained by the Vitek GPS-SA Card. These investigations in 1992 reported 14.2% very major (false-susceptible) errors (20). In contrast, Knapp et al. (10) examined 67 oxacillin-resistant isolates of S. aureus with the Vitek GPS-SA Card and found nearly complete agreement with the mecA results. More recently, Frebourg et al. (4) evaluated the ability of three automated systems to detect oxacillin resistance. In that study, the Vitek system with the GPS 503 Card was judged acceptable, but it failed to detect 3 of 64 mecA-positive S. aureus isolates (4.7% false-susceptible results) (4). Only 2% of mecA-positive isolates were misclassified as oxacillin susceptible in our evaluation, and we confirmed the Vitek system accuracy using the latest GPS card and associated software.

Nevertheless, there remain several concerns regarding the accuracy of detection of oxacillin resistance in heterogeneously resistant populations of clinical S. aureus isolates using phenotypic methods. Difficulties in the differentiation of MRSA from borderline oxacillin-resistant isolates may still occur (2, 13). Although we did not evaluate the accuracy of the Vitek system and the MRSA-Screen with such rare but challenging isolates, previous studies indicated that both tests are able to accurately detect heteroresistant isolates (1, 4). MRSA-Screen has also been found to be able to differentiate borderline oxacillin-resistant S. aureus from true MRSA (14). However, one study of mecA-negative S. aureus strains with borderline susceptibility to oxacillin revealed that 10% of such strains were classified as MRSA by earlier Vitek cards (11), and the Vitek GPS 106 Card (tested here) must be assessed in this area. Thus, the MRSA-Screen could emerge as a useful and technically simple test for the direct assessment of mecA in clinical laboratories where PCR for this gene or the Vitek system is not readily available.

In conclusion, we found the MRSA-Screen and the Vitek system with the modified GPS 106 Card to be reliable for the detection of oxacillin resistance in S. aureus. Both tests were rapid and easy to perform, and each has the potential for wider use in clinical microbiology laboratories.