Laboratory Detection of Vancomycin-Intermediate/Resistant Staphylococcus aureus (VISA/VRSA)

Most isolates of S. aureus are susceptible to vancomycin. The concentration of vancomycin required to inhibit these strains (called the minimal inhibitory concentration or MIC) is typically between 0.5 and 2 micrograms/mL (μg/mL). In contrast, S. aureus isolates for which vancomycin MICs are 4-8 μg/mL are classified as vancomycin-intermediate, and isolates for which vancomycin MICs are ≥16 μg/mL are classified as vancomycin-resistant. The revised definitions for classifying isolates of S. aureus are based on the interpretive criteria published in January 2006 by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS)*.

CLSI lists only susceptible disk diffusion interpretive criteria (in mm) for vancomycin and Staphylococcus spp. There has not been a sufficient number of non-susceptible isolates to develop resistant and intermediate breakpoints. Organisms for which the vancomycin zone diameters are >=15mm are considered susceptible, although several studies show that this breakpoint is unreliable for detecting VISA strains.

* Clinical and Laboratory Standards Institute/NCCLS. Performance Standards for Antimicrobial Susceptibility Testing. Sixteenth informational supplement. M100-S16. Wayne, PA: CLSI, 2006.

CLSI is a global, interdisciplinary, nonprofit, standards-developing and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the healthcare community. Go to: CLSI.

The term glycopeptide refers to a group of antimicrobial agents that includes vancomycin and teicoplanin. Since the first two VISA isolates in the United States were also resistant to teicoplanin, the term glycopeptide-intermediate S. aureus (GISA) was used to indicate this broader resistance profile. While GISA may be a more specific term for strains intermediate to both vancomycin and teicoplanin, not all VISA strains are intermediate to teicoplanin; therefore, VISA is a more accurate and widely used term.

Vancomycin continues to be an important antimicrobial agent for treating infections caused by S. aureus strains that are resistant to oxacillin (MRSA) and other antimicrobial agents. The reduced susceptibility of VISA and VRSA strains to vancomycin leaves clinicians with relatively few therapeutic options for treating these infections.

Not all susceptibility testing methods detect VISA and VRSA isolates. Three out of six confirmed VRSA isolates were not reliably detected by automated testing systems in a recent report. Subsequently, some manufacturers have optimized their systems for VRSA detection, so laboratories should check with manufacturers to determine if their system has FDA clearance for VRSA detection. VRSA are detected by reference broth microdilution, agar dilution, Etest®, MicroScan® overnight and Synergies plus™; BD Phoenix™ system, Vitek2™ system, disk diffusion, and the vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].

VISA isolates are not detected by disk diffusion. Methods that typically detect VISA are non-automated MIC methods including reference broth microdilution, agar dilution, and Etest® using a 0.5 McFarland standard to prepare inoculum. Automated methods and vancomycin screen agar plates usually detect VISA for which the vancomycin MICs are 8 µg/ml, but further studies are need to define the level of sensitivity of these methods for S. aureus for which the vancomycin MICs are 4 µg/ml.

Laboratories that use automated methods that are not validated for VRSA detection should also include a vancomycin screen agar plate for enhanced detection of VRSA. If possible, laboratories should incorporate the vancomycin agar screen plate for testing all S. aureus. Alternatively, the screening may be limited to MRSA isolates, since nearly all VISA and all VRSA reported to date (i.e., April 2006) were also MRSA. Laboratories using disk diffusion to determine vancomycin susceptibility should consider adding a second method for VISA detection. The vancomycin screen plate is useful for detecting VISA (MIC = 8 µg/ml). Reliable detection of VISA (MIC = 4 µg/ml) may require a non-automated MIC method.

Information about susceptibility testing methods and their limitations should be made available to clinicians additional testing can be requested when reduced susceptibility to vancomycin is suspected.

The vancomycin agar screen test uses commercially prepared agar plates to screen pure cultures of bacteria for vancomycin. These plates contain BHI agar and 6 µg/ml of vancomycin. A 10µl inoculum of a 0.5 McFarland suspension should be spotted on the agar using a micropipette (final concentration=106 CFU/ml). Alternatively, a swab may be dipped in the McFarland suspension, the excess liquid expressed, and used to inoculate the vancomycin agar screen plate. For quality control, laboratories should use Entercococcus faecalis ATCC 29212 as the susceptible control and E. faecalis ATCC 51299 as the resistant control. Up to eight isolates can be tested per plate; quality control should be performed each day of testing. Growth of more than one colony is considered a positive result. All staphylococci that grow on these plates should be inspected for purity, and the original clinical isolates should be tested using an FDA-cleared MIC method for confirmation. Plates prepared in-house using various lots of media performed inconsistently and were inferior to those obtained commercially (CDC unpublished data). Performance of commercially prepared plates varies by individual manufacturer.

Testing should incorporate the following CLSI recommendations:

• Inoculum: Use direct colony suspension
• Incubation: 35°C, ambient air, for a full 24 hr.
• Endpoint: Examine very closely for any indication of growth

According to the newest CLSI (formerly NCCLS) standards, a vancomycin-intermediate or –resistant result for staphylococci isolate should be verified by repeating a validated MIC method and the organism identification.

Please refer to the VISA/VRSA testing algorithm, which presents a strategy of detecting and confirming VISA and VRSA using appropriate test methods.

Yes. The following additional antimicrobial agents should be tested against VISA and VRSA isolates: Clindamycin, daptomycin, linezolid, quinupristin/dalfopristin, rifampin, and trimethoprim-sulfamethoxazole. The laboratories of the Division of Healthcare Quality Promotion of the Centers for Disease Control and Prevention (CDC) will perform tests for these additional antimicrobial agents. It is essential to send probable isolates of VISA and VRSA to CDC as quickly as possible, even if the laboratory has the capability to test additional agents in-house; to facilitate organism confirmation and enhance infection control efforts. Isolates should be sent to CDC via your local or state health department. CDC may be notified of presumptive VISA/VRSA by sending an email to SEARCH@cdc.gov. Visit Surveillance for Emerging Antimicrobial Resistance Connected to Healthcare (S.E.A.R.C.H.) for more information.

It is best to freeze isolates at -60°C or lower in standard stock culture medium, such as a broth containing 15% glycerol, or in defibrinated sheep blood. However, if a laboratory cannot do this, the isolate should be subcultured to a non-selective agar slant (e.g., trypticase soy agar) and incubated overnight at 35°C. The following day, the caps should be tightened and the slants stored at 2-8°C. Repeated subcultures should be kept to a minimum prior to storage.

All VRSA isolates to date have been oxacillin-resistant (i.e., MRSA) and contained the resistance gene mecA. Most VISA isolates were also oxacillin-resistant. However, two VISA isolates became oxacillin-susceptible upon repeat isolation from the patient and one was oxacillin-susceptible but contained mecA.

All VRSA isolates to date contained the vanA vancomycin resistance gene. The vanA gene is usually found in enterococci and typically confers high-level vancomycin resistance (MICs= 512-1024µg/ml) to these organisms. Most VRSA-positive patients had a history of infections caused by both vancomycin-resistant enterococci (VRE) containing vanA and MRSA. It is likely that the vanA determinant was transferred via plasmids or transposons from the VRE to the MRSA strain, resulting in the VRSA.

The mechanism of reduced vancomycin susceptibility in VISA strains is not fully understood. VISA cells have thicker cell walls that contain many cell wall subunits capable of binding vancomycin extracellularly and changes in several metabolic pathways. Vancomycin must reach the cell membrane and bind to the growing cell wall complex to inhibit cell growth.

Yes. There is significant concern about the spread of VISA and VRSA among patients because of limited treatment options. If a VISA or VRSA is suspected, specific infection control precautions need to be initiated by infection control personnel to decrease the risk of VISA/VRSA transmission to other patients. It is critical for laboratory workers to contact the infection control team immediately when a VISA or VRSA is suspected. Additionally, laboratories should notify the local and/or state health department and the Division of Healthcare Quality Promotion, Coordinating Center for Infectious Diseases, CDC, by sending an email to SEARCH@cdc.gov. The isolate should be saved and sent to the health department and CDC for confirmatory testing.

CDC developed a training tool for laboratorians to enhance their understanding and improve their proficiency in performing antimicrobial susceptibility testing (M.A.S.T.E.R.).

To report or request testing of suspected VISA or VRSA isolates, send an email to SEARCH@cdc.gov with your contact information (i.e., name, title, telephone number, laboratory or facility name, and a description of your testing methods and results facility and/or laboratory name, telephone number). Visit Surveillance for Emerging Antimicrobial Resistance Connected to Healthcare (S.E.A.R.C.H.) for more information.