The clinical significance of hVISA has been debated since its initial description in 1997, and the uncertainty is likely to continue for the foreseeable future (2
). Clinical MRSA infections with hVISA strains have been associated with treatment failure and increased length of hospital stay (5
). Since no commonly used clinical laboratory methods, including automated susceptibility testing systems, can identify hVISA, a practical, real-time method for detection of reduced susceptibility to vancomycin is imperative. However, given the continuum of resistance illustrated in Fig. , finding the perfect test to detect hVISA will be a challenge. In this survey of clinical MRSA isolates with vancomycin MICs of 2 μg/ml by the reference broth microdilution method, a BHI screen agar method was shown to have the highest sensitivity and specificity in detecting hVISA compared to the PAP-AUC reference method. Heteroresistance has been seen in strains with vancomycin MICs as low as 0.5 μg/ml; however, we chose to focus our study on a collection of clinical MRSA isolates with vancomycin MICs of 2 μg/ml since these strains demonstrate heteroresistance at a greater rate (1
), and in a previous study we found no heteroresistance by PAP-AUC among MRSA isolates with vancomycin MICs of 0.5 and 1.0 μg/ml by the reference broth microdilution method (32
). All methods evaluated reliably detected heteroresistance among a collection of MRSA isolates with vancomycin MICs of ≥4 μg/ml by standard Etest (data not shown).
The screen agar approach is an inexpensive and efficient way to test multiple clinical isolates on a daily basis. However, several screen agar methods differing in medium composition, inoculum density, and choice of antibiotic and antibiotic concentration have been evaluated for the detection of hVISA and demonstrated poor sensitivity (12
). Currently, the only CLSI vancomycin screen agar method in place for clinical isolates for the detection of vancomycin-resistant S. aureus
(VRSA) and possibly VISA is BHI agar containing 6 μg/ml vancomycin (BHIA6V), a method originally established for detection of vancomycin resistance in enterococci (39
), and several studies showed this method to have a very low sensitivity for the detection of hVISA (44
). The CDC recommends this as a supplemental test for VISA detection, with the caveat that strains with vancomycin MICs of ≤4 μg/ml will not be reliably identified (http://www.cdc.gov/ncidod/dhqp/ar_visavrsa_labFAQ.html
) and screen agar plates with a lower concentration of 3 μg/ml vancomycin have a very high false-positive rate (20
In addition to the vancomycin concentration, the base medium of the screen agar appears to be important. The addition of supplements that enhance growth of hVISA could potentially improve the detection of hVISA by screen agar methods. Willey et al. found the addition of pancreatic digest of casein to BHI agar and 4 μg/ml vancomycin an improvement to other screen agars for the detection of VISA, and 97.7% of VISA strains in their study were successfully detected with high specificity within 24 h (42
). The addition of 20% horse serum to BHI has also been suggested as a means to differentiate between hVISA and VSSA (14
Although increasing the inoculum from a 0.5 to a 2.0 McFarland standard improved the sensitivity of the BHI screen agar method at 48 h (91% and 100%, respectively), there were 39 false positives with the higher inoculum when using the criterion for hVISA of two or more colonies growing in at least one of the four 10-μl droplets. Using a higher cutoff for the 2.0 McFarland inoculum (growth of 10 or more colonies from at least one of the four 10-μl droplets), the BHI screen agar method was 62% sensitive and 98% specific at 24 h and 81% sensitive and 90% specific at 48 h. The quadruplicate testing method (i.e., use of four 10-μl droplets and looking for growth in any one of the four droplets) enhanced sensitivity; up to 8/19 (42%) hVISA isolates might have been missed if only one droplet was inoculated on the screen agar plate.
The BHI screen agar method failed to identify only two isolates found to be hVISA by PAP-AUC. However, both isolates had a standard Etest teicoplanin MIC of 3 μg/ml (above the European Committee on Antimicrobial Susceptibility Testing [EUCAST] breakpoint of ≤2 μg/ml; www.eucast.org
) and were hVISA by the Etest GRD teicoplanin MIC. Historically, S. aureus
acquired resistance to teicoplanin, a glycopeptide that is widely used outside the United States, much earlier than resistance to vancomycin (18
) and teicoplanin resistance is frequently accompanied by a small increase in vancomycin resistance (10
). Additionally, Shlaes et al. (33
) demonstrated that penicillin binding protein 2 (PBP 2) is overproduced in a teicoplanin-resistant S. aureus
mutant strain compared with the level of production by its parent strain, and overproduction of PBP 2 is also observed in hVISA strain Mu3. Although MRSA strains that are resistant to teicoplanin can still remain susceptible to vancomycin and cross-resistance does not necessarily occur, all VISA strains identified to date have shown reduced susceptibility to teicoplanin (34
), making teicoplanin a useful marker for VISA and, potentially, hVISA. Ultimately, in the absence of molecular determinants specific to hVISA, the use of more than one method may be necessary for optimal screening sensitivity.
The Etest macromethod and the Etest GRD incorporate steps to improve detection of hVISA, such as use of enriched media, prolonged incubation, both vancomycin and teicoplanin, and a higher inoculum (Etest macromethod). Use of these methods as routine clinical laboratory tests for hVISA detection has been increasing, yet no standardized diagnostic methodology exists for either. Additionally, interpreting the results of both Etest methods for hVISA can be subjective and variable, and performing replicates is costly. Initial studies suggested that the Etest macromethod was a potentially effective screening test for detection of hVISA (24
); however, the accuracy of this method varies significantly between different studies (12
), probably due to the criteria used to define hVISA (29
) and the variability of the inoculum size utilized (24
). Though we followed the manufacturer's recommended method and inoculum size, we found low sensitivity, similar to results reported by Adam et al. (1
In conclusion, we found a BHI screen agar method that was easy to perform using standard media and inoculum sizes commonly utilized in clinical microbiology laboratories to be a useful method to identify hVISA. BHI agar plates with the addition of casein and 4 μg/ml vancomycin had the highest sensitivity and specificity in identifying hVISA compared to the two Etest methods when using PAP-AUC as the gold standard method. The sensitivity of detection improved significantly with incubation for 48 h, underscoring the apparent slow growth of hVISA isolates and the importance of longer incubation even at the expense of slightly lower specificity. Although the positive predictive value for this test was only 72%, 4/8 (50%) of the false positives were near the cutoff for hVISA by PAP-AUC (ratios of 0.83, 0.85, 0.86, and 0.89) and might have biologic relevance in the continuum of emerging resistance. Larger-scale studies to further evaluate the value of the BHI screen agar method for identification of hVISA in a clinical setting are warranted.