The relationship between hvisa, VISA, high vancomycin MIC and outcome in

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1 JCM Accepts, published online ahead of print on 16 May 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved The relationship between hvisa, VISA, high vancomycin MIC and outcome in serious Staphylococcus aureus infection. Natasha E. Holmes 1,2, Paul D. R. Johnson 1,2,4, Benjamin P. Howden 1,3,4,5, Austin Centre for Infection Research (ACIR), Department of Infectious Diseases, Austin Health, Heidelberg VIC 2 Department of Medicine, University of Melbourne, Parkville VIC 3 Department of Microbiology, Austin Health, Heidelberg VIC 4 Department of Microbiology, Monash University, Clayton VIC 5 Department of Microbiology and Immunology, University of Melbourne, Parkville VIC Corresponding author: A/Prof Benjamin Howden Department of Infectious Diseases Austin Health PO Box Studley Road Heidelberg VIC 3084 Australia Tel: Fax: benjamin.howden@austin.org.au 25 1

2 Abstract Vancomycin has been used successfully for over 50 years for the treatment of Staphylococcus aureus infections, particularly methicillin-resistant S. aureus. It has proved remarkably resilient but its efficacy is now being questioned with the emergence of strains of S. aureus that display heteroresistance, intermediate resistance and occasionally complete vancomycin resistance. More recently an association has been established between poor outcome and infections with strains of S. aureus with elevated vancomycin minimum inhibitory concentration within the susceptible range. This mini-review summarizes the definitions, mechanisms, clinical impact and laboratory identification of reduced vancomycin susceptibility in S. aureus, and discusses practical issues for the diagnostic laboratory in testing and interpreting vancomycin susceptibility for S. aureus infections. History and definition of reduced vancomycin susceptibility in Staphylococcus aureus Vancomycin, a glycopeptide with activity against a wide range of Gram-positive organisms, was discovered in 1952 and by 1958 was registered by the United States Food and Drug Administration for treatment of penicillin-resistant and methicillinresistant Staphylococcus aureus. Unlike the rapid appearance of S. aureus resistant to penicillin and semi-synthetic penicillins, reduced susceptibility or resistance to vancomycin took over three decades to emerge. S. aureus strains displaying vancomycin heteroresistance (hvisa) and vancomycin intermediate resistance (VISA) were first isolated in Japan in 1996 (8). hvisa strains are phenotypically susceptible using broth microdilution (BMD) however more detailed testing reveals 2

3 subpopulations of cells with reduced susceptibility to vancomycin. Vancomycinresistant S. aureus (VRSA) due to acquisition of the vana resistance determinant from enterococci was subsequently reported in the United States in VRSA has also been described in Iran and India, although it remains rare worldwide. Differences in vancomycin breakpoints In the United States, the Clinical and Laboratory Standards Institute (CLSI) reduced the vancomycin minimum inhibitory concentration (MIC) susceptibility breakpoint for S. aureus from 4 µg/ml to 2 µg/ml in 2006 (3). Therefore any literature published before this date needs careful appraisal as the definitions of susceptible, intermediate and resistant have now changed. VISA and VRSA are currently defined as vancomycin MIC using BMD of 4-8 µg/ml and 16 µg/ml respectively (3). In contrast, both the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the British Society for Antimicrobial Chemotherapy (1) simply define S. aureus strains as vancomycin susceptible (MIC 2 µg/ml) or resistant (MIC > 2 µg/ml) (1, 6). The rationale cited for this definition are difficulties in confirming heteroresistance and avoiding confusion for clinicians because there is a likelihood of clinical treatment failure when vancomycin is used to treat infections where the MIC is above 2 µg/ml. Definition of hvisa Although it is accepted that hvisa strains are phenotypically susceptible to vancomycin using routine laboratory methods but contain vancomycin-intermediate subpopulations on culture, there is no precise definition for hvisa. These subpopulations are typically present at frequencies of 10-6 to Population analysis 3

4 profile (PAP) testing remains the gold standard for detection of these subpopulations (7, 8, 33). One example is the modified PAP method by Wootton et al. (33) in which the area under the curve (5) of a PAP test (PAP/AUC) between a test organism and the hvisa reference strain Mu3 (ATCC ) is calculated. Using this method, vancomycin-susceptible S. aureus (VSSA) is defined as a PAP/AUC ratio < 0.9 and hvisa as a PAP/AUC ratio 0.9. While earlier studies focused on the role of VISA and hvisa in vancomycin treatment failure, there are now increasing reports of poor clinical outcomes in S. aureus infections where the vancomycin MIC is in the upper end of the susceptible range ( 2 µg/ml) and hvisa has been excluded (29). The proportion of S. aureus isolates that are hvisa increases as the vancomycin MIC increases, and the differences between VSSA, hvisa and VISA are accordingly subtle and may represent a continuum of small incremental changes in MIC and other characteristics. In contrast VRSA is a separate entity with acquisition of the vana gene conferring complete vancomycin resistance in the majority of reported strains. Mechanisms of reduced vancomycin susceptibility Cell wall Sequential mutations in VSSA lead to the emergence of hvisa and ultimately VISA. The hallmark changes are alterations in the bacterial cell wall resulting in reduced autolytic activity and wall thickening. This is thought to result in an impaired ability of vancomycin to reach its binding site, and occurs specifically during the cell cycle when the division septum is being formed. These changes are particularly noted after prior exposure to vancomycin (4, 13). Multiple genetic mutations have been 4

5 implicated in the pathogenesis of these cell wall modifications, usually occurring in genes important for cell wall metabolism such as vrars (15) and grars, however recent attention has focused on the essential S. aureus regulator walkr (14) Accessory gene regulator locus Differences in the accessory gene regulator (agr) quorum-sensing system locus have also been associated with vancomycin heteroresistance. The agr types I and II have been associated with vancomycin resistance (32), while altered agr function leading to reduction in RNA III transcription and delta-haemolysin production has also been linked with resistance (26). Other changes in hvisa Often multiple small sequential changes lead to step-wise generation of hvisa and VISA (12). A number of other changes have been connected with vancomycin heteroresistance, including metabolic changes, altered surface proteins or muropeptides, reduced growth kinetics, and attenuated virulence (12, 23). While the genetic determinants of hvisa and VISA are partially understood, the relative contributions of these mutations and altered cellular processes that contribute to hvisa and VISA in determining the vancomycin MIC are unknown. Clinical impact of hvisa, VISA and elevated vancomycin MIC Clinical features of hvisa and VISA The spectrum of clinical disease caused by hvisa and VISA is similar to VSSA but the prevalence and clinical impact is difficult to determine due to the lack of a 5

6 standardized definition and absence of controlled prospective studies. High inoculum infections such as bacteremia, endocarditis and osteomyelitis (2, 19), and persistent bacteremia (13, 16, 19) have been associated with vancomycin heteroresistance, and this may lead to vancomycin treatment failure (2) or other complications (19). However it is difficult to determine whether hvisa is the cause or corollary of treatment failure in some studies, especially if it is not clear when the hvisa or VISA isolate was detected in the course of the infection. Interestingly, one study noted reduced rates of shock in patients with heteroresistant infections compared with VSSA (28), possibly demonstrating a clinical correlate of attenuated virulence noted in animal models of VISA infection. Interestingly, pooled data from a recent meta-analysis demonstrated similar mortality rates between VSSA and hvisa infections, however treatment failure was more common in the hvisa group (30). Other studies that include less serious infections have reported comparable outcomes and question the clinical relevance of hvisa (11). Elevated vancomycin MIC Clinical factors associated with elevated vancomycin MIC are similar to those associated with the development of hvisa, and include prior vancomycin exposure (18), prior methicillin-resistant S. aureus bacteremia (MRSA) and increased patient age. Although rates of hvisa detection by PAP/AUC analysis increase as the vancomycin MIC by BMD increases (22, 25), increased mortality and treatment failure has also 6

7 been reported in infections with VSSA isolates with elevated vancomycin MIC in the fully susceptible range (10, 27-29). Typically these isolates have MICs near the susceptibility breakpoint such as 1.5 or 2 µg/ml using different MIC methodologies. This has created debate about whether vancomycin susceptibility breakpoints should be reduced further, however it is difficult to ascertain an appropriate new breakpoint from these studies as there is significant heterogeneity in clinical features and infection types, different MIC testing methods, and different MIC values associated with inferior outcomes. In addition hvisa was not assessed in many of these studies. The underlying mechanism by which elevated vancomycin MIC in VSSA causes inferior outcomes has not yet been elucidated. It has been suggested that vancomycin treatment be avoided in these situations as it is presumed that the continuum of changes that lead to reduced vancomycin susceptibility and hvisa is implicated in the development of resistance. In addition, reduced vancomycin bactericidal activity has been noted in vitro in VSSA isolates with elevated vancomycin MIC (27). However differences in bacterial genotype may be an alternative explanation, with recent publications highlighting associations between genotype and elevated vancomycin MIC. This is particularly relevant as elevated vancomycin MIC and inferior outcomes in VSSA are not merely confined to MRSA, but also occur in methicillin-susceptible S. aureus (MSSA) (10), including patients receiving betalactam therapy. Elevated vancomycin MIC may therefore be a surrogate marker for an unknown mechanism leading to treatment failure. Laboratory identification of hvisa, VISA and elevated vancomycin MIC 7

8 The lack of a precise definition and standardized testing makes optimum detection of hvisa difficult, as these strains will be missed by routine susceptibility tests and relying on vancomycin MIC alone will not be sufficient Colony morphology There may be clues when evaluating growth characteristics on conventional agar plates as hvisa strains may have altered growth kinetics. Careful observation may reveal smaller-sized colonies or mixed small colony variants (SCV) amongst normal colonies in a pure culture, reduced pigmentation and hemolysis, and slower growth (12, 20). These changes may be subjective and are not diagnostic and their observation should not replace confirmatory testing. Screening tests for hvisa Due to the low frequency of vancomycin-intermediate subpopulations in hvisa, the low inoculum required for BMD MIC testing is insufficient to detect these subpopulations. Consequently methods for hvisa detection use a higher inoculum, prolonged incubation (to promote growth of resistant subpopulations), or more nutritious agar (12). The macromethod Etest (MET) is a screening test for hvisa using a higher inoculum (2 McFarland standard) and a longer incubation (48h). A positive test is reported if the teicoplanin MIC is 12 µg/ml or if the teicoplanin MIC is 8 µg/ml with vancomycin MIC also 8 µg/ml. The actual MIC result cannot be reported because the method differs from standard MIC calculation. An alternative screening test is the Glycopeptide Resistance Detection (GRD) Etest which is a double-ended Etest strip 8

9 using a standard inoculum (0.5 McFarland) on Mueller-Hinton + 5% blood agar. A positive GRD is reported if the vancomycin or teicoplanin MIC is 8 µg/ml. A number of screening agar plates have also been developed, however the sensitivity has been variable (12). These Etest screening tests have been compared with the gold standard PAP and shown to have good sensitivity and specificity (34), although a recent report suggested that MET and GRD did not perform as well as expected (31). Ultimately any screening test should undergo confirmatory testing with PAP/AUC before reporting any positive result. A novel method for rapid identification of hvisa or VISA within 4 hours from clinical isolates has recently been described using multiplexed automated digital microscopy (21). Results were correlated with PAP analysis and further studies are required to determine its clinical utility. Confirmatory tests for hvisa The definitive method for identifying hvisa is the population analysis profile (PAP), however it is time consuming, labour-intensive and difficult to apply in real-time clinical decision-making due to delayed test results. The modified PAP/AUC described by Wootton et al. (33) compares the PAP/AUC of a reference hvisa strain Mu3 with the test organism, with a PAP/AUC of <0.9, and >1.3 for VSSA, hvisa and VISA respectively. 9

10 As the genetic determinants for hvisa are still being delineated and involve different mechanisms, there is no specific molecular assay for diagnosis or detection currently available either in the research or diagnostic laboratory VISA and VRSA Detection of VISA and VRSA is easier as there are defined CLSI MIC criteria. It is important to note that these have been defined using the reference BMD method. Results using other MIC methods should always be confirmed with BMD prior to reporting. Vancomycin MIC The reference method for vancomycin MIC is BMD. As this is labour-intensive and time consuming, many diagnostic laboratories assess vancomycin susceptibility using other methods such as agar dilution, Etest, disc diffusion and commercial instrumentbased assays (for example, Vitek-2, Phoenix, Microscan). Unfortunately these methodologies produce subtle differences in MIC results compared with BMD, with Etest consistently one-fold dilution (or log 2 dilutions) higher than reference BMD (10, 17, 24). Note that automated methods are required to align with BMD for United States Food and Drug Administration licensing. Many studies that have evaluated vancomycin MIC and outcome have noted that elevated Etest MIC predicts poor outcome and the hvisa phenotype better than BMD, often around 1.5 µg/ml (constituting a half-dilution step when using traditional BMD) (9, 29). The implications of vancomycin MIC and methodology are relevant and crucial when considering vancomycin pharmacodynamics. The area under the concentration curve 10

11 divided by the minimum inhibitory concentration (AUC/MIC) ratio is the best predictor of vancomycin efficacy. Small variations in MIC will therefore lead to significant changes in the AUC/MIC ratio. In addition, the optimal vancomycin AUC/MIC targets were devised using BMD MIC methodology. How should the diagnostic laboratory test and interpret vancomycin susceptibility in S. aureus? Vancomycin MIC From a practical viewpoint, the relevance of different vancomycin susceptibility breakpoints according to CLSI, EUCAST or BSAC adopted by individual laboratories may be less important than other information provided in a laboratory report or by direct liaison with the clinician. The MIC method should always be stated, because clinicians need to be mindful of MIC methodology if using the MIC result for therapeutic decisions (for example, pharmacodynamic and dosing considerations). As a number of studies have shown that Etest MIC seems to predict treatment failure better than BMD, this may be a convenient and practical MIC method. Interpretation of susceptibility results should occur in discussion with relevant clinical personnel such as infectious diseases pharmacists and physicians, clinical microbiologists, and hospital antimicrobial committee members. It may be prudent to recommend caution about increased risk of vancomycin treatment failure in isolates with MIC > 1 µg/ml (regardless of the test method and irrespective of heteroresistance) (9, 29). However it is also important to remember that the clinical context is important, and patients who are responding to vancomycin may not 11

12 automatically need to be switched to an alternative agent merely in response to an elevated vancomycin MIC laboratory result hvisa The ideal manner for detection of vancomycin heteroresistance is dependent on many factors such as laboratory workload, number of specimens, capacity to perform more detailed tests, and cost. More importantly, the selection of isolates that will undergo hvisa testing must be considered carefully as it is not feasible to test all S. aureus clinical isolates. Clinically-based and laboratory-based algorithms have been proposed to guide the screening and confirmatory testing process (12). Patients at increased risk of heteroresistance and vancomycin treatment failure (such as older age, prior vancomycin treatment, previous MRSA infection, high bacterial burden infections) should be prioritized for hvisa screening as early as possible in order to facilitate results being available in a clinically meaningful timeframe. Again, the decision to change therapy from vancomycin to alternative agents needs to be based on the clinical context and not purely on laboratory susceptibility results. Although several screening tests have reasonable sensitivity and specificity, and can be helpful in a more timely fashion before PAP/AUC, a confirmatory test should always be performed on any isolate referred for hvisa testing for a definitive result. Table 1 summarizes some of these key points including the clinical impact of vancomycin susceptibility and implications for the diagnostic laboratory. Vancomycin has been the workhorse antibiotic for MRSA infections for over 50 years. Although the area of vancomycin heteroresistance and susceptibility testing has 12

13 become complicated, rates of VRSA and VISA are still relatively low while hvisa is more common. In order to facilitate more rapid testing for hvisa and VISA, further understanding of the molecular changes associated with heteroresistance is required. Additional knowledge about the relationship between elevated vancomycin MIC and outcome in VSSA is urgently required to clarify the ongoing role of vancomycin in the treatment of serious S. aureus infections. Acknowledgments This work was supported by National Health and Medical Research Council (NHMRC) Australia. Downloaded from on November 30, 2018 by guest 13

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16 Howden, B. P., P. D. Johnson, P. B. Ward, T. P. Stinear, and J. K. Davies Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob. Agents Chemother. 50: Howden, B. P., C. R. McEvoy, D. L. Allen, K. Chua, W. Gao, P. F. Harrison, J. Bell, G. Coombs, V. Bennett-Wood, J. L. Porter, R. Robins-Browne, J. K. Davies, T. Seemann, and T. P. Stinear Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR. PLoS pathogens 7:e Howden, B. P., T. P. Stinear, D. L. Allen, P. D. Johnson, P. B. Ward, and J. K. Davies Genomic analysis reveals a point mutation in the twocomponent sensor gene gras that leads to intermediate vancomycin resistance in clinical Staphylococcus aureus. Antimicrob. Agents Chemother. 52: Howden, B. P., P. B. Ward, P. G. Charles, T. M. Korman, A. Fuller, P. du Cros, E. A. Grabsch, S. A. Roberts, J. Robson, K. Read, N. Bak, J. Hurley, P. D. Johnson, A. J. Morris, B. C. Mayall, and M. L. Grayson Treatment outcomes for serious infections caused by methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility. Clin. Infect. Dis. 38: Hsu, D. I., L. K. Hidayat, R. Quist, J. Hindler, A. Karlsson, A. Yusof, and A. Wong-Beringer Comparison of method-specific vancomycin minimum inhibitory concentration values and their predictability for treatment outcome of meticillin-resistant Staphylococcus aureus (MRSA) infections. Int. J. Antimicrob. Agents 32:

17 Lodise, T. P., C. D. Miller, J. Graves, A. Evans, E. Graffunder, M. Helmecke, and K. Stellrecht Predictors of high vancomycin MIC values among patients with methicillin-resistant Staphylococcus aureus bacteraemia. J. Antimicrob. Chemother. 62: Maor, Y., M. Hagin, N. Belausov, N. Keller, D. Ben-David, and G. Rahav Clinical features of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia versus those of methicillin-resistant S. aureus bacteremia. J Infect. Dis. 199: Marlowe, E. M., M. D. Cohen, J. F. Hindler, K. W. Ward, and D. A. Bruckner Practical strategies for detecting and confirming vancomycinintermediate Staphylococcus aureus: a tertiary-care hospital laboratory's experience. J. Clin. Microbiol. 39: Metzger, S., C. S. Price, W. M. J. Dunne, and D. Howson Automated 4- hour detection of heteroresistant vancomycin-intermediate Staphylococcus aureus (hvisa) (poster 083), 111th General Meeting American Society for Microbiology. American Society for Microbiology, New Orleans. 22. Musta, A. C., K. Riederer, S. Shemes, P. Chase, J. Jose, L. B. Johnson, and R. Khatib Vancomycin MIC plus heteroresistance and outcome of methicillin-resistant Staphylococcus aureus bacteremia: trends over 11 years. J. Clin. Microbiol. 47: Peleg, A. Y., D. Monga, S. Pillai, E. Mylonakis, R. C. Moellering, Jr., and G. M. Eliopoulos Reduced susceptibility to vancomycin influences pathogenicity in Staphylococcus aureus infection. J. Infect. Dis. 199:

18 Prakash, V., J. S. Lewis, II, and J. H. Jorgensen Vancomycin MICs for Methicillin-Resistant Staphylococcus aureus Isolates Differ Based upon the Susceptibility Test Method Used. Antimicrob. Agents Chemother. 52: Rybak, M. J., S. N. Leonard, K. L. Rossi, C. M. Cheung, H. S. Sader, and R. N. Jones Characterization of vancomycin-heteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year period (1986 to 2007). J. Clin. Microbiol. 46: Sakoulas, G., G. M. Eliopoulos, R. C. Moellering, Jr., C. Wennersten, L. Venkataraman, R. P. Novick, and H. S. Gold Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob. Agents Chemother. 46: Sakoulas, G., P. A. Moise-Broder, J. Schentag, A. Forrest, R. C. Moellering, Jr., and G. M. Eliopoulos Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant Staphylococcus aureus bacteremia. J. Clin. Microbiol. 42: Soriano, A., F. Marco, J. A. Martinez, E. Pisos, M. Almela, V. P. Dimova, D. Alamo, M. Ortega, J. Lopez, and J. Mensa Influence of vancomycin minimum inhibitory concentration on the treatment of methicillin-resistant Staphylococcus aureus bacteremia. Clin. Infect. Dis. 46: van Hal, S. J., T. P. Lodise, and D. L. Paterson The Clinical Significance of Vancomycin Minimum Inhibitory Concentration in Staphylococcus aureus Infections: A Systematic Review and Meta-analysis. Clin. Infect. Dis. 54:

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20 452 Table 1. Summary of key points addressing vancomycin susceptibility in S. aureus and implications for the diagnostic laboratory. Vancomycin susceptibility Definition Clinical impact Laboratory implications Reference VSSA BMD MIC 2 µg/ml ß-lactams always preferred if Perform MIC if invasive infection (1, 3, 6) methicillin-susceptible VSSA VSSA with elevated vancomycin MIC Note variable method and value for elevated MIC * BMD MIC 2 µg/ml plus Modified PAP/AUC < 0.9 Potential vancomycin treatment failure and increased mortality Optimize vancomycin dosing Source control Consider alternative antibiotic if clinical failure^ hvisa BMD MIC 2 µg/ml plus Potential vancomycin treatment failure Modified PAP/AUC 0.9 Optimize vancomycin dosing Source control Screening : MET or GRD Etest Consider alternative antibiotic if clinical failure^ VISA BMD MIC = 4-8 µg/ml Avoid glycopeptides Perform MIC if invasive infection (9, 29, 33) MIC results differ according to method; always report method Consider early hvisa screening Perform MIC if invasive infection (33) Prioritize hvisa screening if risk factors for heteroresistance (refer text) Facilitate prompt confirmatory testing at reference laboratory Facilitate susceptibility testing for alternative (3, 33) Source control antibiotics 20

21 VRSA BMD MIC 16 µg/ml Avoid glycopeptides Confirmatory testing for resistance (3) mechanism, e.g. van gene testing * An Etest > 1 µg/ml appears to be the MIC method and value most predictive of vancomycin treatment failure and increased mortality in several publications. ^ There is no standardized definition of clinical failure, however criteria may include: persistent or relapsed bacteremia, development of new hemodynamic instability, or persistent fevers with worsening inflammatory markers. Laboratory report should include MIC method and value with a comment such as for further information, please discuss this result with the laboratory or with clinical infectious diseases or microbiology staff. Downloaded from hvisa screening method dependent on laboratory work-flow, preferences and practices, e.g. Etest-based methods (MET, GRD). BMD, broth microdilution MIC, minimum inhibitory concentration PAP/AUC, area under the curve of standard population analysis profile graph MET, macromethod Etest GRD, glycopeptide resistance detection 21 on November 30, 2018 by guest

22 VSSA, vancomycin-susceptible S. aureus hvisa, heterogeneous vancomycin-intermediate S. aureus VISA, vancomycin-intermediate S. aureus VRSA, vancomycin-resistant S. aureus Downloaded from 22 on November 30, 2018 by guest