Diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus in Latin America.
Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections globally and a growing problem across Latin America. (1-3) Epidemiologic studies in the region have charted a significant rise in MRSA infections both in hospital and community settings. (1) A key step in the successful treatment of these infections is early and accurate diagnosis.
In clinical settings, diagnosis is based on a combination of epidemiologic information, clinical symptoms and characterization of the infecting MRSA strain. The Monitoring/Surveillance Network for Resistance to Antibiotics, set up with the support of the Pan-American Health Organization (PAHO), provides epidemiologic information on bacterial resistance across Latin America. In some countries, including Argentina, Chile, Ecuador, Uruguay and Venezuela, an organized quality control system is present to support local surveillance, but in others, the capacity for microbiologic diagnosis is limited to a few large university hospitals in the major cities, and limited data, especially regarding community-acquired MRSA, are available in these regions.
Various international guidelines are available that provide recommendations for best practices in MRSA diagnosis and treatment. However, adoption of these recommendations can be sporadic, especially at regional levels where resources may be a significant limiting factor. Most guidelines provide a range of options for MRSA diagnosis that can be adapted for different regional requirements. However, it may not always be clear which tests are appropriate and sufficient in specific circumstances. Additional guidance is therefore required to establish consistency of approach across the region.
GUIDELINES FOR DIAGNOSIS OF MRSA
Guideline documents have been published in a number of countries outlining recommended protocols and procedures for the identification of MRSA (Table 1 (4-9)).
The Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee on Clinical Laboratory Standards, NCCLS) in the USA has developed a range of best practice documents covering all aspects of microbiologic testing, including recent publications entitled 'Performance Standards for Antimicrobial Susceptibility Testing' (4) and 'Surveillance for Methicillin-Resistant Staphylococcus aureus: Principles, Practices, and Challenges'. (5)
The European Antimicrobial Resistance Surveillance System (EARSS), funded by the European Commission, provides a comprehensive surveillance and information system on the spread of antimicrobial resistance in Europe. EARSS has published protocols for diagnostic testing of various organisms with antibiotic resistance traits, including MRSA, VISA and VRSA. (6,10) Similarly, the Sociedad Espanola de Infectologia y Microbiologia Clinica (SEIMC), based in Spain, has published recommendations for the identification of various bacterial strains with antimicrobial resistance, including MRSA. (7)
In the UK, the British Society for Antimicrobial Chemotherapy (BSAC) published their first guidelines on microbial sensitivity testing in 1991, including minimum inhibitory concentration (MIC) breakpoints for clinically relevant bacteria, and more recently provided standardized methods for disc susceptibility testing for a range of organisms, including MRSA. (8) A Joint Working Party of the BSAC, the Hospital Infection Society (HIS) and the Infection Control Nurses Association (ICNA) recently published evidence-based guidelines on the laboratory diagnosis of MRSA. (9) These guidelines include recommendations on the identification of MRSA and methods of susceptibility testing and screening.
Since the various guidelines differ in their scope and detail, and generally do not apply specifically to infections in Latin America, infection control teams are advised to choose guidelines to follow and to adapt them to their local situation, considering such factors as epidemiology, available antibiotics and resources, likely sources of infection, and risk factors associated with their specific patient population and environment. The CLSI guidelines are the guidelines of choice in most Latin American countries. Evaluation of the implementation of guidelines is also important, as is education of healthcare workers, in order to ensure that consistent best practices are maintained.
IDENTIFICATION OF S. AUREUS
S. aureus causes a wide range of clinical infections, resulting in direct invasion of bacteria into different organs and consequent tissue damage. The clinical manifestations of infection result from the release of various toxins, either locally or systemically, and include a range of diseases dependent on the location of the infection (Table 2).
For localized infections, a clinical diagnosis is often sufficient without the need for analysis of cultures. However, for systemic infections, proper and prompt detection of S. aureus strains and their susceptibility to different antibiotics is of paramount importance in order for healthcare workers to provide appropriate treatment, and to initiate relevant control measures.
Initial, rapid assessment of clinical samples is typically achieved using conventional microscopy, by which staphylococci appear as rounded, Gram-positive cocci growing in clusters. It is important to distinguish S. aureus isolates from other staphylococcal species, such as coagulase-negative staphylococci (CoNS), and various tests are available to achieve this (Table 3). While several of these tests can be used interchangeably under appropriate circumstances, the relative benefits and limitations of each should be understood by microbiologists and healthcare professionals in order that appropriate conclusions can be drawn.
A number of factors influence the choice of S. aureus identification tests employed in a given situation, including cost, speed of result, facilities available, sensitivity and specificity. The joint BSAC/HIS/ICNA guidelines (9) recommended that a tube coagulase or latex agglutination test should be used for routine identification of S. aureus or for confirmation after DNase tests, or after negative results in a slide coagulase test. Although the readout from the slide test is much quicker than for the tube test (15 s vs. 4-24 h), the former has a higher false-negative rate (~15%). Consequently, the tube test is considered more definitive, and is the preferred coagulase test for identification of S. aureus.
Under circumstances where clinicians require a rapid assessment of MRSA, a slide coagulase test may be confirmed by latex agglutination, automated approaches or molecular tests. An international multicenter study, in which various commercial agglutination kits for identification of S. aureus were assessed using 892 staphylococcal isolates, found reliable detection of S. aureus (> 98% sensitivity and > 98% selectivity for Slidex Staph Plus). (11) Automated tests provide a similar level of accuracy for identification of S. aureus and are used across Latin America, but these may not be available in smaller local centers.
More sophisticated approaches to speciation of staphylococci are available that can provide identification of most species, but tend to involve a greater battery of tests. In a Brazilian study, Iorio et al. (12) demonstrated a scheme for the rapid identification of 198 staphylococcal isolates (including 17 of S. aureus) using a simplified battery of phenotypic tests. Staphylococci were initially identified using Gram stain, the catalase test, acid production from glucose in Hugh and Leifson's OF base medium, and susceptibility to bacitracin. Nine phenotypic tests were then used to distinguish staphylococcal species, achieving 98.5% accuracy across species (100% for S. aureus) in 72 h. Such schemes may be useful in routine laboratories, and particularly in developing countries where costs and resources are significant issues.
Methicillin sensitivity testing
There are many options for testing methicillin susceptibility of S. aureus, including disc diffusion, MIC measurements (in broth or by Etest), chromogenic agar, latex agglutination, automated methods, rapid screening methods and molecular approaches (see Table 4 (4,5,8,9,13-20) for details).
For media-based methods, test conditions such as media type, incubation times and temperature, play an important role in determining the outcome of methicillin sensitivity tests, as reflected in many of the published guidelines, and these factors should be considered carefully when designing appropriate tests. The BSAC recommends Columbia or Mueller Hinton agar supplemented with NaCl (2%) for dilution and disc diffusion methods, (4,8) and addition of up to 5% NaCl to media has been shown to improve detection of resistance for most strains. (21,22) Typically, methicillin resistance is detected more reliably at lower temperatures (30-35[degrees]C), (23-25) although some rare strains may grow slowly at 30[degrees]C when 5% NaCl is present. Both the CLSI and BSAC recommend that incubations are performed for 24 h, (4,8) but for some heterogeneous strains, resistant sub-populations may grow more slowly, and incubations of 48 h may be required to improve detection. Cefoxitin has now taken over as the antibiotic of choice for methicillin sensitivity testing, with methicillin itself no longer produced. Oxacillin remains a second option, but several publications have demonstrated that cefoxitin is more reliable than oxacillin. (13,14,26,27)
In Latin America, the methodology used for identification of MRSA differs between countries. The disc diffusion method, using oxacillin or cefoxitin discs, is popular in some countries, whereas Etest strips are generally considered too expensive for routine use. Confirmation tests, such as the methicillin screen plate test, are not widely used and molecular analysis of MRSA strains is restricted to a few centers in Brazil, Argentina, Chile, Mexico and Colombia. In cases of nosocomial outbreaks, the identity of MRSA strains is usually assumed from the phenotypic pattern of antibiotic resistance. Many laboratories in Latin America use automated methods, and these offer a convenient and, in some cases, rapid approach to identification of MRSA. The VITEK GPI system and more recent VITEK2 (Biomerieux[R]), the Microscan[R] Rapid POS COMBO (Dade/Microscan) and the Phoenix system (BD Biosciences), are all widely used for detection of MRSA, and the Vitek system will also soon include a screening test for vancomycin susceptibility.
The joint BSAC/HIS/ICNA guidelines (9) recommended that "a standard, recognized method, such as those published by the BSAC or the CLSI, should be used for routine susceptibility testing of S. aureus', but that 'other tests should be considered acceptable if they give equivalent or better performance". Disc diffusion, MIC determination and latex agglutination are all sufficient and affordable methods for routine methicillin sensitivity testing. Latex agglutination to detect penicillin-binding protein 2a (PBP2a) may also be used as a confirmatory method.
Rapid detection of MRSA is especially important in settings where quick preventive or therapeutic measures are needed, such as in intensive care units and in some surgical interventions where prosthetic material substitutions are required. Latex agglutination and ChromAgar are reliable methods for detection of MRSA and the results are available more quickly than other methods.
Importantly, consistent protocols should be introduced for all of the above tests where possible, and should be carried out using appropriate susceptible and resistant control strains, such as those outlined in the BSAC and CLSI guidelines. (4,8) For MIC and disc diffusion studies, reference values for MIC and zone of inhibition are provided in the CLSI guidelines to define susceptibility, intermediate resistance and resistance to specific antimicrobial agents (Table 5). (4) MRSA should be reported as resistant to all currently-available -lactam agents (penicillins, -lactamase/-lactamase inhibitor combinations, cephems and carbapenems), since activity of -lactam agents against MRSA in in vitro tests does not necessarily translate into clinical efficacy.
DETECTION OF REDUCED SUSCEPTIBILITY TO VANCOMYCIN (VRSA AND VISA)
MRSA infections are commonly treated with glycopeptide antibiotics such as vancomycin and teicoplanin. However, MRSA isolates with reduced susceptibility or resistance to vancomycin have emerged in recent years, (28) including in Latin America. (29) Globally, these isolates have been termed vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) depending on their level of resistance. Although VISA/VRSA strains have not been identified frequently in Latin America, and the incidence does not appear to be increasing, (30) the potential importance of these organisms is reflected in the inclusion of vancomycin susceptibility testing within guidelines for the diagnosis of MRSA. (8-10)
The current 'gold standard' for the diagnosis of VISA or VRSA is the screen test. Here, plates made up of brain-heart infusion agar and 6 mg/mL vancomycin are spotted with a 10 [micro]l inoculum of a 0.5 McFarland bacterial suspension and incubated for 24 h, with the growth of more than one colony signifying a positive result. (31) S. aureus ATCC 25923 and Enterococcus faecalis ATCC 51299 may be used as negative and positive controls, respectively. Mueller Hinton agar containing vancomycin or teicoplanin may also be used in the screen test, (8,9) but a longer incubation time (48 h) is suggested.
Most guidelines recommend MIC methods for confirmation of positive screen test results. (9,10) However, care should be taken in choosing appropriate tests for VISA and VRSA, since not all methods are appropriate for both strains (Table 6). VISA and VRSA, for example, are not reliably detected using automated methods, (31,32) whereas disc diffusion is inappropriate for VISA, but can be used for VRSA. Generally, a non-automated MIC method (e.g. broth dilution, agar dilution or Etest) with a 24-hour incubation is appropriate. (8,10) Strains with a MIC of [less than or equal to] 2 [micro]g/ mL are considered susceptible to vancomycin (Table 5), (4) although increasing vancomycin MICs within this 'susceptible' range have been linked to an increased risk of clinical failure. (33) VISA with heterogeneous sensitivity to vancomycin (h-VISA) should be confirmed by a population analysis profile (PAP) method, since MICs for these strains may be similar to those for susceptible strains. (9)
It has been proven that h-VISA significantly complicates the treatment of bacteremia patients and that it is frequently not identified by clinical laboratories. The best detection method for h-VISA is the measurement of the area under the curve (AUC) from a PAP test, however it is very labour-intensive, costly and is not appropriate in a clinical setting. There are currently three reasonable alternatives to PAP that are highly sensitive and specific and that must be used in all MRSA isolates with a vancomycin MIC of 1-2 [micro]g/mL.
1) "New strip" Etest detection method for resistance to glycopeptides (vancomycin, 32-0.5 [micro]g/mL; teicoplanin, 32-0.5 [micro]g/mL; bioMerieux AB) (34)
2) Mueller-Hinton agar supplemented with 5 [micro]g/mL teicoplanin
3) Plates made up of brain heart infusion agar supplemented with 6 [micro]g/mL vancomycin, as described in the literature (28,35)
Following positive tests for VISA or VRSA, samples should be forwarded to a reference laboratory for population analysis using appropriate control strains (for example, ATCC 700698, ATCC700699 and Oxford strain, or an alternative control (10)). Organisms that can be used as controls for sensitivity testing, for the evaluation of low levels of glycopeptide resistance (glycopeptide intermediate S. aureus [GISA]) and heterogeneous glycopeptide resistance (heterogeneous GISA [h-GISA]) are: S. aureus ATCC 29213, ATCC 700698 (Mu3; h-GISA) and ATCC 700699 (Mu50; GISA).
The heteroresistance phenomenon has been in MRSA strains which, despite having a vancomycin MIC below that of the breakpoint of susceptible strains, had subpopulations growing in the presence of 4-8 [micro]g/mL of vancomycin. (28,36) Since their description, these strains have been called vancomycin-heteroresistant and have been detected in several countries, (37,38) including some countries in Latin America. In a study carried out in Venezuela, Colombia, Ecuador and Peru, Reyes et al. described nine strains of h-VISA in 1,570 S. aureus (0.57%). (39) This heterogeneity in vancomycin resistance is similar to that described for methicillin in MRSA, where only 1x[10.sup.-6] bacteria express this characteristic.
Vancomycin heteroresistance has been considered as a potential cause of therapeutic failure. (38) However, to know the real incidence and importance of this strain type, it is necessary to establish a reliable and reproducible detection method. Some authors suggest that current vancomycin heteroresistance detection methods induce, rather than detect, resistance to vancomycin, and thus it will be impossible to establish its clinical relevance until we better understand the control mechanisms of vancomycin resistance. (40,41)
Susceptibility testing should also be performed for erythromycin, clindamycin (including detection of the inducible mechanism in erythromycin resistant strains), daptomycin, linezolid, rifampin, quinupristin/dalfopristin and trimethoprim/sulfamethoxasole. These tests can be performed in-house if appropriate facilities are available or, more frequently, in a reference laboratory. It is recommended that probable isolates of VISA and VRSA are sent to a reference laboratory as quickly as possible, even if there is a capability to test additional agents in-house, in order to facilitate organism confirmation and enhance infection control efforts.
IMPLICATIONS FOR THE REGION
The growing incidence and awareness of MRSA across Latin America has been met by an extensive effort towards early diagnosis, appropriate intervention and widespread surveillance. As would be expected from a region with such diversity in resources, a wide variety of diagnostic tests are used routinely in clinical practice, illustrated in this review, and various quality control measures are applied. As MRSA is likely to be a continuous threat to public health in Latin America for the foreseeable future, it is timely that current processes are reviewed and measures to ensure consistent practices are adopted across the region.
Existing guidelines covering MRSA diagnosis and treatment are thorough, and these should be used as a basis to standardize practices. A tiered set of recommendations may be required to accommodate both well-funded, larger centers, and local clinics with limited resources. Also, recommendations may need to be adapted for individual countries based on local resources, epidemiology and specific clinical requirements.
A coordinated system for quality control is a key requirement for successful MRSA diagnosis, and centralized, accessible reference facilities should be developed to support local centers. Collaboration within individual countries and across the region is important in this regard.
Education is a key factor in providing consistency of approach. Microbiology laboratories should participate in the education of medical and healthcare students and workers to perform procedures appropriately, and regional support networks should be set up to provide longer-term support and to facilitate the introduction of new techniques. Finally, systems to evaluate the implementation of guidelines should be introduced in order to ensure that consistent and best practices are adopted and maintained across the region.
While these recommendations are unlikely to halt the spread of antibiotic resistant S. aureus strains across Latin America, they should assist healthcare workers in achieving the most appropriate balance between the management of local resources and the provision of high quality diagnostics, both in hospitals and in the local communities.
IMPLICATIONS FOR CLINICAL PRACTICE
* Diagnosis of MRSA should be carried out according to specific guidelines.
* Guidelines should be selected and adapted to take account of local resources and needs.
* A coordinated system for quality control should be established to support microbiologic diagnosis, including regional access to central reference facilities.
* Medical and healthcare professionals should be educated in best practice diagnosis.
* Systems should be established to evaluate the implementation of guidelines and best practices across Latin America.
Pfizer Inc., New York, NY, USA, provided support for meetings of the Latin American Working Group on Gram Positive Resistance. Pfizer Inc. had no involvement in the collection, analysis and interpretation of data, in the writing of the manuscripts, or in the decision to submit the articles for publication.
The support provided by Choice Pharma (Hitchin, UK), funded by Pfizer Inc., consisted of manuscript formatting and writing assistance.
J. Zurita: Advisory Board member and consultant for Pfizer; received research grant from Wyeth.
C. Mejia: Advisory Board member for Pfizer and Abbott; consultant for Pfizer; received funding from Tibotec for HIV research, from Avexa for studies in HIV treatment and from Merck for participation in the SMART study.
M. Guzman-Blanco: Advisory Board member for Pfizer, Merck and BD; consultant for Pfizer, Wyeth and Janssen; received research funding from Wyeth and Merck.
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Jeannete Zurita  Carlos Mejia  Manuel Guzman-Blanco  on behalf of the Latin American Working Group on Gram Positive Resistance.
 Hospital Vozandes, Quito, Ecuador.
 Hospital Roosevelt, Guatemala City, Guatemala.
 Centro Medico de Caracas, Caracas, Venezuela.
Correspondence to: Jeannete Zurita Directora del Servicio de Microbiologia y Tuberculosis Hospital Vozandes Villalengua Oe2-37 Quito, Ecuador
E-mail: jzurita@hcjb. org.ec
Table 1. Guidelines for diagnosis of MRSA (4-9) Source Guidelines Clinical and Laboratory Performance Standards Standards Institute for Antimicrobial (CLSI) Susceptibility Testing (4) Surveillance for Methicillin-Resistant Staphylococcus aureus: Principles, Practices, and Challenges (5) European Antimicrobial New and updated protocols Resistance Surveillance for antimicrobial System (EARSS) susceptibility testing of pathogens under EARSS surveillance 2005 (6) Sociedad Espanola Protocolos de diagnostico de Infectologia y en Microbiologia (7) Microbiologia Clinica (SEIMC) British Society for BSAC standardized disc Antimicrobial susceptibility Chemotherapy (BSAC) testing method (version 7) (8) Joint Working Party of Guidelines for the the British Society laboratory diagnosis for Antimicrobial and susceptibility Chemotherapy, the testing of Hospital Infection methicillin-resistant Society and the Staphylococcus aureus (MRSA) (9) Infection Control Nurses Association (BSAC/HIS/ICNA) Table 2. Clinical manifestations of S. aureus infection Source of infection Disease Skin and soft tissue Impetigo, boils, carbuncles, abscesses, cellulitis, fasciitis, pyomyositis, surgical and traumatic wound infections Foreign body-associated Intravascular catheter, urinary catheter Intravascular Bacteraemia, sepsis, septic thrombophlebitis, infective carditis Bone and joints Septic osteomyelitis, septic arthritis Respiratory Pneumonia, empyema, sinusitis, otitis media Other invasive infections Meningitis, surgical space infection Toxin-mediated disease Staphylococcal toxic shock, food poisoning, staphylococcal scalded skin syndrome, bullous impetigo, necrotizing pneumonia, necrotizing osteomyelitis Table 3. Methods for identification of S. aureus isolates Principle Method Test: Coagulase Coagulase produced by Tube test certain Gram-positive Detects free coagulase cocci, including S. aureus either in bound Bacterial suspension form (attached to the added to diluted bacterial cell wall) rabbit plasma and or as free enzyme, incubated at 37[degrees]C converts fibrinogen to insoluble fibrin in Free coagulase causes the presence of plasma, plasma to clot into resulting in clotting a gel, usually within 4 h Presence of coagulase Slide test Detects distinguishes S. aureus cell-bound coagulase from coagulase-negative staphylococci (CoNS) Bacterial suspension mixed with plasma on a slide Presence of bound coagulase causes cocci to clump together rapidly (5-10 s) Test: Latex agglutination Proteins on the surface S. aureus bacterial of S. aureus (e.g. suspension mixed with protein A, clumping factor specific latex spheres group-specific antigens, capsular polysaccharides), Aggregation results are recognized by in a clearing of specific latex spheres, background media resulting in aggregation Test: Catalase S. aureus produce abundant 3% hydrogen peroxide catalase, which can added to colonies interact with hydrogen on agar plate peroxide to produce oxygen Catalase-positive colonies produce oxygen and bubble almost immediately Test: Mannitol salt agar (MSA) S. aureus strains grow Bacteria plated onto well in high salt and MSA consisting of are able to ferment mannitol, ~ 7.5-10% mannitol to produce acid, salt and the pH which can be detected indicator phenol using a pH indicator red Pathogenic staphylococci, such as S. aureus, grow well in high salt and turn MSA yellow through the release of acid Test: Polymyxin B and novobiocin susceptibility Polymyxin B is a cationic Discs of polymyxin B detergent antibiotic-specific (10 U) added to agar for Gram-negative bacilli plate S. aureus are resistant to polymyxin B Novobiocin is an amino-coumarin Discs of novobiocin antibiotic which can be (5 [micro]g) added used to differentiate S. to agar plate aureus from some CoNS S. aureus and some CoNS (e.g. S. epidermis) are sensitive to novobiocin, whereas most other CoNS (e.g. S. saprophyticus) are resistant Test: DNase and heat-stable nucleases Identification of S. aureus Methods include based on detection of anti-nuclease test specific DNase and to measure antibody heat-stable nucleases to nucleases common common to S. aureus strains to all strains of S. aureus, and metachromatic agar diffusion for heat-stable nucleases Test: Automated methods Automated systems employ Commercial systems a battery of tests to include: VITEK/VITEK2 achieve S. aureus (bioMerieux[TM]) identification Phoenix (BD Biosciences) Microscan (Dade Behring) Test: Molecular methods S. aureus contain a number Methods include PCR of genes that allow (simple, multiplex them to be distinguished and real-time), DNA from other staphylococcal sequencing, and species hybridization-based techniques Species-specific genes commonly used include nuclease (nuc), coagulase (coa), protein A (spa), femA and femB, Sa442, and S. aureus specific 16S rRNA Principle Considerations Test: Coagulase Coagulase produced by Tube test certain Gram-positive cocci, including S. Some staphylococcus aureus either in bound species not commonly form (attached to the found in human isolates (e.g. S. bacterial cell wall) schleiferi and S. or as free enzyme, intermedius) may converts fibrinogen to test positive insoluble fibrin in the presence of plasma, Occasionally, rare resulting in clotting S. aureus may test negative Some strains require up to 24 h incubation Tests should not be incubated beyond 24 h Presence of coagulase Slide test distinguishes S. aureus from coagulase-negative Rapid result staphylococci (CoNS) 10-15% of S. aureus strains test negative Some other staphylococcus species test positive (e.g. S. schleiferi, S. lugdunensis) Test: Latex agglutination Proteins on the surface Latex agglutination of S. aureus (e.g. kits specific for protein A, clumping factor S. aureus available group-specific antigens, commercially (e.g. capsular polysaccharides), Staphaurex, Staphaurex are recognized by Plus; Wellcome Diagnostics, specific latex spheres, UK) resulting in aggregation Detect both MSSA and MRSA Sensitivity > 98% for all S. aureus strains Cheap, rapid test Some other staphylococcus species may test positive (e.g. S. schleiferi, S. lugdunensis) Test: Catalase S. aureus produce abundant Cheap, rapid result catalase, which can interact with hydrogen Distinguishes catalase-producing peroxide to produce oxygen cocci (e.g. staphylococci) from non-producers (e.g. streptococci) Cannot be performed on blood agar because blood contains catalase Test: Mannitol salt agar (MSA) S. aureus strains grow Usually detects MRSA well in high salt and and MSSA MSA can are able to ferment be supplemented mannitol to produce acid, with other antimicrobials, which can be detected including cefoxitin, using a pH indicator for MRSA screening Test: Polymyxin B and novobiocin susceptibility Polymyxin B is a cationic Polymyxin B and detergent antibiotic-specific novobiocin discs for Gram-negative bacilli are relatively cheap and easy to use They should be used as part of a series of diagnostic tests for S. aureus Novobiocin is an amino-coumarin antibiotic which can be used to differentiate S. aureus from some CoNS Test: DNase and heat-stable nucleases Identification of S. aureus Some rare CoNS can be based on detection of positive in heat-stable specific DNase and nuclease tests heat-stable nucleases common to S. aureus strains S. aureus can be detected on deoxyribonuclease (DNase) plates used to screen isolates Since various amounts of DNase are produced by CoNS, positives should be confirmed with an additional test Test: Automated methods Automated systems employ Convenient and reliable a battery of tests to for S. aureus achieve S. aureus identification Resource issues limit use in smaller laboratories Test: Molecular methods S. aureus contain a number Robust and simple to of genes that allow perform them to be distinguished from other staphylococcal Many local laboratories do not species have facilities for molecular approaches Table 4. MRSA susceptibility testing (4,5,8,9,13-20) Principle Recommended method Test: Disc diffusion (Kirby-Bauer) Discs containing Disc diffusion (4,8,9) antimicrobial agent placed on the surface 2% NaCl in MH or Columbia agar of agar plate inoculated with bacterial Incubation 30-35[degrees]C for suspension, and zone [greater than or equal to] 24 h of inhibition assessed around colonies Cefoxitin discs are more reliable than oxacillin discs. (13,14,26,27) Zone for oxacillin or cefoxitin should be read by holding plate up to light source Test: MIC determinations following antibiotic dilution in broth or agar Growth in different Broth (4) dilutions of antimicrobial agent in broth or agar 2% NaCl in MH broth + oxacillin to reveal MIC or cefoxitin Inoculum of 5x[10.sup.5] cfu/mL Incubation 33-35[degrees]C for 24 h Agar (4,8,9) Tests on MH or Columbia agar with 2% NaCl + oxacillin Inoculum of [10.sup.4] cfu/mL Incubation at 30-35 [degrees]C for 24 h Test: Etest Etest[R] (bioMerieux), Etest (15-18) based on plastic strips containing a 2% NaCl in MH agar predefined gradient of 15 antibiotic Inoculum density concentrations equivalent to 0.5-1.0 McFarland standard Incubation at 35[degrees]C for 24 h Strips laid out on agar plate, and growth assessed along gradient to determine MIC Test: Plate screening Agar plates for Plate screening (4) screening 4% NaCl in MH agar + oxacillin 6 mg/L Inoculum density equivalent to 0.5 McFarland standard Incubation at [less than or equal to] 35[degrees]C for 24 h > 1 colony is indicative of resistance Test: Latex agglutination detection of PBP2a MRSA strains produce PBP2a extracted from a penicillin-binding suspension of colonies protein 2a (PBP2a) Latex particles coated with monoclonal antibodies to PBP2a added to extract Presence of PBP2a causes particle agglutination Addition of a penicillin may induce PBP2a production and give a stronger reaction Also available in slide format Test: Chromogenic agar Chromogenic agars Several commercial agars specific for MRSA, available, (19,20) allowing rapid detection including: of colonies through colored reactions S. aureus ID (bioMerieux) CHROMagar MRSA (CHROM agar) Chromogenic MRSA Agar (Oxoid) Test: Automated methods Automated systems employ Commercial systems a battery of tests to provide a convenient, VITEK GPI (bioMerieux[R]) and often rapid, approach to S. aureus Phoenix (BD Biosciences) and MRSA identification Microscan (Dade Behring) Test: Molecular methods MRSA strains possess mecA Methods include PCR gene, which encodes (simple, multiplex penicillin-binding and real-time), DNA protein 2a (PBP2a) sequencing and hybridization-based techniques Detection of mecA for methicillin resistance is combined with species-specific genes such as nuclease (nuc), coagulase (coa), protein A (spa), femA and femB, Sa442, 16S rRNA fibrinogen-binding protein genes Principle Considerations Test: Disc diffusion (Kirby-Bauer) Discs containing Some strains show antimicrobial agent heteroresistance, which placed on the surface is apparent as an inner of agar plate inoculated zone within a larger with bacterial zone of inhibition suspension, and zone of inhibition assessed On oxacillin discs, some around colonies hyper-producers of penicillinase give no zone of inhibition and will be incorrectly reported as MRSA Test: MIC determinations following antibiotic dilution in broth or agar Growth in different Relatively cheap dilutions of antimicrobial agent in broth or agar More cumbersome than disc to reveal MIC diffusion, Etest and latex agglutination methods Test: Etest Etest[R] (bioMerieux), Easy to set up based on plastic strips containing a Not widely used in Latin America due predefined gradient to cost of 15 antibiotic concentrations Test: Plate screening Agar plates for Cheap, reliable screening Recommended for screening and confirming resistance identified by disc diffusion Test: Latex agglutination detection of PBP2a MRSA strains produce Rapid, cheap and reliable penicillin-binding method for detection of MRSA protein 2a (PBP2a) Suitable for confirmation of resistance Isolates producing small amounts of PBP2a may give weak agglutination reactions, or agglutinate slowly Some rare MRSA isolates may test negative May not be reliable for colonies grown on media containing NaCl Test: Chromogenic agar Chromogenic agars Chromogenic agars can specific for MRSA, have high sensitivity allowing rapid detection for MRSA (> 98%) of colonies through colored reactions Appropriate for rapid MRSA screening Test: Automated methods Automated systems employ Convenient and reliable a battery of tests for S. aureus to provide a convenient, and often rapid, Some false-positives may be identified approach to S. aureus and MRSA These methods can be used for identification detection of MRSA, but do not detect VRSA Test: Molecular methods MRSA strains possess mecA Robust and simple to perform gene, which encodes penicillin-binding Rapid and unambiguous protein 2a (PBP2a) characterization of MRSA Reference method of choice for MRSA confirmation May detect occasional susceptible strains carrying a non-functional or non-expressed mecA Resource issues reduce widespread use Table 5. CLSI-recommended reference MIC and zone diameter breakpoints for S. aureus (4) Antimicrobial agent Disc content ([micro]g) * Methicillin 5 Oxacillin 1 Cefoxitin 30 Vancomycin -- Teicoplanin 30 Clindamycin 2 Daptomycin -- Linezolid 30 Rifampin 5 Quinupristin- 15 dalfopristin Trimethoprim- 1.25/23.75 sulfamethoxazole Zone diameter breakpoints (nearest mm) * Antimicrobial agent S I R Methicillin [greater than or 10-13 [less than or equal to] 14 equal to] 9 Oxacillin [greater than or 11-12 [less than or equal to] 13 equal to] 10 Cefoxitin [greater than or -- [less than or equal to] 22 equal to] 21 Vancomycin -- -- -- Teicoplanin [greater than or 11-13 [less than or equal to] 14 equal to] 10 Clindamycin [greater than or 15-20 [less than or equal to] 21 equal to] 14 Daptomycin -- -- -- Linezolid [greater than or -- -- equal to] 21 Rifampin [greater than or 17-19 [less than or equal to] 20 equal to] 16 Quinupristin- [greater than or 16-18 [less than or dalfopristin equal to] 19 equal to] 15 Trimethoprim- [greater than or 11-15 [less than or sulfamethoxazole equal to] 16 equal to] 10 MIC standard ([micro]g/mL) * Antimicrobial agent S I R Methicillin [less than or -- [greater than or equal to] 8 equal to] 16 Oxacillin [less than or -- [greater than or equal to] 2 equal to] 4 Cefoxitin [less than or -- [greater than or equal to] 4 equal to] 8 Vancomycin [less than or 4-8 [greater than or equal to] 2 equal to] 16 Teicoplanin [less than or 16 [greater than or equal to] 8 equal to] 32 Clindamycin [less than or 1-2 [greater than or equal to] 0.5 equal to] 4 Daptomycin [less than or -- -- equal to] 1 Linezolid [less than or -- -- equal to] 4 Rifampin [less than or 2 [greater than or equal to] 1 equal to] 4 Quinupristin- [less than or 2 [greater than or dalfopristin equal to] 1 equal to] 4 Trimethoprim- [less than or -- [greater than or sulfamethoxazole equal to] 2/38 equal to] 4/76 S-Susceptible; I-Intermediate resistance; R-Resistant * Reproduced, with permission, from CLSI publication M100-S19, Performance Standards for Antimicrobial Susceptibility Testing, Table 2C. Copies of the current edition may be obtained from Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. www.clsi.org Table 6. VISA and YRSA detection Suitable for: Test VISA VRSA Screen test Yes Yes Broth dilution Yes Yes Agar dilution Yes Yes Etest Yes Yes Diffusion method No Yes (but results may be equivocal) Automated methods No No
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|Author:||Zurita, Jeannete; Mejia, Carlos; Guzman-Blanco, Manuel|
|Publication:||The Brazilian Journal of Infectious Diseases|
|Date:||Nov 1, 2010|
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