In Many Tests It Is Acceptable to Read a Positive Result Before the Incubation Time Is Completed
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Abstract
An of import task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of meaning bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of selection for particular infections. The most widely used testing methods include broth microdilution or rapid automated instrument methods that use commercially marketed materials and devices. Manual methods that provide flexibility and possible cost savings include the disk diffusion and gradient improvidence methods. Each method has strengths and weaknesses, including organisms that may be accurately tested by the method. Some methods provide quantitative results (eg, minimum inhibitory concentration), and all provide qualitative assessments using the categories susceptible, intermediate, or resistant. In general, current testing methods provide accurate detection of common antimicrobial resistance mechanisms. Nevertheless, newer or emerging mechanisms of resistance crave constant vigilance regarding the ability of each test method to accurately observe resistance.
Emergence of Antimicrobial Resistance and the Rationale for Performing Susceptibility Testing
The performance of antimicrobial susceptibility testing by the clinical microbiology laboratory is important to confirm susceptibility to called empirical antimicrobial agents, or to detect resistance in individual bacterial isolates. Empirical therapy continues to be effective for some bacterial pathogens because resistance mechanisms have non been observed e.g., connected penicillin susceptibility of Streptococcus pyogenes . Susceptibility testing of private isolates is important with species that may possess caused resistance mechanisms (eg, members of the Enterobacteriaceae, Pseudomonas species, Staphylococcus species, Enterococcus species, and Streptococcus pneumoniae ).
Overview of Usually Used Susceptibility Testing Methods
Broth dilution tests. One of the earliest antimicrobial susceptibility testing methods was the macrobroth or tube-dilution method [ane]. This procedure involved preparing two-fold dilutions of antibiotics (eg, 1, 2, 4, 8, and sixteen µg/mL) in a liquid growth medium dispensed in test tubes [1, 2]. The antibiotic-containing tubes were inoculated with a standardized bacterial suspension of 1–5×105CFU/mL. Following overnight incubation at 35°C, the tubes were examined for visible bacterial growth every bit evidenced past turbidity. The everyman concentration of antibiotic that prevented growth represented the minimal inhibitory concentration (MIC). The precision of this method was considered to be plus or minus ane two-fold concentration, due in big part to the practice of manually preparing serial dilutions of the antibiotics [3]. The advantage of this technique was the generation of a quantitative consequence (ie, the MIC). The principal disadvantages of the macrodilution method were the tedious, manual chore of preparing the antibiotic solutions for each test, the possibility of errors in preparation of the antibiotic solutions, and the relatively large amount of reagents and space required for each test.
The miniaturization and mechanization of the test by use of small, disposable, plastic "microdilution" trays (Figure 1) has made broth dilution testing practical and pop. Standard trays contain 96 wells, each containing a volume of 0.ane mL that allows approximately 12 antibiotics to be tested in a range of 8 two-fold dilutions in a single tray [2, 4]. Microdilution panels are typically prepared using dispensing instruments that aliquot precise volumes of preweighed and diluted antibiotics in broth into the individual wells of trays from large volume vessels. Hundreds of identical trays tin be prepared from a unmarried main set of dilutions in a relatively brief period. Few clinical microbiology laboratories prepare their own panels; instead frozen or dried microdilution panels are purchased from one of several commercial suppliers. The cost of the preprepared panels range from approximately $10 to $22 each. Inoculation of panels with the standard 5×x5CFU/mL is accomplished using a disposable device that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the microdilution tray or by use of a mechanized dispenser. Post-obit incubation, MICs are determined using a manual or automatic viewing device for inspection of each of the panel wells for growth [ii].
Figure 1
A goop microdilution susceptibility panel containing 98 reagent wells and a disposable tray inoculator
Figure 1
A broth microdilution susceptibility panel containing 98 reagent wells and a disposable tray inoculator
The advantages of the microdilution procedure include the generation of MICs, the reproducibility and convenience of having preprepared panels, and the economic system of reagents and space that occurs due to the miniaturization of the exam. At that place is also aid in generating computerized reports if an automatic console reader is used. The main disadvantage of the microdilution method is some inflexibility of drug selections available in standard commercial panels.
Antimicrobial gradient method. The antimicrobial slope improvidence method uses the principle of establishment of an antimicrobial concentration slope in an agar medium as a means of determining susceptibility. The Etest (bioMérieux AB BIODISK) (Figure 2) is a commercial version available in the U.s.. It employs thin plastic test strips that are impregnated on the underside with a stale antibiotic concentration slope and are marked on the upper surface with a concentration calibration. As many as 5 or half-dozen strips may be placed in a radial mode on the surface of an appropriate 150-mm agar plate that has been inoculated with a standardized organism suspension like that used for a disk diffusion examination. Afterwards overnight incubation, the tests are read by viewing the strips from the top of the plate. The MIC is adamant past the intersection of the lower function of the ellipse shaped growth inhibition area with the exam strip.
Figure 2
A Staphylococcus aureus isolate tested by the Etest gradient improvidence method with vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar. The minimum inhibitory concentration of each agent is determined by the intersection of the organism growth with the strip equally measured using the scale inscribed on the strip.
Figure 2
A Staphylococcus aureus isolate tested by the Etest slope diffusion method with vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar. The minimum inhibitory concentration of each amanuensis is adamant by the intersection of the organism growth with the strip equally measured using the calibration inscribed on the strip.
The gradient diffusion method has intrinsic flexibility by being able to test the drugs the laboratory chooses. Etest strips toll approximately $2-$3 each and can stand for an expensive approach if more than a few drugs are tested. This method is all-time suited to situations in which an MIC for only 1 or 2 drugs is needed or when a fastidious organism requiring enriched medium or special incubation atmosphere is to be tested (eg, penicillin and ceftriaxone with pneumococci) [5-7]. By and large, Etest results accept correlated well with MICs generated by goop or agar dilution methods [five-9]. Even so, at that place are some systematic biases toward college or lower MICs determined past the Etest when testing sure organism-antimicrobial agent combinations [6, x]. This tin represent a potential shortcoming when standard MIC interpretive criteria derived from broth dilution testing [x] are applied to Etest MICs that may not exist identical.
Disk diffusion test. The disk diffusion susceptibility method [two, 11, 12] is simple and practical and has been well-standardized. The test is performed past applying a bacterial inoculum of approximately ane–2×10eightCFU/mL to the surface of a big (150 mm diameter) Mueller-Hinton agar plate. Upwards to 12 commercially-prepared, fixed concentration, paper antibody disks are placed on the inoculated agar surface (Effigy 3). Plates are incubated for 16–24 h at 35°C prior to decision of results. The zones of growth inhibition around each of the antibiotic disks are measured to the nearest millimeter. The diameter of the zone is related to the susceptibility of the isolate and to the diffusion rate of the drug through the agar medium. The zone diameters of each drug are interpreted using the criteria published by the Clinical and Laboratory Standards Found (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS) [13] or those included in the Us Food and Drug Administration (FDA)-approved product inserts for the disks. The results of the disk diffusion examination are "qualitative," in that a category of susceptibility (ie, susceptible, intermediate, or resistant) is derived from the test rather than an MIC. Nevertheless, some commercially-available zone reader systems claim to calculate an judge MIC with some organisms and antibiotics past comparison zone sizes with standard curves of that species and drug stored in an algorithm [14, fifteen].
Figure 3
A disk diffusion test with an isolate of Escherichia coli from a urine culture. The diameters of all zones of inhibition are measured and those values translated to categories of susceptible, intermediate, or resistant using the latest tables published by the CLSI.
Figure 3
A deejay diffusion exam with an isolate of Escherichia coli from a urine culture. The diameters of all zones of inhibition are measured and those values translated to categories of susceptible, intermediate, or resistant using the latest tables published by the CLSI.
The advantages of the disk method are the test simplicity that does not require any special equipment, the provision of categorical results easily interpreted by all clinicians, and flexibility in selection of disks for testing. It is the to the lowest degree plush of all susceptibility methods (approximately $2.50-$v per test for materials). The disadvantages of the disk test are the lack of mechanization or automation of the exam. Although not all fastidious or slow growing leaner can be accurately tested by this method, the disk test has been standardized for testing streptococci, Haemophilus influenzae, and N. meningitidis through use of specialized media, incubation conditions, and specific zone size interpretive criteria [12].
Automated instrument systems. Use of instrumentation can standardize the reading of stop points and often produce susceptibility test results in a shorter period than manual readings considering sensitive optical detection systems allow detection of subtle changes in bacterial growth. There are 4 automated instruments presently cleared past the FDA for use in the U.s.a.. Three of these can generate rapid (3.5–16 h) susceptibility examination results, while the fourth is an overnight system [xvi]. The MicroScan WalkAway (Siemens Healthcare Diagnostics) is a large self-contained incubator/reader device that can incubate and analyze xl–96 microdilution trays. The WalkAway utilizes standard size microdilution trays that are hydrated and inoculated manually and and so placed in i of the incubator slots in the instrument. The instrument incubates the trays for the appropriate flow, examining them periodically with either a photometer or fluorometer to determine growth development. Gram-negative susceptibility test panels containing fluorogenic substrates can be read in 3.v–seven h. Split up gram-positive and gram-negative panels read using turbidimetric finish points are ready in 4.v–18 hours.
The BD Phoenix Automated Microbiology Organization (BD Diagnostics) has a large incubator reader with a chapters to process 99 test panels that contain 84 wells devoted to antibiotic doubling dilutions and are inoculated manually. The Phoenix monitors each console every 20 min using both turbidometric and colorimetric (oxidation-reduction indicator) growth detection. Test panels for gram-negative, gram-positive, S. pneumoniae, β-hemolytic, and viridans group streptococci are available. MIC results are generated in 6–16 h.
The Vitek ii Organisation (bioMérieux) is highly automated and uses very meaty plastic reagent cards (credit carte size) that incorporate microliter quantities of antibiotics and test media in a 64-well format. The Vitek two employs repetitive turbidimetric monitoring of bacterial growth during an abbreviated incubation menses. The instrument can be configured to accommodate thirty–240 simultaneous tests. The susceptibility cards allow testing of mutual, apace growing gram-positive, and gram-negative aerobic bacteria, and South. pneumoniae in a menstruum of iv–10 h. An older, less automated, Vitek 1 Organisation is all the same used in some laboratories. The system is more limited with a 45-well card and does not include S. pneumoniae .
The Sensititre ARIS 2X (Trek Diagnostic Systems) is an automated, overnight, incubation and reading system with a 64-panel chapters. The examination panels are standard 96-well microdilution plates that tin be inoculated with a Sensititre Autoinculator. Growth is determined by fluorescence measurement after 18–24 h of incubation. Test panels are available for gram-positive and gram-negative bacteria, Due south. pneumoniae, Haemophilus species, and nonfermentative gram-negative bacilli.
The Phoenix, Sensititre ARIS 2X, Vitek 1 and 2, and WalkAway instruments have enhanced computer software used to interpret susceptibility results including "adept systems" for analyzing exam results for singular patterns and unusual resistance phenotypes [sixteen]. Two studies [17, 18] have shown that providing rapid susceptibility exam results can pb to more timely changes to appropriate antimicrobial therapy, substantial direct cost savings attributable to ordering of fewer boosted laboratory tests, performance of fewer invasive procedures, and a shortened length of stay. These benefits are best realized when coupled with extended laboratory staffing schedules, and real-time, electronic transmission of verified results. One of the early shortcomings of rapid susceptibility testing methods was a lessened ability to notice some types of antimicrobial resistance including inducible β-lactamases and vancomycin resistance. However, the recently FDA-cleared instruments have fabricated meaning improvements in big part through modifications of the instruments' computer software to either provide extended incubation for problematic organism-drug combinations, or past editing of susceptibility results using proficient software to preclude unlikely results from being reported. In some cases these modifications upshot in prolonged incubation (ie, >ten h) of test panels to clinch accurate results, thus rendering them less "rapid."
Selection of Drugs for Routine Testing
The laboratory must test and report the antimicrobial agents that are well-nigh advisable for the organism isolated, for the site of the infection, and the institution's formulary [thirteen, 19]. The CLSI provides tables that list the antimicrobial agents appropriate for testing members of the Enterobacteriaceae, Pseudomonas, and other gram-negative glucose nonfermenters, staphylococci, enterococci, streptococci, Haemophilus species, etc. [13]. The listings include recommendations for agents that are important to examination routinely, and those that may exist tested or reported selectively based on the establishment'south formulary.
The availability of antimicrobial agents for testing by the laboratory'due south routine testing methodology must next be adamant. The deejay diffusion and gradient diffusion procedures offer the greatest flexibility including testing of newly available drugs. Virtually broth microdilution or automatic exam panels contain ⩽96 wells, finer limiting the number of agents tested or the range of dilutions of each drug that can be included. Manufacturers of commercially prepared panels have attempted to bargain with this trouble past offering a number of dissimilar standard console configurations, or by including fewer dilutions of each drug in a unmarried panel [19]. Another solution to this problem is testing antimicrobial agents that accept activities that are essentially the aforementioned as the desired formulary drugs. The CLSI susceptibility testing document [13] lists groups of some antimicrobial agents with nearly identical activities that tin can provide practical alternatives for testing.
Interpretation of Susceptibility Test Results
The results of a susceptibility test must be interpreted by the laboratory prior to communicating a report to a patient's physician. Optimal interpretation of MICs requires knowledge of the pharmacokinetics of the drug in humans, and information on the likely success of a particular drug in eradicating bacteria at diverse body sites [twenty]. This is best achieved by referring to an expert source such equally the CLSI, which publishes interpretive criteria for MICs of all relevant antibiotics for nigh bacterial genera [xiii]. Indeed, both MIC values and disk diffusion zone diameters must be interpreted using a tabular array of values that relate to proven clinical efficacy of each antibiotic and for various bacterial species [12]. The CLSI zone size and MIC interpretive criteria are established by assay of iii kinds of information: (1.) microbiologic data, including a comparison of MICs and zone sizes on a large number of bacterial strains, including those with known mechanisms of resistance that have been divers either phenotypically or genotypically; (two) pharmacokinetic and pharmacodynamic data; and (3) clinical studies results (including comparisons of MIC and zone diameter with microbiological eradication and clinical efficacy) obtained during studies prior to FDA approval and marketing of an antibiotic [20].
A "susceptible" result indicates that the patient's organism should respond to therapy with that antibiotic using the dosage recommended commonly for that type of infection and species [thirteen, 20]. Conversely, an organism with a MIC or zone size interpreted equally "resistant" should not be inhibited by the concentrations of the antibody achieved with the dosages commonly used with that drug [thirteen, 20]. An "intermediate" effect indicates that a microorganism falls into a range of susceptibility in which the MIC approaches or exceeds the level of antibody that tin can ordinarily be achieved and for which clinical response is likely to be less than with a susceptible strain. Exceptions can occur if the antibiotic is highly concentrated in a body fluid such as urine, or if higher than normal dosages of the antibiotic can exist safely administered (eg, some penicillins and cephalosporins). At times, the "intermediate" result can likewise hateful that certain variables in the susceptibility test may not have been properly controlled, and that the values have fallen into a "buffer zone" separating susceptible from resistant strains [13, 20]. Generally, reporting of a category result of susceptible, intermediate, or resistant provides the clinician with the data necessary to select appropriate therapy. Reporting of MICs could assistance a physician is selecting from among a group of similar drugs for therapy of infective endocarditis or osteomyelitis, in which therapy is likely to be protracted.
It is important that the tables used for susceptibility test interpretations correspond the most electric current criteria. Indeed, the CLSI documents are reviewed and updated oftentimes, usually once per yr. Use of onetime or outdated information from the original editions of FDA-approved drug labels or older CLSI tables could represent a serious shortcoming in the reporting of patients' results.
What Is the Acceptable Accurateness of a Susceptibility Test Method?
When assessing the accuracy of various susceptibility testing methods as compared to standard reference methods, the terms very major and major errors have been used to describe false-susceptible or imitation-resistant results, respectively. In evaluations of new susceptibility testing methods it is important to examine a representative number of strains that are resistant to various drugs to verify the ability of the new test to detect resistance and to test a number of susceptible strains to decide the rate of major errors that might be expected in a typical clinical laboratory setting [16, 21]. To exist cleared for marketing in the United states, the FDA requires that very major errors attributable to a test device should be <1.five% for individual species/drug comparisons, major errors should not exceed 3%, and an overall essential MIC understanding of >90% of device MICs within one doubling dilution of a CLSI reference MIC [22]. A recent, international standard on susceptibility examination device evaluation proposes similar but non identical criteria for acceptable accuracy [23]. The emergence of new antimicrobial resistance mechanisms, including some that may exist difficult to notice (eg, vancomycin intermediate susceptibility in S. aureus and carbapenemase product in some gram-negative organisms) requires that the performance of susceptibility devices be constantly reassessed and updated when needed. In some cases, it has been necessary to apply special ancillary testing methods (eg, unmarried concentration screening agars, modified Hodge test for carbapenemase production) [13] to supplement routine testing by a commercial instrument system.
Current Test Methods and Future Directions
The antimicrobial susceptibility testing methods described in this article provide reliable results when used according to the procedures defined by the CLSI or by the manufacturers of the commercial products. Withal, there is considerable opportunity for improvement in the surface area of rapid and accurate recognition of bacterial resistance to antibiotics. There is a need for evolution of new automated instruments that could provide faster results and besides relieve coin by virtue of lower reagent costs and reduced labor requirements. To attain this, it will likely be necessary to explore different methodologic approaches for detection of bacterial growth. The directly detection of resistance genes by polymerase chain reaction or similar techniques has limited utility, because only a few resistance genes are firmly associated with phenotypic resistance (eg, mecA, vanA, and vanB ) [24]. In that location are hundreds of β-lactamases, and numerous mutations, acquisitions, and expression mechanisms that event in fluoroquinolone, aminoglycoside, and macrolide resistance [25]; too many to be hands detected past current molecular techniques. Thus, it seems likely that phenotypic measures of the level of susceptibility of bacterial isolates to antimicrobial agents will go along to be clinically relevant for years to come up.
Acknowledgments
Potential conflicts of involvement. J.H.J. and M.J.F. disembalm their membership on microbiology advisory committees for BD Diagnostics and bioMérieux. J.H.J. has advised Accelr8 Engineering and has received enquiry support from BD Diagnostics, bioMérieux, Merck, Pfizer, and Siemens Healthcare.
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