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WS/T 639-2018: Technical specification on antimicrobial susceptibility tests
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Technical specification on antimicrobial susceptibility tests
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WS/T 639-2018
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Basic data | Standard ID | WS/T 639-2018 (WS/T639-2018) | | Description (Translated English) | Technical specification on antimicrobial susceptibility tests | | Sector / Industry | Health Industry Standard (Recommended) | | Classification of Chinese Standard | C59 | | Word Count Estimation | 34,317 | | Date of Issue | 2018-12-11 | | Date of Implementation | 2019-06-01 | | Older Standard (superseded by this standard) | WS/T 125-1999; WS/T 248-2005 | | Regulation (derived from) | State-Health-Communication (2018) No.23 | | Issuing agency(ies) | National Health Commission | WS/T 639-2018: Technical specification on antimicrobial susceptibility tests---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Technical specification on antimicrobial susceptibility tests
ICS 11.020
C 50
WS
People's Republic of China Health Industry Standard
Replacing WS/T 125-1999, WS/T 248-2005
Technical requirements for antimicrobial susceptibility testing
Published on.2018 - 12 - 11
2019 - 06 - 01 implementation
National Health and Wellness Committee of the People's Republic of China
Content
Foreword...II
1 Scope...1
2 Terms and definitions...1
3 Drug selection and reporting for routine susceptibility testing...4
4 susceptibility test method...6
5 Various bacterial susceptibility tests...11
6 Quality Control...16
7 Performance verification of commercial drug susceptibility testing system...17
Appendix A Clinical susceptibility test methods and test conditions for various bacteria of the genus ...21
Appendix B Uncommon bacterium and non-harvest bacteria susceptibility test breakpoints...25
References...30
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard replaces WS/T 125-1999 "Standard Test Standard for Antibacterial Drugs by Paper Method" and WS/T 248-2005 "Anti-microbial Resistance to Anaerobic Bacteria"
Drug sensitivity test method. This standard integrates the contents of WS/T 125-1999 and WS/T 248-2005 with the following changes.
-- Increased the principles and reporting formats for routine susceptibility testing reports;
-- Added other susceptibility test methods such as dilution method, gradient diffusion method and automated instrument method;
-- Increased performance verification of rare drug susceptibility criteria and commercial drug susceptibility testing systems;
-- Revised the detection and quality control requirements for common resistance phenotypes of common bacteria.
This standard was drafted. Peking University People's Hospital, Anhui Provincial Hospital, Beijing Friendship Hospital affiliated to Capital Medical University, Beijing Hospital.
The main drafters of this standard. Wang Hui, Zhang Zheng, Ma Yuling, Hu Yunjian, Su Jianrong, Wang Xiaojuan, Chen Hongbin, Li Henan.
As of the date of implementation of this standard, WS/T 125-1999 and WS/T 248-2005 are abolished.
Technical requirements for antimicrobial susceptibility testing
1 Scope
This standard specifies the technical requirements for clinical antibiotic susceptibility testing, including drug selection and reporting for routine susceptibility testing, and drug susceptibility testing.
Test methods, various bacterial susceptibility tests, special resistance phenotype detection of common bacteria, quality control of drug susceptibility test, commercial drug susceptibility test
System performance verification.
This standard applies to clinical laboratories at all levels for clinical microbiological testing.
2 Terms and definitions
The following terms and definitions apply to this document.
2.1
Antimicrobial susceptibility testing
Detecting the in vitro sensitivity of microbes (this document specifically refers to bacteria) against microbial drugs (specifically referred to as antibacterial drugs) to guide
The bed is rationally selected for the microbiological test of the drug, referred to as the drug sensitivity test.
2.2
Minimum inhibitory concentration Minimal inhibitory concentration; MIC
The minimum antibacterial concentration that inhibits macroscopic growth of microorganisms in agar or broth dilution drug sensitivity test.
2.3
Breakpoint Breakpoint
Can predict clinical treatment effects to determine sensitive, intermediate, dose-dependent, resistant, non-sensitive minimum inhibitory concentrations (MIC)
Or the value of the diameter of the inhibition zone (mm).
2.3.1
Sensitive Susceptible;S
When the antibacterial drug is in the sensitive range of the MIC value or the inhibition zone diameter of the isolate, the recommended dose is used for treatment, and the drug is infected.
The concentration usually reached at the site can inhibit the growth of the tested bacteria, and clinical treatment may be effective.
2.3.2
Intermediary Intermediate; I
When the MIC value of the strain or the diameter of the zone of inhibition is intermediate, the value is close to the concentration reached by the drug in the blood and tissue, thereby treating
The response rate is lower than the sensitive flora. This classification means that the treatment may be higher than the conventional dose or at the site where the drug is physiologically concentrated.
effective. This classification can also be used as a “buffer domain” to prevent significant deviations caused by minor, uncontrollable technical factors, especially toxicity.
A narrower range of drugs.
2.3.3
Dose-dependent sensitive Susceptible-dose dependent;SDD
The sensitivity of bacterial strains to antimicrobial agents depends on the dose of antimicrobial agents. When a drug has a MIC or inhibition zone diameter for the strain in SDD
In the scope, the clinical efficacy can be achieved by modifying the dosage regimen by increasing the dose and/or increasing the frequency of administration.
2.3.4
Resistant Resistant; R
When the MIC value or the inhibition zone diameter of the antibacterial agent is in the classification range, the conventional treatment plan is used, and the drug is in the infection department.
The concentration of the drug achieved does not inhibit bacterial growth, and/or the test strain acquires a special resistance mechanism, and therapeutic studies have shown that
The clinical efficacy of the drug is not clear.
2.3.5
Non-sensitive Nonsusceptible; NS
For those antibacterial drugs that have only sensitive refracts due to unrepresented or rare resistance, when the MIC value of the drug is higher or lower than that of an isolate
This classification is non-sensitive when the diameter of the circle is below the sensitive point.
2.4
Epidemiological cutoff value; ECV
Differentiating the microbial population into MIC values or zone of inhibition with or without acquired resistance is the upper limit of population sensitivity. According to ECV,
The strains were divided into wild type and non-wild type.
2.4.1
Wild type Wild-type; WT
According to the ECV value, a strain that has not obtained a resistance mechanism or a decrease in sensitivity in the evaluation of antibiotics (including antifungal drugs) is defined as
Wild type.
2.4.2
Non-wild type Non-wild-type; NWT
According to the ECV value, the antibiotics (including antifungal drugs) are evaluated in a drug resistance mechanism or a strain with reduced sensitivity.
It is non-wild type.
2.5
Potency Potency
The antibacterial activity of the antibacterial component is determined by the same standard substance. The unit is mg/g, IU/g or expressed as a percentage.
2.6
Basic agreement; EA
The MIC value of the MIC system to be tested differs from the reference method by no more than one dilution factor. When the method to be evaluated is the disk diffusion method, the EA is not calculated.
2.7
Classification consistency Categorical agreement; CA
The drug sensitivity method and the reference method were evaluated to judge the test results as consistency of sensitivity, mediation and drug resistance.
2.8
Very significant error; Very major error; VME
Misjudgment of resistance is sensitive, that is, false sensitivity.
2.9
Major error, major error, ME
Sensitive mistakes are judged as drug resistance, that is, false drug resistance.
2.10
Minor error
The agent is judged to be sensitive or resistant, or to be resistant or sensitive.
2.11
Routine test
A paper diffusion method, broth or agar dilution method for routine clinical testing.
2.12
Supplementary (unconventional) drug sensitivity test (Supplemental (not routing) test
Sensitivity or drug resistance of a certain type of drug is detected by methods other than conventional disk diffusion method, broth or agar dilution method, and
The method does not require additional testing to confirm the sensitivity or drug resistance of the drug.
2.13
Screening test
Provide a test of the assumed results. When the screening result is positive, additional tests are required to confirm.
2.14
Alternative drug test Surrogate agent test
When the drug sensitivity of the target antibacterial drug cannot be detected or the drug sensitivity detection performance of the substitute drug is superior to the target drug, the drug can replace the target drug
The drug was tested for drug susceptibility.
2.15
Equivalent agent test
A drug can predict the drug susceptibility results of similar drugs that are closely related to it, and the drug susceptibility test for the drug can reduce the detection of other related drugs.
Quantity to improve detection efficiency.
3 Drug selection and reporting for routine susceptibility testing
3.1 susceptibility test test grouping of drugs
Group A. Basic antibacterial drugs routinely tested and reported for specific strains.
Group B. routine testing, but only selective reporting of basic antibacterials, such as when resistant to Group A drugs of the same type. Selective reporting indication
Also included. specific site isolates (eg, third-generation cephalosporins for Enterobacteriaceae in cerebrospinal fluid), mixed infections, multiple site infections, and patient-to-patient
Group A drug allergy/intolerance/non-response, for infection control purposes.
Group C. Includes alternative or complementary antibacterials. In some medical institutions, local or epidemic strains are resistant to multiple drugs in group A/group B
When testing this group of drugs; when it is allergic to basic test drugs, or when testing rare bacteria, or when epidemiology and infection control are needed,
Test supplements.
Group U. Includes those antibacterial drugs that are only or primarily used to treat urinary tract infections.
Laboratorys at all levels determine the different strains of the laboratory based on local pathogen characteristics, drug representation, clinical needs, and laboratory conditions.
Drug testing and reporting combination. The above-mentioned principles are combined to select an automated drug sensitive plate, which is supplemented when the drug sensitive plate cannot meet the demand.
3.2 Interpretation of results
The results of laboratory testing and reporting of the MIC value of the antibacterial drug or the diameter of the inhibition zone (mm) are divided into. sensitive (S), dose dependent
Lymphatic sensitivity (SDD), intermediate (I), drug resistance (R) or non-sensitive (NS); according to ECV, divided into wild type (WT) or non-wild type (NWT).
3.3 Reporting principles
3.3.1 Basic principles
Susceptibility testing can only be reported when the isolate may have clinical significance rather than colonization or contamination. Susceptibility testing to detect acquired resistance
Non-naturally resistant, the laboratory should identify the natural resistance spectrum of various species of bacteria, and avoid false positives of natural resistance. Individual genus
Often sensitive, or the infection caused by the bacteria does not require the use of antibacterial drugs (such as diarrhea caused by Escherichia coli O157), routine susceptibility test is not required.
The laboratory selectively reports drug susceptibility results based on drug grouping, specimen type, and the like.
3.3.2 Drug sensitivity methods and selection of vertices
For different drug/species combinations, the laboratory should select appropriate susceptibility methods and develop procedures for susceptibility testing (including
Quality control standards, interpretation of results, etc.). The laboratory should adopt the breakpoint issued by the domestic and foreign authorities in the past two years, and review the drug sensitivity test of the room every year.
Measuring the range of the breakpoint of the system; because the concentration range of the drug sensitive strip is limited, when the new breakpoint cannot be used, the corresponding drug resistance phenotype test should be supplemented.
And inform the clinic.
Determine the applicability of the breakpoint and the strain, the drug sensitivity method, the test conditions, the paper content, the infection site, the drug (type, dose, frequency,
Way) related. When the above conditions meet the corresponding requirements, the discount point applies. For drugs that are not given a breakpoint by the International Committee on Drug Abuse, it is recommended
Use domestic and foreign expert consensus and authoritative literature to conduct operations and judgments, and inform the clinic.
3.3.3 Special notes on drug sensitivity report
a) Salmonella and Shigella isolated from the intestine need only report ampicillin, a fluoroquinolone and trimethoprim/sulfa
Oxazole. Salmonella isolated from the gut, need to increase the test and report a third-generation cephalosporin, if necessary, test and report
Prime. Salmonella typhimurium (S. typhimurium and Salmonella paratyphi A, B, S, S. typhimurium) should be tested for drug susceptibility, intestinal separation
Non-typhoid Salmonella is generally not tested for susceptibility. Salmonella and Shigella to the first and second generation cephalosporins, cephalosporins
Both amino and aminoglycosides may be sensitive in vitro, but clinical treatment is ineffective, so these drugs are not tested and reported.
b) Oxacillin-resistant Staphylococcus is resistant to the currently available β-lactams (except ceftaroline). Testing penicillin and
The sensitivity of other β-lactams can be inferred by oxacillin or cefoxitin.
c) Enterococcus to aminoglycosides (except for high concentrations of aminoglycosides), cephalosporins, clindamycin and trimethoprim/sulfonate
Amoxazole may be sensitive in vitro, but clinical treatment is ineffective and should not be reported as sensitive.
d) Cerebrospinal fluid isolates are not routinely tested and reported for the following drugs. only oral dosage forms, first and second generation cephalosporins
Enterin (except cefuroxime injection) and cephalosporins, clindamycin, macrolides, tetracyclines and fluoroquinolones.
e) Respiratory isolates should not be tested and reported for daptomycin, urine isolates do not report chloramphenicol, clindamycin and erythromycin, non-urinary
The liquid isolate did not test and report nitrofurantoin.
f) Reports of drug susceptibility may be issued after a rare drug-resistant phenotype, extensive drug resistance or total drug resistance bacteria should be reviewed. Once confirmed, hospital infection control should be reported
Office. The laboratory retains these strains for epidemiological investigations or for additional clinical susceptibility testing.
3.3.4 Report Content and Form
3.3.4.1 General information
The report includes. patient information (name, age, gender, medical record number, etc.), clinical information (such as departments, clinical diagnosis, antibacterial drugs)
Material use, specimen type, etc.), laboratory information (including specimen collection time, inspection time, receiving time, and audit report time, operation)
People and reviewers double signature) and so on.
3.3.4.2 Drug sensitivity test
3.3.4.2.1 Names of bacteria and drugs
The name of the bacteria should be standardized, see the bacterial naming standard. The Latin name should be marked. The drug name should use the chemical name of the specification,
Do not use the product name. It is advisable to display the Chinese and English names of the drugs at the same time, and the different types of drugs of the same class are arranged in a concentrated manner.
3.3.4.2.2 Results presented
a) The report should clearly list the breakpoints of the various drugs for the strain, the version number or publication time of the standard used in the laboratory.
b) If there is no breakpoint and borrow other artificial species breakpoints or use authoritative document breakpoints, note the remarks.
c) MIC method reports MIC value (in mg/L or μg/mL) and interpretation of results; disc diffusion method reports inhibition zone diameter value (should be
Is an integer in mm). The results are interpreted according to the vertices or ECV as S, I, R, SDD, NS, WT, NWT, and so on.
d) Do not report susceptibility results for alternative drugs. For example, cefoxitin for judging the resistance of Staphylococcus aureus to oxacillin should not be reported
Report.
3.3.4.2.3 Special resistance phenotype
It is advisable to clearly label the special resistant phenotype in the report and to explain the special resistant phenotype professionally, including meaning, mechanism and drug use limit.
System and recommendations. For rare drug resistance, review and indicate the status of the results, such as review, review, etc. Rare and contradictory resistance
The phenotype is confirmed.
3.3.4.3 Remarks or notes on the report
a) Formal notes. A detailed explanation for symbols, abbreviations, etc.
b) Professional notes. including conceptual explanations (such as natural resistance of special bacteria), clinical significance and treatment recommendations, etc.
Scientific basis.
c) Importance tips. such as "high pathogenicity/high transmission".
d) Timeliness Tip. If “If you have doubts, please contact the laboratory within 48 hours”.
e) Management tips. such as “need to report infectious disease card”.
f) Disclaimer. If “The result is only responsible for the specimen”, “The interpretation of the results should be combined with the patient's clinical manifestations and treatment response, only
For reference" and so on.
g) Hospital address and laboratory contact methods.
3.3.5 Report issuance
Drug susceptibility reports should be issued as soon as possible. In an emergency, some results should be issued first, such as blood culture and cerebrospinal fluid and other sterile body fluid culture.
Preliminary drug susceptibility test results. Regardless of advance or delay, the report should show the time and indicate the report level (preliminary or final report).
Preserve antibiotic susceptibility test data and report epidemiological analysis results to the clinic at least annually.
4 drug sensitivity test method
4.1 Classification of drug susceptibility test methods
Drug susceptibility test methods include. dilution method (including agar dilution method and broth dilution method), disk diffusion method, gradient diffusion method and automation
Instrument method. The dilution method is a quantitative test method for detecting the sensitivity of an antibacterial drug, and is a reference method for a drug sensitivity test.
4.2 Dilution method
4.2.1 Agar dilution method
4.2.1.1 Materials
4.2.1.1.1 Mueller-Hinton Agar (MHA)
a) Weigh MHA dry powder according to product instructions.
b) Autoclave sterilization at 103.4 Kpa, 121.3 °C for 15 min.
c) Immediately put in a water bath at 40 °C ~ 50 °C for use.
d) After the preparation of the medium, the pH is adjusted to 7.2-7.4 at room temperature (25 °C).
e) Some nutrient-demanding bacteria with high nutritional requirements such as Haemophilus influenzae, Neisseria gonorrhoeae and Streptococcus, MHA medium must be added to the phase
Supplemental substances or special media should be used as detailed in Appendix A.
4.2.1.1.2 Antimicrobial gradient dilution
a) The entire formulation process of the antimicrobial dilutions is strictly sterile. See the light-decomposable tigecycline protected from light, easy to degrade,
Unstable drugs such as cefaclor, ampicillin, clavulanic acid and imipenem are now available.
b) Antimicrobial powders can be obtained from standard products or commercial powders produced by pharmaceutical companies.
c) The analytical balance is called the drug, and the active ingredients of the antibacterial drug are calculated as follows.
Actual weighing of antibacterial powder (mg) = volume of actual antibacterial drug solution (mL) × stock concentration (mg/L) /
Antimicrobial potency (μg/mg)
The volume (mL) of the actual preparation of the antibacterial drug stock solution = the actual weight of the antibacterial drug powder (mg) × the titer of the antibacterial drug
(μg/mg)/stock concentration (mg/L)
d) Formulation of drug stock solution. The concentration of the drug stock solution is not less than 1000 mg/L or higher than 10 times of the highest detection concentration of the antibacterial drug to be tested.
For most stable and non-degradable antimicrobials, dispense unused stock to sterile tubing at -70 °C or lower.
Temperature storage, the time limit is 6 months. Take one tube each time to avoid repeated freezing and thawing to affect its activity.
e) Dilution of antibacterial drug stocks. A series of required antimicrobial drug detection gradients were prepared by the dilution method.
4.2.1.1.3 Drug-containing MHA plate
a) Add the gradient to the diluted dilution of the antibacterial drug (growth control MHA plate plus sterile water) in a ratio of 1.9 to 4.2.1.1.1.
It has been autoclaved and the temperature is between 40 °C and 50 °C in MHA agar.
b) Mix the agar and antibacterial drug thoroughly.
c) Pour the plate, the thickness of the MHA-containing agar plate is 3 mm to 4 mm.
d) Allow to cool at room temperature until solidified. Use or place the plate in a closed plastic bag on the same day, store at 2 °C ~ 8 °C, and store it.
Limited to 5 days.
e) The plate is equilibrated at room temperature and placed in the incubator for 30 min to dry the agar surface.
4.2.1.2 Operation steps
4.2.1.2.1 Preparation of initial inoculum suspension
The tested strain and the quality control strain were prepared with a suspension of 0.5 gram turbidity unit, about 1×108 CFU/mL～2×108 CFU/mL, as the initial
Inoculum and place in a test tube rack in a certain order.
4.2.1.2.2 Dilution of inoculum suspension
Dilute the initial inoculum 1.10 with sterile broth or saline, mix and dilute the diluted inoculum in the order of 0.1
mL to 96-well microplate.
4.2.1.2.3 Inoculation of bacterial suspension
a) Mark the direction of the drug-containing MHA plate inoculation point.
b) Inoculate the diluted inoculum in the order of 1 μL ~ 3 μL to the non-antibacterial drug by using a multi-point inoculant or a standard inoculating loop.
The growth of the control plate, the gradient drug MHA plate surface (from low to high concentration) and the second growth control plate. most
The final inoculum inoculation amount was 104 CFU/point (point diameter 5 mm to 8 mm).
c) Place the plate at room temperature until the spot is dry, but leave it for no more than 30 minutes.
4.2.1.2.4 Incubation
Incubate for 16 h to 20 h in an air environment at 35 °C ± 2 °C. The incubation conditions and time of the susceptibility test plate for the bacterium are shown in Appendix A.
4.2.1.2.5 Interpretation of results
The plate was placed on a black, non-reflective background to read the results. The lowest concentration to inhibit visible growth of bacteria in the naked eye is the MIC.
4.2.2 Broth dilution method
4.2.2.1 Materials
4.2.2.1.1 Cation-adjusted Mueller-Hinton broth (CAMHB)
a) Weigh the CAMHB dry powder according to the product instructions.
b) Autoclave at 103.4 Kpa, 121.3 °C for 15 min, cool to room temperature and set aside.
c) After the preparation of the medium, the pH should be adjusted to 7.2-7.4 at room temperature (25 °C).
4.2.2.1.2 Antimicrobial gradients
a) Preparation, storage and dilution of antibacterial drug stocks with agar dilution method.
b) Dispense the prepared gradient diluted antibacterial solution solution into a 96-well microplate at a concentration of 0.05 mL/well (micro broth dilution)
Method) or dispense 1 mL/tube into a 13 mm × 100 mm test tube with a nut (macro broth dilution method), use or freeze on the same day
Store at ≤ -20 °C for use.
4.2.2.2 Operation steps
4.2.2.2.1 Preparation of initial inoculum suspension
The tested strain and the corresponding quality control strain, such as Escherichia coli ATCC 25922, prepare a 0.5 gram turbidity unit suspension for initial vaccination
Suspension of bacteria.
4.2.2.2.2 Dilute the inoculum suspension
Use the CAMHB broth prepared in 4.2.2.1.1 to dilute the initial inoculum 1.100 times (micro broth dilution method) or 1.150 times (macro broth)
Diluted and diluted, and the concentration of the bacteria solution is about 1×10 6 CFU/mL.
4.2.2.2.3 Inoculation of bacterial suspension
a) Pipette the diluted inoculum in order to absorb 0.05 mL to the 4.2.2.1.2 prepared 96-well microplate or equivalent
Pipette 1 mL to the equal-volume test tube prepared in step 4.2.2.1.2 (final inoculum concentration is 5 × 105 CFU/mL).
b) The diluted bacterial solution is inoculated within 15 minutes.
c) Simultaneously inoculate the diluted inoculum into a non-selective agar plate...
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