Antimicrobial sensitivity testing

1. Dr. Dinesh Jain
SMS MC Jaipur

2. ANTIMICROBIAL SUSCEPTIBILITY
TESTING (AST)
• AST is an in-vitro
laboratory procedure
for determining the
susceptibility of
bacteria to different
antimicrobial agents

3. GUIDELINES
Standard setting organizations
include:-
- Clinical and Laboratory
Standards Institute (CLSI),
formerly NCCLS (National
Committee on Clinical
Laboratory Standards)
- European Union Committee on
Antimicrobial Susceptibility
Testing (EUCAST)
- British Society for Antimicrobial
Chemotherapy (BSAC)
- CLSI standards are followed all
over the world

4. Objective of AST
 To guide the clinician in selection of most
appropriate antimicrobial therapy for a particular
clinical disease in an easily interpretable manner

5. Uses of AST
1. Control the use of inappropriate
antimicrobial agents in clinical
practice
2. The accumulated sensitivity results
on different pathogens will guide
physician in choosing ‘empirical’
treatment in serious patients before
laboratory results are available
3. Reveals the changing trends of
resistance in local isolates
4. Aids in development of antibiotic
policy

6. Indications for AST
1. When organism contribute to an infectious process
(i.e. for pathogens only)
 AST should not be performed on normal biota or
colonizing organisms
2. When pathogen belong to a species capable of
exhibiting resistance to common antimicrobial
agents

7. Standardization in AST
Purpose of standardization
- Optimizes bacterial growth conditions
- Optimizes conditions for maintaining antimicrobial
integrity & activity
- Maintains reproducibility & consistency in resistance
profile of an organism

8. Standardized components of AST :
1. Bacterial inoculum size
2. Growth medium (Mueller Hinton base) : pH,
cation conc., blood & serum supplements,
thymidine content
3. Incubation atmosphere, temperature, duration
4. Antimicrobial concentrations

9. Inoculum preparation
 Requires pure culture
 Two methods:
1. Direct colony suspension method
2. Growth method

10. 1. Direct colony suspension method
- Direct suspension of colonies in broth/saline from overnight growth
from non selective culture medium
2. Growth method (Log phase method)
- Pick 4-5 well isolated colonies of same morphology from overnight
growth culture on agar medium
- Inoculate into 4-5 ml of suitable broth media (usually trptic soy broth)
- Incubate at 350C until visually turbid (usually 2-6 hrs)
- Adjust turbidity with sterile saline/broth to match turbidity of
0.5 McFarland barium sulphate standard (1.5X10 8 CFU/ml)
- Verified by- visually comparing inoculum & 0.5 Mc Farland std. against
Wickerhams card (white background and contrasting black lines ) , or
by spectrophotometer with 1cm light path; for 0.5 McFarland
standard, absorbance at 625nm should be 0.08- 0.10

11. Growth Medium
 Recommended medium: Mueller-Hinton
broth & agar
 Other media : MHBA, HTM, GC agar base with
supplements (for fastidious org.)
 MHA is considered best medium for routine
susceptibility testing of most non-fastidious
bacteria (as per CLSI)
Reasons : - Shows good batch to batch
reproducibility for susceptibility testing
- Low in sulphonamide, trimethoprim
& tetracycline inhibitors
- Supports growth of most non-fastidious
bacterial pathogens
- Years of data & clinical experience on
its performance

12.  Depth of media : 4mm thick
- A 9cm plate requires 25ml medium (4mm depth)
- A 15cm plate requires 60-70ml medium (4mm depth)
 pH of medium : 7.2-7.4
 Cation concontration of medium affects susceptibility
results e.g divalent cations Ca2+ & Mg2+ affects
susceptibility of P.aeruginosa against aminoglycosides
 Increased thymidine/thymine content of media can reverse
the inhibitory effects of sulfonamides & trimethoprim,
giving smaller or no zone of inhibition
 Freshly prepared plates may be used the same day or stored
in refrigerator(2°-8°C) for 1-2weeks
 Incubation at 35°C in ambient air for 16-18 hrs for most
non-fastidious organisms

13. Antimicrobial Agent Discs
 Antimicrobial impregnated Whatman No.1
filter paper discs with 6mm diameter
 Commercially supplied in containers with
desiccant e.g. bottles/ cartridge/ multi-disc
panel
 Antibiotic discs should be stored in refrigerator
(2°-8°C) or frozen at -20°C or lower until
needed
 Before use, allow discs to come to room
temperature (1-2hrs)
 Commercially available mechanical disc-
dispensing apparatus should also be
refrigerated
 Do not use antibiotic discs beyond their expiry
date

14. Selection of antimicrobial test
batteries
 Each laboratory should have a battery/panel of antibiotics
ordinarily used for testing (using CLSI guidelines)
 Drug formulary decided by clinicians, pharmacists and medical
technologists
 Generally laboratories choose 10-15 antibiotics to test
susceptibility for GP organisms and another 10-15 for GN
organisms
 Primary objective
- Use the least toxic, most cost effective, and most clinically
appropriate narrow spectrum agents
- Refrain from more costly, broader spectrum agents

15. Criteria for selection of antimicrobial panel
Content :
1. Organism identification or Group
2. Acquired resistance patterns common to local microbial
flora
3. Antimicrobial susceptibility method used
4. Site of infection
5. Availability of Antimicrobial agents

16. Interpretive Categories of Results
(Qualitative )
CLSI charts are used to interpret the measured zone sizes as Susceptible,
Intermediate or Resistant
 Susceptible(S) : An organism is called susceptible to an antibiotic when
the infection caused by it is likely to respond to treatment with this
antibiotic, at the recommended dosage
 Resistant(R) : An organism is called resistant if it is expected not to
respond to a given antibiotic, irrespective of the dosage & of the
location of the infection
 Intermediate(I) : An organism may be inhibited by attainable
concentrations of certain drugs (e.g. beta-lactams) if higher dosages can
be used safely or if the infection involves a body site where that drug is
physiologically concentrated (e.g. urinary tract)
 Non-susceptible : This term is used for isolates for which only a
susceptible interpretive criteria has been defined because of absence or
rare occurrence of resistant strains (e.g. daptomycin & staphylococci)

17. Methods for AST
1. Conventional susceptibility testing methods
- Dilution methods: -Broth dilution
- Agar dilution
- Diffusion methods: - Disc diffusion method
(Kirby-Bauer method)
- Stokes comparative method
2. Commercial susceptibility testing systems
- Broth microdilution methods
- Antimicrobial gradient test (E-Test)
- Automated AST systems

18.  CLSI reference methods are :-
- Broth macrodilution
- Broth microdilution
- Agar dilution
- Disc diffusion test
 Choice of methods to be used by individual laboratory is
based on factors like :-
- Relative ease of performance
- Cost
- Flexibility in selection of drugs for testing
- Availability of automated & semi automated
devices for testing
- Perceived accuracy of the methodology

19. DILUTION METHODS
(Quantitative Susceptibility Method)
 Dilution sensitivity tests usually measures the minimum inhibitory
concentration (MIC) & minimum bactericidal conc. (MBC)
MIC : The lowest antimicrobial conc. that completely inhibits
visible bacterial growth after overnight incubation, as detected
visually or with an automated or semiautomated method
MBC : The lowest antimicrobial conc. that kills 99.9% of original
inoculum after subculture of organism to antibiotic free medium
 Achieved by adding dilution of antimicrobials in either agar or
broth medium
 Types- 1. Broth Macrodilution (Tube dilution)
Broth Microdilution
2. Agar dilution

20. Broth Dilution Method
 Two types:
1. Broth Macrodilution :
uses 1ml or more broth
in test tubes
2. Broth Microdilution :
uses about 0.05-0.1ml
broth in microtitre trays
 Microdilution method is
now considered
International reference
susceptibility testing
method

21. Broth dilution
 Principle : It involves challenging the organism of interest with antimicrobial
agents in a liquid environment
 Each antimicrobial agent is tested using a range of concentrations (µg/ml),
which includes safest therapeutic conc. possible in patient’s serum
 A series of doubling twofold dilutions of antimicrobial agents is
incorporated into the broth medium (M/C Cation Adjusted Mueller-Hinton
Broth i.e. CAMHB with pH of 7.2-7.4)
 Inoculate with standardized bacterial suspension (0.5 McFarland) to make
final inoculum of 5 X10 5CFU/ml
 Incubated at 35°C for 16-20hrs & examined for growth
 Growth control & Sterility control should always be included
 Growth is indicated by turbidity or sedimented button formation
 Lowest antimicrobial conc. that completely inhibits visible bacterial growth
is recorded as MIC
 Tube showing no visible growth is subcultured & colonies counted
 The lowest dilution showing less than 0.1% growth compared to control is
MBC

23. Broth Micro-dilution Method
 It combines precision of preparing dilutions in large volumes
with ease of testing in microtitre plates
 The microtitre plates containing a panel of several
antimicrobial agents (tested simultaneously), may be
prepared in-house or available commercially either frozen or
freeze-dried (lyophilized)
 The antimicrobial-microdilution panels are stored at -20°C or
lower (preferably -70°C), thawed before use; once thawed
should never be refrozen
 Autospense (formerly Quickspense) is commercially available
automated instrument that dispense replicate aliquots from
tubes in which large volumes of antimicrobial agent has been
prepared
 To facilitate examination of growth end points, parabolic
magnifying mirror is used

24. Uses of MIC & MBC
 Clinical conditions when MIC are useful:-
1. Endocarditis
2. Meningitis
3. Septicemia
4. Osteomyelitis
5. Immunosuppressed patients (HIV, cancer etc.)
6. Prosthetic devices
7. Patients not responding despite “S” reports
8. Critically ill patients
 MBC is not routinely performed, except in management of
endocarditis, when dose & combination of antibiotics
adjusted according to MBC

25. Advantages & Disadvantages of
Broth Dilution Method
 Broth dilution methods provide both quantitative results
(MIC) & qualitative results (category interpretation)
1. Broth macrodilution :
- Laborious & time consuming
- Useful for research purposes only (tests one drug with a
bacterial isolate)

26. 2. Broth microdilution :
 Advantages :-
- Convenient to use
- Economy of reagents & space
- Inter-laboratory reproducibility
- Allows simultaneous testing of multiple antimicrobial agents
with individual isolates
- Results may be obtained by visual examination,
semiautomated or automated instruments
 Disadvantages :-
- High cost
- May suffer from degradation of antibiotic during storage
- Some inflexibility of drug selections in commercial panels

27. Agar Dilution Method
 Standard media : Mueller-Hinton Agar
 Each doubling dilution of an antimicrobial agent is
incorporated in single agar plate
 Surface of each plate is inoculated with 1 µl of
0.5 McFarland bacterial suspension with final
inoculum of 1X104 CFU/spot
 After incubation (35°C for 16-20hrs), plates examined
for growth
 MIC is lowest conc. of antimicrobial in agar that
completely inhibits visible growth
 To facilitate testing of large number of cultures, an
instrument called Steers replicator (simultaneously
delivers upto 36 different isolates) is used for
inoculation of agar plates
 Test results may be reported as the MIC only, the
interpretive category only or both

28. Advantages & Disadvantages of
Agar dilution method
 Advantages:-
- Well standardized reference method
- Large no. of isolates can be tested
simultaneously
- Microbial contamination easily detected
(as compared with broth methods)
- Determines MIC for N. gonorrhoeae
which does not grow sufficiently in broth
(broth dilution methods)
 Disadvantages:-
- Time consuming
- Labor intensive method

29. DISC DIFFUSION METHOD
(Qualitative Susceptibility Method)
Involves detection of antimicrobial resistance by
challenging bacterial isolates with antibiotic discs
placed on surface of an agar plate that has been
seeded with a lawn of bacteria
Two methods : -1. Kirby-Bauer method
2. Stokes Comparative method
Kirby-Bauer method is usually the recommended
method of disc diffusion testing
 Suitable for organisms that grow rapidly overnight
at 35°-37°C

30.  Basic Principle : As soon as the antibiotic impregnated disc comes
in contact with moist agar surface, it absorbs moisture from agar &
antibiotic diffuses into agar medium.
- The rate of extraction of antibiotic from the disc is greater than
rate of diffusion in medium
- As the distance from disc increases, there is logarithmic reduction
in the antibiotic concentration
- Visible growth of bacteria occurs on surface of agar where the
conc. of antibiotic has fallen below its inhibitory level for the test
strain
- The point at which critical cell mass is reached appear as sharply
marginated circle of bacterial growth with disc forming centre of
circle (Zone of Inhibition)
- The conc. of diffused antibiotic at this interface of growing &
inhibited bacteria is called critical concentration & approximates
MIC obtained in dilution tests

31. TEST PROCEDURE
 Once isolated colonies of potential pathogen are available ,
proceed as follows :-
1. Inoculum prepared & standardized to 0.5 McFarland
2. Inoculation (Lawn culture): MHA plates inoculated by
streaking with sterile cotton swab (3times in
3 quadrants rotating 60° each time)
3. Predetermined battery of antimicrobial discs is
applied within 15min of inoculating the media; using
sterile forceps, template or mechanical disc dispensing
apparatus; press firmly to ensure uniform contact with
agar
- Discs should not be relocated once it comes in contact
with agar surface (as some drugs diffuses almost
instantaneously)

32. - To avoid overlapping zones: Apply upto
5discs to 90mm diameter plate & upto 12
discs on a 150mm plate
- Distance between two adjacent discs be at
least 2.5cm (centre to centre) & from egde
of plate to disc should be 1.5cm
4. Within 15min of disc application ,invert the
plates & incubate at 35°C in ambient air for
16-18hrs (for most non-fastidious
organisms)
- Some fastidious organisms require
incubation in ambient air with 5% CO2
5. Measure the inhibition zone diameters
(using reflected light) to the nearest
millimeter with a ruler or calipers

33. - Use transmitted light when measuring zones for :
- Staphylococci with oxacillin
- Enterococci with vancomycin
6. Zone diameters are interpreted (CLSI guidelines) &
organisms reported as S, I, R (Qualitative)
7. Control strains used for quality control are-
- S.aureus ATCC 25923
- E.coli ATCC 25922
- P.aeruginosa ATCC 27853

34. DISC DIFFUSION METHOD
Special situations in interpretation of
results
1. If unusual double zone occur, measure the innermost zone
2. If distinct colonies present within the zone, it can be due to
either mixed culture (not pure culture) or resistant mutants
of the test bacterium. To resolve this problem, isolation,
identification & susceptibility testing of resistant colonies is
required
3. Proteus mirabilis or P. vulgaris may swarm producing thin
veil that may penetrate the zone of inhibition. Ignore
swarming in zone & outer clear margin should be measured
4. With trimethoprim & sulphonamide discs, growth may not
be completely inhibited at outer margin, resulting in light
haze of growth within the zone. The clear zone of approx.
80% inhibition should be read as zone diameter

35. 5. Haze around an oxacillin disc when testing
S.aureus is significant & should not be ignored
6. Readings should not be attempted on poorly
inoculated plates (zone margins are indistinct) &
test should be repeated
7. When beta-lactamase producing S.aureus is tested
with penicillin, zones are often large but growth
just outside the zone of inhibition is
characterstically heaped up; regardless of the size
of inhibition zone, strains be reported as resistant
8. When testing streptococci on blood supplemented
media, zone of growth inhibition should be
measured, not the zone of haemolysis

36. DISC DIFFUSION METHOD
Factors influencing Zone size
1. Inoculum density :
- Too light the inoculum, zone size will be larger; relatively resistant
strains falsely reported as susceptible & vice versa
2. Depth of agar medium : Ideally 4mm depth
- Very thin media : excessively large zones
- Very thick media : smaller zones
3. Timing of disc application : Within 15min of plate inoculation
- If plates after inoculation, left at room temp for longer than
standard time, multiplication of inoculum may occur before discs
are applied, causing reduction in zone diameter & susceptible
strains will be reported as resistant

37. 4. Potency of antibiotic discs :
- If potency of drug is reduced (deterioration during
storage), zone size will show corresponding reduction
in size
5. Temperature & time of incubation :
- If temp is lowered, time required for effective growth
is extended & larger zones result
6. Composition of media : Affects rate of growth,
diffusion of antibiotics & activity of antibiotics
7. pH of medium :
- If pH too low, low potency of certain drugs
(e.g. aminoglycosides, macrolides) & excessive activity of
some (e.g. tetracycline)

38. Advantages & Disadvantages of
Disc diffusion method
 Advantages :-
- Simple to perform , inexpensive & very reproducible
- Does not require any special equipment
- Provides susceptibility category results, easily understood by clinicians
- Flexible regarding selection of antimicrobial agents for testing
- Ability to respond quickly to changes in interpretive breakpoints or
when new agents are introduced
 Disadvantages :-
- Applies only to bacterial species for which this method has been
standardized (rapidly growing non-fastidious isolates), so
not suitable for slow growing fastidious pathogens
- Quantitative MIC results indicating degree of susceptibility are
needed in some cases; not provided by this method

39. PRIMARY SENSITIVITY TESTS
 Specimen serves as the inoculum
e.g. Urine, a positive blood culture or a
swab of pus
 Little value for specimens from patients
already receiving antibiotics or specimen
from sites likely to be heavily
contaminated.eg. Bedsores, vaginal swabs,
abdominal wounds
 Gram stain of the specimen should be
done to select probable antibiotic panel

40.  Advantages :-
- Results are available 1 day earlier & additional labor
and cost of performing subculture tests is avoided
- May help to identify bacteria that have constant pattern
of sensitivity to the antibiotics tested
- In mixed cultures, may help to separate bacterial
species with different sensitivities
- May reveal presence of small no. of resistant variants
 Disadvantages :-
- Bacterial inoculum cannot be controlled (often too light)
- As the identity of any pathogen present is unknown,
choice of drugs to be tested is based on organism
thought most likely to be present

41. STOKE’S COMPARATIVE METHOD
 Same plate comparative disc diffusion test
 Medium used- Iso-sensitest agar (ISA) or ISA
with supplements (lysed horse blood)
 Use NCTC controls e.g. NCTC 10418 E.coli
NCTC 6571 S.aureus,
NCTC 10602 P.aeruginosa
 In original Stoke’s method, inoculum of control
strain is spread in two bands on either side of the
plate (upper & lower 1/3rd of plate).
- The test organism is inoculated onto the central
1/3rd of plate
- An uninoculated gap 2-3mm wide should separate
the test & control areas, for applying antibiotic
discs

42.  In Rotary plating method, control is
inoculated in centre of the plate, leaving
15mm band around the plate edge
 Inoculate test organism to the 15mm band,
leaving 2-3mm gap between test & control
inocula where antibiotic discs are applied
(maximum 6 per plate)
 Interpretation based on comparison between
zones seen with test organisms & those of
known sensitive control
• Sensitive : zone size of test larger ,equal or
not more than 3mm smaller than control
• Intermediate : zone size of test at least 2mm
but more than 3mm smaller than control
• Resistant : zone size of test less than 2mm

43. E-tests : Gradient Diffusion
 Epsilometer test or E-test (AB biodisk) is
an antimicrobial gradient diffusion test
 “MIC on a strip”
 The test combines principle of disc
diffusion & agar dilution methods
 A predefined, continuous & exponential
gradient of antibiotic concentrations is
immobilized along a rectangular plastic
test strip (6x0.5cm), on one side
 The other side of strip contains a numeric
scale (MIC scale) that indicates the drug
concentration
 The range corresponds to fifteen 2-fold
dilutions

44.  The test strip is applied on surface of an
inoculated agar plate & incubated overnight
 Several strips may be placed radially on the
same plate so that multiple antimicrobials
tested against a single isolate
 After incubation, MIC is read from the point
on the graded strip where zone of growth
inhibition (tear drop/elliptical shaped)
passes
 Most E-test require examination with a
hand lens to look for minute colonies
intersecting the strip
 This Quantitative method provides on-scale
MIC data in situations where level of
resistance can be clinically significant & in
treatment decisions for critical infections
like sepsis

45. Uses of E-test :-
1. Determine MIC of anaerobic & fastidious slow growing organisms, or
for a specific type of patient or infection
2. Detecting – Glycopeptide resistant Enterococci (GRE)
- Glycopeptide intermediate S.aureus (GISA)
- Resistant Mycobacterium tuberculosis
- Extended spectrum beta-lactamases (ESBL)
3. Detecting low level of resistance
4. Testing an antibiotic not performed in routine use or a new recently
introduced antibiotic agent
5. Confirming an equivocal AST result
 High cost of test makes it unsuitable for routine testing of multiple
antimicrobial agents against most non-fastidious organisms
 Short shelf life, storage at -20°C

46. Automated Susceptibility Testing
Systems
Automated AST systems available for use include:
1. Biomerieux Vitek 2
2. MicroScan WalkAway
3. BD Phoenix
4. Sensititre ARIS 2X
 These systems vary with respect to :
- Extent of automation of inoculum preparation &
inoculation
- Methods used to detect growth
- Algorithms used to interpret & assign MIC
values and categorical findings (S,I,R)
 Detects growth in microvolumes of broth with
various dilutions of antimicrobials
 Detection via photometric, turbidimetric or
fluorometric methods

47. VITEK SYSTEMS
 The Vitek 1 was originally
designed by NASA for use as an on-
board space exploration test system
 It is based on use of small thin plastic
cards each containing many wells
linked by capillaries
 These cards are available as
susceptibility & identification
cards(ID)
 Biomerieux has stopped further
development of new cards &
software updates for Vitek 1
 The more automated Vitek 2 is
widely used automated method

48. Components of VITEK 2 system :-
1. A Robotic Filling module whereby a standard suspension of
the organism in saline is drawn up via a vacuum into the
card
2. An Incubator/Reader module containing a carousel to hold
the cards & a photometer to measure optical density of the
sensitivity cards and the biochemical color changes of the
identification cards
3. A Computer module analyses growth curve & generates an
algorithm derived MIC value. It also analyses the
biochemical id & compares it to a database
4. An “Advanced Expert System” (AES) software analysis
module recognizes new/ unusual/ inconsistent results &
highlights “alert” organisms (e.g. MRSA, VRE, Gentamicin
resistance)
- The Advanced Expert System (AES) has built-in antibiotic
interpretation rules

49. VITEK 2
 Fully automated system for bacterial/fungal identification &
antibiotic susceptibility testing
 Reduces set up time & minimizes manual steps
 AST based on broth susceptibility with closed plastic cards
with 64 wells containing specified conc. of antibiotics
 Inoculum is automatically introduced via a filling tube into
plastic cards
 Cards are incubated in temperature controlled environment
 Optical readings are performed every 15min to measure the
amount of light transmitted through each well & analyzed by
system’s software (AES) to derive the MIC data, category
interpretation and organism’s antimicrobial resistance
patterns
 Results in 6-8hrs
 ID currently biochemical

50. MicroScan WalkAway System
 AST based on conventional broth
microdilution MIC methods
 96 well microdilution plates manually
inoculated with multiprong device
 Bacterial growth is detected
spectrophotometrically or
fluorometrically
 The Walkaway system incorporates a
fluorescence detection system that
provides same day identification &
susceptibility testing for some organisms
 Fluorogenic approach provides AST
results in 3.5 to 5.5 hours
 ID currently biochemical

51. BD Phoenix
 AST based on broth susceptibility with
specific cards
 Automated adjustment of inoculum & AST
dilution
 Readings for each well done every 20
minutes using both colorimetric change in
redox indicator & turbidity to determine
organism’s growth
 Results available in 8-12 hrs
 ID currently biochemical
 Growth/ reactions “continuously”
monitored
 Supplemental testing (e.g. confirmatory
ESBL test for E.coli) is included in each
panel, reducing need for additional or
repeat testing

52. Advantages & Disadvantages of
Automated Systems
 Advantages :-
- Labor saving
- Rapid results (3.5 to 16 hrs)
- Increased reproducibility
- Data management with expert system analysis
- “Expert system” software
- Detects multi-drug resistances
- Detects ESBL
- Correlates bacterial ID with sensitivity
 Disadvantages :-
- Higher cost for equipment & consumables
- Predetermined antimicrobial panels (inflexibility)
- Inability to test all clinically relevant organisms
- Problems with detection of some resistance phenotypes

53. Limitations of AST
 Laboratory test conditions cannot reproduce the in vivo environment
at the infection site where antimicrobial agent & bacteria actually
interact
 Factors determining patient outcome, are not taken into account by
AST, which includes-
- Antibiotic diffusion into tissues & host cells
- Serum protein binding of antimicrobial agents
- Drug interaction & interference
- Status of patient defence & immune systems
- Multiple simultaneous illnesses
- Virulence & pathogenicity of infecting bacterium
- Site & severity of infection
 DESPITE THESE LIMITATIONS, “AST” AIDS IN OPTIMAL MANAGEMENT
OF PATIENTS

54. Genotypic/Molecular Methods
• Application of genotypic methods can allow rapid
detection of resistance genes direct from the
sample
• Examples include :-
- mecA gene detection by PCR denotes resistance
to methicillin in Staph aureus (MRSA)
- Rifampicin & isoniazid resistance in MDR
Mycobacterium tuberculosis can be detected
using DNA probe (rpoB)
- Antiviral drug resistance due to genetic point
mutations can be examined by PCR for HIV, CMV
& HCV

55. Quality Control (QC) in AST
Laboratory quality can be defined as accuracy,
reliability, and timeliness of reported test results
 QC includes procedures to monitor the test
system to ensure accurate & reproducible results
 Goals of QC program are to monitor :-
- Precision & accuracy of susceptibility test
procedures
- Performance of reagents used in test
- Performance of persons who carry out the tests
& report the results

56. Quality Assurance (QA)
 A QA program helps to ensure that testing
materials & processes consistently provide quality
results
 QA includes :-
- QC
- Calibration & maintenance of equipment
- Proficiency testing
- Training & competency assessment
- Monitoring, evaluation & taking corrective actions
(if necessary)
- Record keeping

57. Responsibilities of Laboratory in QC :
 Laboratories perform QC to ensure that testing materials &
reagents are maintained properly & testing is performed
according to established protocols
1. Use of current CLSI standards & manufacturer’s instructions
 CLSI recommends use of ATCC strains for QC in AST
2. Adherence to the established procedures
- Inoculum preparation
- Incubation conditions
- Determination of end points
- Interpretation of results
3. Drug storage under environmental conditions recommended
by manufacturer (to prevent deterioration)
4. Proficiency of personnel performing tests

58. Frequency of QC Testing
 Routine QC performed each day the test is performed
 A 20 or 30 day testing plan if successfully completed
(i.e. less than 10% inaccuracy), allows the user to
convert from daily to weekly QC
 Media (e.g. MHA) : QC of each batch or lot with
appropriate QC strains before their first use for testing
patient isolates
 Antibiotic discs tested daily or weekly (if satisfactory
performance with daily QC) for their activity
 Maintain records: lot #, expiry date, date of QC and
results

59.  Out of range QC results : More than 1 in 20 or 3 in
30 results outside the accuracy limits
 Catagories of out of range QC results-
- Random: due to “chance”
- Identifiable error (can easily be corrected)
- System related error
 Every out-of-range QC result must be investigated
& corrective action taken
 Daily QC tests must be continued until final resolution
of the problem is achieved
 Suppress reporting patient results when QC is out-of-
range; use alternative test method or a reference
laboratory until the problem is resolved

61. Application of Computers in AST
What is “WHONET”
 Antimicrobial resistance (AMR) monitoring will help to
know current status of AMR locally, nationally & globally
 This will help minimizing consequences of drug
resistance & limit emergence and spread of drug
resistant pathogens
 Thousands of laboratories worldwide need to be linked to
integrate data on most clinically relevant organisms, to
obtain accurate picture of “real resistance”
 For this purpose, a software developed by WHO, called
WHONET (a global networking programme) ,focuses on
data of AST results
 Use of WHONET software :
- Help local laboratory in efficient networking with national
reference laboratory & international bodies like WHO,
NCCLS etc.
- Help in establishing antibiotic policies, treatment guidelines
& infection control policies

62. Thank you