3. Quality Assurance

Quality assurance (QA) measures in the CDS Test are incorporated in the method and references to these measures are included in each section of the description. The most important QA measures are strict compliance with the described CDS method and with the manufacturer’s directions for the use and storage of proprietary consumables. Many of the QA checks used in the CDS test since its introduction are now part of the general routine of NATA accredited laboratories; monitoring and documentation of refrigerator and incubator temperatures, stock batch numbers and receipt times. In this chapter we have included a trouble shooting guide or checklist to which we gave the catchy name “CDS‑QANTAS” hoping that this would encourage laboratories to copy it and use it where necessary.

3.1. Regulatory requirements for in-house in vitro diagnostic medical device (IVD)

In the 7th edition we reported that, according to NATA, antibiotic susceptibility testing by the disc method is regarded as an in house IVD and as such Australian laboratories performing antibiotic susceptibility testing need to comply with the regulatory framework as laid down by the Therapeutic Goods Administration (TGA) by 30th June 2014. More recently, in March 2016, TGA has issued an update “Regulatory requirements for in-house IVDs Version 2.0.” In this 8th edition we have retained as Appendix 1 the detailed explanation of the situation by NATA included in the 7th edition but added the update by TGA as Appendix 2. . In essence the susceptibility test must be contained in the list of in-house IVDs put forward for accreditation and include the approximate date of introduction and a copy of the corporate validation procedure. Any modification of the procedure introduced since 2007 must be supported with a full validation report. More details of the regulatory requirements can be found on the TGA website at https://www.tga.gov.au/, then search for “in-house IVDs”.

The CDS fully complies with these requirements as the method was introduced before 2007, the validation procedure is included in this edition (page 8) and any modification (application to a new antibiotic) is fully validated by the CDS Reference Laboratory. To ensure compliance with the TGA regulatory framework laboratories must maintain meticulous records of all aspects of testing including the results of the QC requirements of the CDS test as set out in this manual.

3.2. Reference strains

The performance of a CDS Test with appropriate reference strains is a critical QA measure. An unsatisfactory result with a reference strain invalidates the results obtained with test strains. It is highly recommended that antibiotic susceptibility testing, with the relevant reference strain, be performed on the same day isolates are tested. In laboratories where antibiotic susceptibility tests are performed infrequently, all discs “in use” should be tested with the relevant reference strain at least once weekly.

Table  3.1. Reference Strains.

CDS quality assurance reference strains for aerobic, micro‑aerophilic bacteria and anaerobic bacteria and yeast susceptibility testing and their Type Culture Reference Number.

.

Reference Strain  Type Culture Reference Number
Bacteroides fragilis ATCC 25285 (β‑lactamase positive)
Campylobacter jejuni NCTC 11168
Candida parapsilosis ATCC 22019
Clostridium perfringens POW 2006
Enterococcus faecalis POW 1994
Escherichia coli NCTC 10418
Escherichia coli NCTC 11560 (β‑lactamase positive)
Haemophilus influenzae NCTC 4560
Haemophilus influenzae NCTC 11315 (β‑lactamase positive)
Neisseria gonorrhoeae WHO C
Neisseria gonorrhoeae WHO O (β‑lactamase positive)
Neisseria gonorrhoeae WHO L
Neisseria gonorrhoeae WHO P
Pseudomonas aeruginosa NCTC 10662
Staphylococcus aureus NCTC 6571
Streptococcus pneumoniae ARL 10582

 

3.2.1. Obtaining reference strains.

The Australian Collection of Micro-organisms (ACM) has suspended its activity and ACM numbering of reference strains is no longer used. However registered CDS users can obtain the equivalent typed reference strains (listed above) from CDS reference laboratory. A certificate of quality is supplied with each strain. Note also that the reference strains POW 2006, POW1994, NCTC 11560 and ARL 10582 have been independently characterised by 16S gene sequencing.

There is no charge for the supply of reference strains to registered CDS users and the address for the laboratory is:

The Antibiotic Reference Laboratory

Department of Microbiology

Clinical Services Building
St. George Hospital
Kogarah NSW 2217Australia
Tel: (02) 9113 3346
Fax: (02) 9113 3349

E‑mail: NSWPATH-SealsCDS@health.nsw.gov.au

dianne.rafferty@health.nsw.gov.au
julie.allerton@health.nsw.gov.au

Neisseria gonorrhoeae WHO C, WHO L, WHO L and WHO P may be obtained from:

 

The WHO Collaborating Centre for STD and Neisseria Reference Laboratory

Department of Microbiology

The Prince of Wales Hospital

Randwick NSW 2031

Australia.

Tel: (02) 9382 9084

Fax: (02) 9398 4275

Email: NSWPATH-WHOCC.SYDNEY@health.nsw.gov.au

 

3.2.2. Handling and storage of reference strains

Upon receipt, immediately subculture reference strains onto suitable media as indicated below.

 

Reference Strain Media
Haemophilus influenzae ACM 5187 & ACM 5188 Chocolate agar
Neisseria gonorrhoeae Chocolate agar
Candida parapsilosis ACM 5283 Sabouraud Dextrose agar
All other reference strains Blood agar

Compliance with NATA Requirements

The handling and storage of reference strains decribed below complies fully with NATA’s Technical Circular 24-January 2016,”The Maintenance of Microbiological Reference Culture Collections (MRCC)”. This document is included in the Manual as Appendix 3.

-70°C Storage

Storage at or below -70°C is the preferred method for maintaining a reliable stock of the reference organisms. Reference cultures used for quality assurance testing ideally should be no more than six passages removed from the received ACM strain.

Preparation

On receipt, culture each reference strain on a suitable medium and incubate overnight. Following overnight incubation, prepare a heavy suspension of the organism in sterile nutrient broth supplemented with 20% glycerol in a cryogenic vial for storage at -70°C or in liquid nitrogen. Multiple suspensions may be created and stored concurrently as backups. These suspensions are one passage removed from the received reference strain.

Recovery

To recover a reference strain from storage, aseptically scrape a small sample of the in‑use frozen suspension using the tip of a Pasteur pipette. Inoculate the scraping onto a suitable medium and immediately return the frozen suspension to -70°C storage. After overnight incubation the recovered culture is now two passages removed from the received reference strain.

The recovered culture, with the exception of Neisseria (see below) and anaerobes, may be used as a temporary stock culture for one week provided it is held at 4°C. Storage at 4°C lessens, but does not suspend, the probability of genetic change. Under no circumstances should 4°C stock cultures be re‑incubated or allowed to stand at room temperature for a prolonged period following the initial overnight incubation. Fresh 4°C stock cultures can be prepared, by successive re‑passaging of the 4°C stock culture at the end of each storage week, a maximum of three more times.

After one week the expired 4°C stock culture should be discarded. These 4°C stock cultures are always between two and five passages removed from the received ACM reference strain.

Fresh Working Cultures

Quality assurance testing should always be performed from fresh 18 to 24 hours cultures. These can be prepared by subculturing from the 4°C stock cultures as required. These fresh working cultures will be at most six passages removed from the received ACM reference strain.

4°C storage

Many smaller laboratories do not have -70°C or liquid nitrogen storage capability. With the exception of Neisseria gonorrhoeae (see below) and anaerobes, it is permissible for these laboratories to maintain reference strain stock cultures at 4°C with successive weekly re‑passaging for a maximum of 30 weeks.

Fresh working subcultures for quality assurance testing may be made from the 4°C stock culture throughout the week, but these should be discarded after use and must not be used for the re‑passaging of 4°C stock culture).

After 30 weeks the laboratory must reacquire the reference organisms from an external ‑70°C or below storage facility.

Storage at 4°C will reduce bacterial metabolism and so will lessen, but not suspend, the probability of genetic change. Laboratories following this protocol need to be aware that this method is not optimal and they must be vigilant in monitoring the cultures for changes that may affect their reliability as reference organisms.

Neisseria gonorrhoeae

Neisseria gonorrhoeae will not remain viable if stored at 4°C, but can be stored -70°C or below, as described above. The Neisseria Reference Laboratory (above) recommends that in the absence of -70°C storage facilities,Neisseria gonorrhoeae be stored on chocolate agar slopes under paraffin oil at 30 to 37°C for one month. Culture the received ACM strain onto a chocolate agar slope and incubate in CO2 at 35 to 36°C. After 24 hours incubation cover the slope with sterile paraffin oil and store at 30 to 36°C for one month. Fresh working subcultures for quality assurance testing can be made as required throughout the month.

New storage slopes can be prepared, from the previous slope at the start of each month for up to six months. After six months the laboratory must reacquire the reference organism from an external ‑70°C or below storage facility. As with 4°C storage, this method is not optimal and laboratories must be vigilant in monitoring the culture for changes that may affect its reliability as a reference organism.

3.2.3. Testing reference strains

It is recommended that quality assurance testing with appropriate reference organisms be performed concurrently (on the same day) with the susceptibility testing of the organisms under investigation to ensure that all components of the test are in good working condition. Quality assurance testing of frequently used discs should be performed at least once weekly. An unsatisfactory result with a reference strain invalidates the results obtained for the organism under investigation. For antibiotic discs used infrequently, perform quality assurance testing concurrently with the testing of the organism under investigation.

Quality assurance testing must be performed with each new batch of antibiotic discs and with each new batch of agar plates. It is not necessary to simultaneously test a given cartridge of antibiotic discs against more than one reference organism. For example, if a cartridge of gentamicin 10 μg is tested against Escherichia coli ACM 5185, there is no need to test the same cartridge against Pseudomonas aeruginosa ACM 5189 on the same day.

The -70°C stock culture must be accessed whenever a reference strain consistently fails to give zone sizes within the recommended ranges as specified in Table 12.3.a to d.

Bacteroides fragilis ATCC 25285 and Clostridium perfringens POW 2006

When performing susceptibility testing on Helicobacter pylori isolates but not anaerobes, Bacteroides fragilis ACM 5196 is the reference strain used for the quality assurance testing of metronidazole 5 μg.

When performing susceptibility testing on anaerobes, Bacteroides fragilis ATCC 25285 (a β‑lactamase producer) is used for the quality assurance testing of Augmentin 3 μg, Tazocin 55 μg and Timentin 85 μg and Clostridium perfringens for the quality assurance testing of all other calibrated antibiotics.

See Chapter 7 (Anaerobes), and Table 12.1.c and Table 12.3.c for more information on the testing of anaerobes.

3.2.4 Measuring and recording reference strain results

CDS Users should record the actual measurement of the annular radius of each zone of inhibition each time a reference strain is tested. If the records are kept in a cumulative fashion they will draw attention to changing conditions that in their early stages may not lead to results outside the acceptable range but will eventually do so. Disc potency deterioration is the most common example of this and it is possible to detect this before it becomes a problem by observing the gradual reduction in zone sizes on successive observations.

When testing an antibiotic against the reference strains, if the annular radii lie outside the acceptable interval on two consecutive testings, retest using a new cartridge from the same batch. If the annular radii from the new cartridge also lie outside the acceptable interval, discontinue the use of this batch. The reason being that with 95% confidence limits, there is a chance of the reading to “occasionally” land outside the acceptable range but is unlikely on two consecutive testings.

Figure 3.1 shows an example of the method of recording quality assurance zone sizes used in the Microbiology Department at SEALS, Randwick and is recommended for use by laboratories using the CDS test. The antibiotic disc, its potency and the acceptable zone sizes are shown in bold type. This data can be used in estimating the Measurement of Uncertainty (MoU) for CDS testing by the laboratory.

Staphylococcus aureus NCTC 6571
Date Annular radii (mm)1,2 Sign Batch
Number
Expiry
Date
P 0.5
8.7–13.5
FOX 10
7.1–10.1
E 5
8.0–10.8
TE 10
11.3–1
4.4
OX 1
7.4–10.4
CIP 2.5
9.2–12.4
2.11.10 12 9 9.5 12 9 11 JP 12345 7/7/2011
9.11.10 11 8.5 10 12.5 9.5 11.5 GF 12345 7/7/2011
  1. Please circle zone sizes outside the acceptable interval.
  2. If an annular radius lies outside the acceptable interval on two consecutive testings, retest using a new cartridge from the same batch. If the annular radii from the new cartridge also lie outside the acceptable interval discontinue use of this batch.

Figure 3.1 Sample quality assurance sheet for antibiotic discs.

3.3. CDS-QANTAS Checklist

The CDS Quality Assurance Notations when Testing Antimicrobial Susceptibility checklist is used in “trouble shooting” to define problems revealed by the results observed with the appropriate reference strains in internal QA i.e. the annular radius of the zone of inhibition is not within the acceptable range (see Section 10.3, Table 10.3.a to d). QA is performed with the reference organisms under the conditions described in Section 3.1. If the QA fails, go through the checklist carefully to define the problem.

CDS‑QANTAS CHECKLIST

Organism tested………………………………………… [Y / N]
Medium Appropriate medium used [    ]
  “90mm diameter” Petri dish used [    ]
  Dehydrated media used within expiry date [    ]
  Manufacturer’s instructions followed [    ]
  20 ml of medium in Petri dish [    ]
  4.0 ± 0.2mm depth of medium (measured externally below meniscus) [    ]
  Poured plates are stored at 2‑8°C [    ]
  Plates used within 4 weeks of preparation [    ]
Inoculum 0.56mm diameter wire used [    ]
  Plastic inoculating needle from Copan Innovations (Cat No: 176CS20) [    ]
  Colony sampled less than 36 hours old [    ]
  Material visible on tip of wire [    ]
  Tip of wire not pointed [    ]
  Tip of wire not corroded [    ]
  Wire allowed to cool before stabbing colony [    ]
  Homogeneous suspension [    ]
  Suspension turbidity visible [    ]
  Whole plate flooded [    ]
  Excess suspension removed [    ]
  Flooded plate should dry within 15 min [    ]
Antibiotic discs Stock discs stored at or below ‑20°C [    ]
  Discs in use stored at 4°C with active desiccant [    ]
  Packaging of discs not damaged [    ]
  Discs used within expiry date [    ]
  Dispenser at room temperature before opening [    ]
  Desiccant in dispenser active* [    ]
  Positions in dispensers not shared [    ]
  Correct disc potencies [    ]
  No more than 6 discs on plate (usually) [    ]
  Antibiotic discs applied within 30 min of flooding [    ]
  Discs flat on medium [    ]
Incubation conditions Correct incubation temperature [    ]
  Correct atmosphere of incubation and humidity [    ]
  Incubated overnight (minimum16 hours) [    ]
  No more than 5 plates per stack when possible [    ]
Measuring zones of inhibition Homogeneous lawn of growth [    ]
Satisfactory growth of organism [    ]
  Measured from edge of disc [    ]
  Measured to edge of confluent growth [    ]
  Measured from back of plate (where possible) [    ]
  Not measured adjacent to another antibiotic disc [    ]
  Check antibiotics with 2 or 4mm cut‑off [    ]

*    Timentin, Augmentin, and Tazocin discs are highly susceptible to inactivation by humidity and ambient temperature. These discs need to be stored at a low temperature (4oC or -20oC) with an active desiccant (Bacto Lab. Ph: 02 9602 5499, Merck Microbiology.Ph: 1800 335 571).

3.4. External quality assurance program

CDS Users are reminded to follow the guidelines listed below when participating in the Royal College of Pathologists of Australasia Quality Assurance program (RCPA, QAP).

  1. Do not test antibiotics or use discs that have NOT been calibrated for use with the CDS Test.
  2. If the antibiotic required is not calibrated, look up Table 12.2.a or b ‘Surrogate Disc Testing’ for the surrogate disc and report S or R based on the results obtained with the surrogate disc.
  3. Do not report the susceptibility of any antibiotic that is not calibrated (Table 10.1.a to d) or is not in the ‘Surrogate Disc Testing’ tables (Table 12.2.a to b).
  4. Read the section relevant to the type of organism or mechanism of resistance when dealing with uncommon mechanisms of resistance.

Example: If the organism is a member of the Enterobacteriaceae such as Enterobacter cloacae (member of the EEC group) expressing an inducible β‑lactamase (flattened zone between cefotaxime 5 μg and imipenem 10 μg). It is known that resistant mutants producing large amounts of the enzyme are present at a high frequency. The report should be resistant for penicillins, penicillin/inhibitor combinations, cephalosporins (except cefpirome and cefepime), cephamycins and monobactams irrespective of the size of the inhibitory zone. Test and report cefpirome, cefepime, imipenem, meropenem and ertapenem, the antibiotics marked as T in Table 10.4.a.

  1. If requested to test an organism not calibrated for the testing by the CDS, reluctantly we can use the method of testing used for an organism with similar growth requirement and behaviour (see section9).For example, the testing and reporting of antibiotic susceptibility of Chromobacterium sp. may be performed using the method used for that of non-fastidious Gram negative bacilli such as Pseudomonas sp. A rider should be added that the CDS has not been calibrated for this organism