What’s New
Newsletter 45 (September 2019)
Proceedings of ASM 2019 Workshop
This year’s ASM was held at the Adelaide Convention Centre and the CDS Reference laboratory conducted its annual SIG workshop on Monday 1 July. The workshop focused on the calibration of ceftolozane-tazobactam 30/10ug discs, polymyxin testing, and an overview of carbapenem testing. We also had three posters on ceftolozane-tazobactam disc calibration, polymyxin disc testing results and evaluation of CDS disc testing and Sensititre broth microdilution methods against reference ISO broth micro dilution for Enterobacteriaceae species.
Calibration of ceftolozane-tazobactam: This cephalosporin/β-lactamase inhibitor combination was calibrated for testing Enterobacteriaceae and Pseudomonas aeruginosa on Sensitest agar in air at 35-37°C.
Disc Potency 30-10 ug
MIC of susceptible Enterobacteriaceae strains ≤ 2.0 mg/L
MIC of susceptible Pseudomonas aeruginosa strains ≤ 4.0 mg/L
Annular radius of susceptible strains ≥ 6 mm
Acceptable range for quality control (annular radius in mm):
EC ATCC 25922: 8.9-11.7mm
PS ATCC 27853: 8.4-11.7mm
Details of the calibration using 114 isolates of Enterobacteriaceae and 82 Pseudomonas aeruginosa were summarised and published via poster.
Polymyxin testing: In December 2018 the Global Antimicrobial Resistance Surveillance System (GLASS), a working group of the World Health Organisation, released a technical note to review current methods for the detection of colistin resistance and to provide a framework for its investigation. The report recognised the need for, and current lack of, reliable tests for phenotypic detection of polymyxin resistance.
After last year’s workshop we sourced isolates with reported increased minimum inhibitory concentrations (MIC) to polymyxin. We also revived a range of isolates within our own collection including wild-type susceptible and those identified as having various resistance mechanisms.
The organisms were studied by agar dilution, agar disc diffusion and broth microdilution set up from a single, initial inoculum prepared on the spectrophotometer. In depth discussion of methods, results and discussion can be found on the poster published at the same meeting.
In conclusion – no definitive answer can be made as no single test was completely free of complications. It is likely that a combination of information may be required to infer any given isolate’s level of susceptibility. The CDS reference laboratory supports the continued testing and reporting of polymyxin 300u discs for Pseudomonas spp and Pseudomonas-like organisms but NOT for the Enterobacteriaceae. A resistant zone, i.e. ≤ 4mm, is a good indicator of resistance but a zone > 4mm cannot be trusted for the Enterobacteriaceae. At this time it is probably most prudent to either run a rapid, colourimetric test such as Rapid Polymyxin NP or refer multi-resistant isolates to a reference laboratory capable of determining an MIC as a guide to antimicrobial therapy.
Carbapenem overview: In the CDS method, users are encouraged to use carbapenems not only for the detection of susceptibility but also to aid in recognition of zone morphology changes.
Meropenem has replaced imipenem as the therapeutic carbapenem of choice in Australia so laboratories should report the susceptibilities to meropenem and not imipenem. The CDS recommendation is to continue to test with both Meropenem 5ug and Imipenem 10ug. Imipenem cannot be used as a surrogate for meropenem as some strains of Proteus spp, Providencia spp & Morganella morganii are resistant to imipenem but susceptible to meropenem. The imipenem 10ug discs are useful in the assessment of zone morphology, for example, inducible cephalosporins are most readily detected by the placement of an imipenem 10ug disc adjacent to the cefotaxime 5ug disc.
Ertapenem is a newer carbapenem with a narrower spectrum of activity. It is active against most Enterobacteriaceae but less active for Pseudomonas aeruginosa and Acinetobacter spp. Ertapenem has high sensitivity to carbapenems but low specificity. It is not recommended for routine testing but is utilised in the recognition of the presence of metallo-β-lactamases as described in section 5.5.8 of 9th edition of the manual.
Summary of Updates in the on-line manual:
Chapter 8: Application of the CDS to Unusual organisms: Adjustment of zone sizes of Ampicillin 5ug to an inhibitory zone of ≥4mm for isolates with decreased susceptibility.
Table 11.1.b: Inclusion of the calibration of ceftolozane-tazobactam for both Enterobacteriaceae & Pseudomonas spp.
Table 11.1.b: Polymyxin testing.
Enterobacteriaceae added notation – for screening purposes only.
Pseudomonas spp etc – adjusted MIC ≤ 2.0mg/L
Table 11.2: Removal of notation of ofloxacin that indicated its use is restricted to veterinary medicine.
Table 11.3.b: Inclusion of QC reference ranges for ceftolozane-tazobactam for EC ATCC 25922 & PS ATCC 27853.
The three presentations and three posters have been uploaded to the website in the ‘Archive’ tab.
As always, if you require help or support in the use of the CDS method please contact the reference laboratory.
Tel: (02) 9113 3346
NSWPATH-SealsCDS@health.nsw.gov.au
dianne.rafferty@health.nsw.gov.au
julie.allerton@health.nsw.gov.au
PratibhaMalini.James@health.nsw.gov.au
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Newsletter 44 (October 2018)
Proceedings of ASM 2018 Workshop
The CDS laboratory held its annual workshop in the main auditorium of the Brisbane Convention Centre on Monday 2 July. There were three presentations covering polymyxin testing, the laboratories performance in UK NEQAS scheme and a short quiz. We also had two posters on display presenting results from our work on polymyxin CDS disc testing and MIC comparisons. The polymyxin poster summarises Dianne’s presentation in the CDS workshop. The MIC comparison poster is the result of several years work. It compares the the results of MIC determinations of several species using CDS, CLSI and Eucast methods.
In response to literature on the uncertain status of colistin testing worldwide, the CDS undertook a review of disc testing with the CDS method. The polymyxin family is notoriously difficult to work with because of its poor diffusion. There is also no agreement in the scientific community about the best and most accurate way to test polymyxins.
Dianne presented the data from our study of both pseudomonads and enterobacteriaceae using agar dilution and 300 u polymyxin discs. The Pseudomonas species performed well with good correlation between MIC and zone sizes. Users are encouraged to continue to report polymyxin results for pseudomonas species according to the 4mm annular radius cut off. The enterobacteriaceae results were less certain with some outliers that warrant further investigation.
Dr James reviewed the laboratory’s performance in the UK NEQAS quality assurance scheme. The presentation began with a brief overview of the scoring system them drilled down to the CDS laboratory’s performance over the past 18 month. Thirty-six isolates were tested with concordance of > 95% the majority of organisms. Four instances of discrepant result were discussed. A good overall performance demonstrates the reference laboratory’s commitment to quality and highlights the sound scientific base of the CDS method.
To conclude the workshop Julie challenged participants with a short quiz. Content was taken from questions put to the reference laboratory over the past twelve months and organisms that have perhaps been neglected in the workshop over the past couple of years.
The three presentations and two posters have been uploaded to the website in the ‘Archive’ tab.
As always, if you require help or support in the use of the CDS method please contact the reference laboratory.
Tel: (02) 9113 3346
NSWPATH-SealsCDS@health.nsw.gov.au
dianne.rafferty@health.nsw.gov.au
julie.allerton@health.nsw.gov.au
PratibhaMalini.James@health.nsw.gov.au
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Newsletter 43 (May 2018)
Summary of Changes in the Ninth edition
This list includes major changes to both the printed and on-line versions of the manual. Some other minor changes were made to general formatting and are not included in this list:
Chapter 3 – Quality Assurance
3.1 Details of the March 2016 TGA update “Regulatory requirements for in-house IVDs version 2.0” are included. Amongst other requirements this important update states that from 1st July 2017 each laboratory must have a list of Class 1-3 IVDs registered with TGA Business services.
3.2 The CDS Reference Laboratory undertook a review of the reference cultures many of which were old and there could be some doubt as to their providence and authenticity. All the reference strains have been replaced with either ATCC or NCTC standard strains (listed in table 3.1) that were acquired from either of the reference laboratories. The contacts for the acquisition of the reference strains are updated in section 3.2.1.
Also included is Figure 3.1, an updated example of record keeping of using the reference strains for quality assurance purposes.
Chapter 4 – Application of the CDS to Gram positive species
4.2.8 The explanation regarding high level aminoglycoside resistance in Enterococcus faecalis is revised.
Chapter 5 – Application of the CDS to Gram negative species
5.5 Now includes details of testing in parallel of ampicillin 25 µg and cephalexin 100 µg discs with all members of Enterobacteriaceae to detect the presence of AmpC mediated resistance.
5.5.8 Metallo-β-lactamases (MBLs)
Some parts of this section, including co-expression of MBL and ESBL were revised.
5.6 Haemophilus influenza ad Haemophilus species
Further information was added to the paragraph on “Other Haemophili”.
Chapter 6 – Application of the CDS to Neisseria spp
6.2. Testing by Reference Laboratory
An update of the section on Neisseria gonorrhoeae was provided by Prof Monica Lahra, WHO Collaborating Centre for STD and Neisseria Reference Laboratory.
Interpretative criteria for Neisseria meningitidis has been removed.
Chapter 8 – Application of the CDS to Unusual organisms.
Reference to testing of yeast was removed and this and all subsequent chapters have been renumbered..
Chapter 9 – Application of the CDS to Veterinary medicine
9.4. This chapter now includes the calibration details of Florfenicol and Pradofloxacin.
Chapter 11 –Tables
All tables have been configured to a new format and the footnotes have been updated. The trade names of the combination discs have been replaced by the generic names of the two antibiotics.
11.3. Quality assurance:
The ATCC reference strains have been tested and annular radii are included for the Gram positive, Gram negative and anaerobic organisms in tables11.3.a and11.3.b. Included in table 11.3. are the reference ranges for Staphylococcus aureus ATCC 9144 on both sensitest agar and blood sensitest agar.
Kanamycin, piperacillin, enoxacin and cefpirome have beenremoved from the tables.
Appendix
The NATA document outlining TGA requirements for registration of Antibiotic Susceptibility Tests as IVDs has been deleted because it is redundant as TGA has updated its requirements. These are now described in the ninth edition under Quality Assurance. The NATA document is still a valuable resource and can be accessed on the CDS website in the archived 8th edition of the CDS Manual.
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Newsletter 42 (February 2018)
Calibration of Florfenicol for use in Veterinary Isolates
Florfenicol is one of the most recent antimicrobial agents exclusively licensed for use in animals, specifically cattle and pigs, and is only approved as an injectable drug for individual animal therapy. Florphenicol is a fluorinated derivative of chloramphenicol. Both are highly potent inhibitor of bacterial protein biosynthesis.
Although chloramphenicol and florfenicol have the same spectrum of activity they differ in their mechanisms of resistance. Over the years, bacteria have developed a number of mechanisms which enable them to circumvent the inhibitory effects of chloramphenicol. Of the chloramphenicol resistance genes known to date, only a small number also mediates resistance to florfenicol.
The results of calibration of florfenicol 30 µg disc is shown below:
MIC of susceptible strains ≤ 8.0 mg/mL
Annular radius of susceptible strains ≥ 6 mm
QC reference ranges S aureus NCTC 6571 9.4 – 12.4
E coli ATCC 25922 8.0 – 10.5
Strep pneumo ATCC 49619 11.4 – 13.9
Calibration of Pradofloxacin for use in Veterinary Isolates
Pradofloxacin is a third generation fluoroquinolone distinguished from enrofloxacin by two structural elements which confer increased potency. Pradofloxacin has been developed for use in veterinary medicine in response to the need for a fluorquinolone with enhanced Gram-positive and anaerobic activity relative to earlier generation fluoroquinolones whilst retaining the same Gram-negative spectrum.
The results of our calibration testing showed that for the vast majority of species moxifloxacin is a suitable surrogate disc for reporting susceptibility to Pradofloxacin. It was notable, however, that several isolates from the Staphylococcus pseudintermedius group used in this calibration were susceptible to Pradofloxacin but resistant to Moxifloxacin. Direct testing of Pradofloxacin therefore may be warranted in this species.
The results of calibration for pradofloxacin 5 µg disc is shown below:
MIC of susceptible strains ≤ 2.0 mg/mL
Annular radius of susceptible strains ≥ 6 mm
QC reference ranges E coli ATCC 25922 12.3 – 15.6mm
S aureus NCTC 6571 11.7 – 15.1mm
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Newsletter 41 (October 2017)
Quality Assurance Update
As stated in our last email to CDS Users in September staff of the CDS laboratory were undertaking a complete review of the Reference Strains used in the CDS and the tables of the range of acceptable zone sizes for each species tested by the CDS. This review is now finished and will be included in the 9th edition of the CDS Manual. However the 9th Edition has a way to go before completion and rather than delaying details of these two important aspects of Quality Assurance we have uploaded them to the Archives on this website and links to them are included in this Newsletter. Details of the Reference Strains are here and tables of acceptable zones sizes are here. Note 1, in the 9th edition as in these PREVIEWS, these tables will be labelled 11.3 ( not 12.3 because Section 8 Yeasts has been deleted ). Note 2, details for Neisseria gonorrhoeae are not yet available but they will be added when they come to hand.
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Newsletter 40 (Sept 2017)
CDS Users Group SIG
In August the CDS Reference Laboratory was formally recognised as a Special Interest Group (SIG) of the ASM. Dianne Rafferty will act as the convenor and Julie Allerton as the secretary. This will not change the way we interact with our users group but by registering as a SIG, it will allow us to streamline our workshops at the annual ASM conference.
This year’s ASM annual conference was held in Hobart where the members of the CDS team held a workshop and took part in the Antimicrobial Special Interest Group workshop. During the CDS workshop Dianne walked us through the harmonisation of agar and broth calibrations and touched on the way forward in terms of standardisation in Australia. Julie looked back on the solid science underpinning the CDS method and how the principles incorporated remain valid to this day. Power Point slides from the presentations are available through the Archives tab on the website.
During the Antimicrobial SIG workshop held on Sunday, the CDS presentation covered issues concerning Haemophilus influenzae and multiple resistance in Gram negative bacteria including a snap shot on Pseudomonas aeruginosa. The phenotypic pattern of aminoglycoside resistance methylase gene (armA) was also discussed. The presentation concluded with a short look at some interesting isolates that contained multiple resistance genes. The slides from this presentation are available through the Archives tab on the website.
Piperacillin 50 µg discs
We have recently been advised by Thermo Fisher Australia that Piperacillin 50 µg discs will no longer be available. As such it has been removed from Tables 12.1.b, 12.2.b & 12.3.b. The updated versions are available online and will be included in future editions of the printed manual.
Quick Update: Sensitest Agar
The most recent batches of Sensitest agar supplied to the CDS Reference Laboratory performed within acceptable parameters. Laboratories using Blood Sensitest may consider returning to Sensitest agar for the testing and reporting of Staphylococcus. Users who encounter difficulties concerning media performance should contact their supplier directly.
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Newsletter 39 (May 2017)
Sensitest Agar- Quality Issues and Continuity of Supply.
The problems with quality and supply of Sensitest Agar have been resolved. The most recent batch of Sensitest that was tested several times by the CDS Laboratory staff did not yield zones that could be as interpreted as indicating susceptibility to Cefoxitin with any of the MRSA test strains. Also the CDS staff has been assured by Oxoid UK that there will no interruption to supply of Sensitest Agar. This has been confirmed by Thermo Fisher Australia and they have supplied contact details for any questions regarding supply. The contact is Christel Helmers, her email is : Christel.Helmers@thermofisher.com . Her telephone number is 61 2 88174294.
The CDS laboratory will monitor all future batches of Sensitest Agar but some laboratories have opted to continue to use Blood Sensitest Agar which does not have the problem that was associated with some batches of Sensitest. We carried out extensive testing as outlined in the footnotes of Tables 12.3, Quality Assurance to establish “acceptable inhibitory zones of inhibition”. There was very little difference in the acceptable zones with blood sensitest and sensitest, the zones either overlapped or were within the sensitest zones. The only exception was Fusidic acid where the zones were reduced but still within the susceptible range (>6mm). The website and future printed editions of the CDS Manual will be modified to contain an updated Table 12.3a Staph. aureus that shows acceptable zones for both agars, in the meantime the modified table can be viewed and downloaded here.
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*Previous Newsletters*
If you are looking for earlier Newsletters these have been relocated to the Archives page here.