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
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.
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.
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
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.
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.
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.
Newsletter 38 (November 2016)
Summary of Changes in the Eighth Edition
This list includes the 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:
Section 3 – Quality Assurance
3.1 Reference to the latest (2016) TGA requirements for in-house in vitro diagnostic medical devices (IVD). This document is included in full in the on-line version as appendix 2.
3.2 The Australian Collection of Micro-organisms (ACM) has ceased its activity therefore the nomenclature of all quality control reference strains have been changed back to their previous Type culture accession numbers.
3.2.2 The handling and storage of reference strains has been updated to comply with NATA’s Technical Circular 24 (2016) which is also included in the on-line version as appendix 3.
Section 5 – Application of the CDS to Gram Negative Species
5.1 Expanded explanation of the effect of combination of β-lactam/β-lactam inhibitors on Acinetobacter species.
5.5 Recommendations regarding the testing and reporting of Meropenem and Imipenem.
5.5.3 Inclusion of Boronic acid procedure for the phenotypic testing for AmpC.
5.5.7 Inclusion of E. cloacae complex and C. freundii complex as part of the EEC subgroup.
5.6 Inclusion of resistance mechanisms found in Haemophilus influenzae including a clarification of the testing and reporting of decreased susceptibility to cefotaxime.
5.9 Inclusion of a description of GES β-lactamase found in P. aeruginosa.
Section 6 – Application of the CDS to Neisseria species
This section has been divided into 2 sections.
6.1 Testing in routine laboratories
6.2 Testing by reference laboratories and is provided in its entirety by the Neisseria Reference Laboratory, Microbiology Department, Prince of Wales Hospital, Randwick.
Section 8 – Application to yeast
This section is deleted as disc testing of yeasts is no longer supported by the CDS Reference Laboratory.
Section 9 – Application to Unusual organisms
Added recommendations for non-fastidious, non-enterobacteriaceae Gram negative Group.
Expanded the fastidious Gram negative organisms to include Aggregatibacter sp.
Section 10 – Application to veterinary medicine
10.3 Added use of Oxacillin with Staphylococcus intermedius group (SIG).
Tables 12.1.a – 12.3.d
Sulphafurazole has been deleted as it is no longer used in clinical practice.
Addition to the table of Morganella and Enterobacteriaceae with an AmpC.
Section 13 – Plates
Inclusion of Plate 13.9.C & Plate 13.9.D that demonstrate the presence of AmpC using the Boronic acid test.
All plates that previously included a sulphafurazole disc have been updated to include instead a co-trimoxazole disc (SXT 25).
Newsletter 37 (July 2016)
Proceedings of ASM 2016 Workshop
The CDS workshop was held in Perth this year, for the first time the workshop was held outside the scheduled program and at a site remote from the principal venue. Not surprisingly the numbers were down on previous years but the three speakers, Dianne Rafferty, Julie Allerton and Dr. Peter Newton presented a number of interesting and important topics. The Powerpoint slides of these presentation have been uploaded to ASM 2016 on the Archive Page. The slides are generally self-explanatory but if you would like any further information on any aspect of the presentations please email the relevant speaker, their email addresses are here. Three other matters were raised at the workshop and further details are set out below:
Formation of the CDS Users Special Interest Group (SIG).
ASM suggested that we form a CDS Users SIG to ensure that that the workshop/SIG meeting would be scheduled at all future ASM Conferences without the need for the CDS Laboratory Staff to arrange this with the organisers. We were told that there would be no fee for membership of the SIG and membership would be open to all CDS Users. Dianne started the process at the workshop and if you would like any further information please contact her.
Stability of Timentin Discs
In our email of 31/05/2016 we reported that we would be studying the stability of Mast Timentin 85 discs. This study is now complete and a summary follows:-The annular radius of the zones of inhibition with a Timentin 85 µg disc and Ps.aeruginosa NCTC 10662 observed in a CDS test performed in a diagnostic Microbiology laboratory were compared daily with those with those performed in the CDS laboratory over a period of 30 days. In the diagnostic laboratory the Timentin disc were contained in the disc dispenser used in routine testing and stored in accordance with the laboratory’s usual practice. In the CDS laboratory the discs were stored and used strictly in accordance with the manufacturer’s specifications. On day 1 the zones were similar in both laboratories (between 9 & 10 mm). By day 8 in the routine laboratory the zones had fallen to 6 mm and at 14 days to 5 mm and at 30 days they measured 4.5 mm. On the other hand the zones sizes of the tests performed in the CDS laboratory remained stable and continued to measure between 9 & 10 mm up to 30 days. This striking difference in the two sets of results reinforces the lead statement of the CDS section on quality assurance i.e “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.”
Surrogate Testing of Pradofloxacin in Veterinary Laboratories
Dianne reported the study of a comparison of moxifloxacin and ciprofloxacin as a surrogate disc for pradofloxacin. Her results can be seen in the report of the 2016 Workshop and they indicated that moxifloxacin was the most appropriate surrogate disc.
Newsletter 36 (August 2015)
Proceedings of ASM 2015 Workshop
The CDS website has been rebuilt and relocated and can be found at http://cdstest.net/. The website contains the latest online version of the manual, access to previous ASM workshops via the Archives section and current news in the What’s New section. We will endeavour to maintain the site with the latest calibrations and updates as they come to hand and appreciate CDS user’s feedback.
As mentioned at ASM 2014 the CDS Reference Laboratory has moved to SEALS – Central at St George Hospital. It has now fully settled in to the new location and with the return of Dianne Rafferty we look forward to a highly productive and informative period. Some CDS users may remember Dianne as she worked alongside Jeanette Pham in the CDS Reference Laboratory from 2000-2006. Any enquiries can be directed to her and the CDS team at firstname.lastname@example.org or email@example.com
Currently in the CDS laboratory we are undertaking a major project that aims to examine the relationship between agar dilution MICs and broth microdilution MICs. This study may take up to 2 years to complete and at the same time we will be comparing the breakpoints set down by the various methods and investigate ways of achieving harmonisation of these values.
Staphylococcus intermedius group (SIG):
This year we undertook a study to determine whether to use Cefoxitin 10µg or Oxacillin 1µg discs to report Methicillin susceptibility for the Staphylococcus intermedius group (SIG). Although this study was primarily aimed at veterinary laboratory practice it is now of interest to microbiologists working in human diagnostic microbiology. The SIG consists of Staphylococcus intermedius, Staphylococcus pseudintermedius and Staphylococcus delphini. This group of organisms are coagulase positive and difficult to differentiate from Staph.aureus and from each other on biochemical testing alone therefore it is recommended that they be reported as the Staphylococcus intermedius group (SIG), the exception occurs where the isolate is obtained from a canine source in which case it is accepted practice to identify the isolate as Staphylococcus pseudintermedius.
Like all Staphylococcus spp the SIG can acquire the mecA gene that confers resistance to methicillin and so we needed to investigate whether we should use Cefoxitin 10µg discs as we do with Staph. aureus or Oxacillin 1µg discs as we do with CoNS. The Powerpoint presentation at ASM 2015 by Dianne Rafferty of these results has been uploaded to the Archive section and can be accessed here.
Summary of the recommendations:
• Methicillin susceptible (mec A gene negative) SIG have a zone of inhibition to oxacillin 1µg discs > 6mm and should be reported as susceptible to methicillin.
• Methicillin resistant (mec A gene positive) SIG have a zone of inhibition to oxacillin 1µg disc <6mm and should be reported as resistant to methicillin.
• When the identification of the isolate is not available at the time of susceptibility testing then both cefoxitin 10µg disc and oxacillin 1µg discs should be tested. If both have a zone >6mm then the isolate should be reported as susceptible to methicillin.
The CDS method: not just about measuring zone sizes
This Powerpoint presentation was given by Dr Peter Newton at ASM 2015 and has been uploaded into the Archive section and can be accessed here. In summary Peter presented that for some organisms and antimicrobials interpretation of the CDS test involves not only measuring the annular radius of the inhibitory zone but an examination of the inhibitory zone morphology is also important in assessing the susceptibility. This aspect of the CDS test is best exemplified by: (1) the sharp zone edge of beta-lactamase producing strains of Staphylococcus aureus and Enterococcus faecalis and (2) the hazy zone edge of vanB phenotype VRE with vancomycin-susceptible enterococci,. The confluent uniform lawn of growth produced by the CDS inoculum preparation is advantageous in the assessment of the inhibitory zone edge.
Discussion arising from the Users Group:
1.Susceptibility Testing of Helicobacter pylori to metronidazole:
Q: Should H. pylori be incubated anaerobically for the first 24hrs to combat the overestimation of resistance to metronidazole?
A: Metronidazole needs to be reduced by the bacterial cell before it is active therefore its use is generally restricted to obligate anaerobes. H. pylori cultures are a mixture of aerobic, microaerophilic and anaerobic cells. Incubation under strict anaerobic conditions suppresses the growth of the aerobic and microaerophilic cells and gives rise to an in vitro phenomenon of apparent susceptibility. However this has not been tested in vivo whereas the results of conventional susceptibility testing (no anaerobic incubation) have been correlated with clinical response in a number of studies. However as metronidazole is invariably used in combination with at least one other antibiotic the correlations are difficult to interpret.
As the CDS test is biased towards specificity (i.e. we try to avoid calling resistant strains susceptible) it is our recommendation that, until further evidence is available to establish a correlation between incubation under anaerobic/microaerophilic conditions, CDS users should adhere to the method described in the manual.
Larsen, A,L. et al. 2012 Resistance rates of metronidazole and other antibacterials in Helicobacter pylori from previously untreated patients in Norway. APMIS 121,353-358.
McNulty, Cliodna., et al. 2002 Helicobacter pylori susceptibility testing by disc diffusion. Journal Antimicrob. Chemother. 49,601-609.
2.Mixed cultures of VRE (vanA and vanB):
Q: If an isolate of VRE was able to have both vanA and vanB genes can we pick this up using the CDS method?
A: As with all disc diffusion tests the CDS method shows the phenotypic result of expression of vanA or vanB or some strains may even show heterogenous resistance. It is important that interpretation of isolates follow the protocol as set out in section 4.2.6 of the manual with mandatory comparison to the reference strain as interpretation of susceptibility is based on characteristics of the inhibitory zone edge as well as the size of the zone.
Q: Is it enough to report R/S or should zone sizes be reported in mm?
A: After discussion with the user’s group it was pointed out that currently it is not a requirement of NPAAC (National Pathology Accreditation Advisory Council) to record zone sizes in mm so reporting R/S should be sufficient.
Reporting of Meropenem for RCPA QAP Survey:
It has been raised with the CDS Reference Laboratory that with RCPA QAP for the Gram negative isolates CDS user’s report Imipenem 10µg. Imipenem is now rarely used to treat patients and has largely been replaced by Meropenem. Imipenem 10µg cannot be used as a surrogate for Meropenem as there have been cases of Proteus spp that are resistant to Imipenem 10µg but sensitive to Meropenem 5µg by mechanisms other than carbapenemase production. Therefore it is recommended that CDS users should test and report Meropenem 5µg susceptibility with all GNR in addition to using Imipenem 10µg adjacent to Cefotaxime 5µg for detection of inducible cephalosporinases.
Postscript: Our attention has been drawn to the fact that the link in Archives to “The CDS Test Method:Fulfilling a Need” published in Microbiology Australia in March 2014 was incomplete. This has been corrected and if you havent read this article you can view or download it here.
Newsletter 35 (October 2013)
Proceedings of ASM 2013 Workshop Uploaded
The Powerpoint presentations by Thanh Nguyen, Peter Newton and Jeanette Pham at the ASM Workshop 2013 have been uploaded to the Archive section and can be accessed here.
Also uploaded in the Archive section is a short article on the Detection of Carbapenemase Enzymes by S.M.Bell and J.N. Pham which appeared in Breakpoint 2013 5. It is reproduced here by the kind permission of Sharon Chen, the editor. It can be viewed or downloaded from the publication section of the Archives.
If you are looking for earlier Newsletters these have been relocated to the Archives page here.