Dataset Information

The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.

ABSTRACT:

Objectives

With increasing antimicrobial resistance, rapid antimicrobial susceptibility testing (RAST) becomes important, especially in patients with bloodstream infections. EUCAST decided to develop a standardized rapid method, based on EUCAST disc diffusion, to offer susceptibility reports within 4-8?h of a positive blood culture (BC).

Methods

BC bottles were spiked with clinical isolates (n?=?332) of the seven most relevant sepsis pathogens with a variety of resistance mechanisms. RAST was performed directly from the bottle and zones read after 4, 6 and 8?h. Several variables were investigated, including the effect of using different BC bottles and of a 0-18?h delay between a positive signal and the performance of RAST.

Results

For five species, most inhibition zones could be read after 4?h. The proportion of results that could be interpreted increased from 75% at 4?h to 84% after 8?h. Categorical agreement against the reference method was good, with error rates of false susceptibility of 0.2%, 0.2% and 0.2% at 4, 6 and 8?h and false resistance of 1.2%, 0.2% and 0.1% at 4, 6 and 8?h, respectively.

Conclusions

With the EUCAST RAST method, reliable AST results can be delivered within 4-8?h of positivity of BC bottles for seven important bloodstream infection pathogens. To reduce the occurrence of errors and to absorb the variability caused by using a non-standardized inoculum, material from different manufacturers and workflow-related delays, we have introduced an area in which interpretation is not permitted, the Area of Technical Uncertainty.

SUBMITTER: Jonasson E

PROVIDER: S-EPMC7069491 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.

Jonasson Emma E Matuschek Erika E Kahlmeter Gunnar G

The Journal of antimicrobial chemotherapy 20200401 4

<h4>Objectives</h4>With increasing antimicrobial resistance, rapid antimicrobial susceptibility testing (RAST) becomes important, especially in patients with bloodstream infections. EUCAST decided to develop a standardized rapid method, based on EUCAST disc diffusion, to offer susceptibility reports within 4-8 h of a positive blood culture (BC).<h4>Methods</h4>BC bottles were spiked with clinical isolates (n = 332) of the seven most relevant sepsis pathogens with a variety of resistance mechanis ...[more]

PMID: 32016342

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Project description:Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these, 184 isolates from a strain collection were tested by an in-house rifaximin disc (40 µg) diffusion test, by an in-house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ≥32 µg ml(-1) as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n = 1) to ≥1024 µg ml(-1) (n = 61); 62 isolates showed a reduced susceptibility (MIC ≥32 µg ml(-1)). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of >1024 µg ml(-1), rifaximin end-point MICs were determined by broth dilution: 4096 µg ml(-1) (n = 2), 8192 µg ml(-1) (n = 6), 16,384 µg ml(-1) (n = 2) and 32,678 µg ml(-1) (n = 1). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ≤0.002 (n = 117) to ≥32 µg ml(-1) (n = 59). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ≥32 µg ml(-1). There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ≥32 µg ml(-1) as breakpoint for reduced rifampicin susceptibility and a <38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin--despite its water-insolubility--may be a suitable candidate for disc diffusion testing.

Project description:BackgroundA variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae.ObjectiveThe aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries.DesignProspective study.SettingMale and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India.ParticipantsN. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge.Material and methodsA total of 295 consecutive N. gonorrhoeae isolates during 2005-2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls.ResultsCDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis.ConclusionsThe CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial resistance surveillance when the WHO Gonococcal Antimicrobial Surveillance Programme is undergoing expansion to meet the ongoing challenges of surveillance and control of gonococcal antimicrobial resistance.

Dataset Information

The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.

Objectives

Methods

Results

Conclusions

Publications

The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.

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