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Etest Application Guide

Etest Application Guide

16273B - en - 2012/07 ETEST APPLICATION GUIDE

AEROBES

RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

Staphylococcus 29213TM ≤ 4 mg/L 35 ± 2°C MHA 0.5 Saline (FX) 18-24h aureus Ambient air S. aureus 43300TM > 4 mg/L - Phenotype interpretation: MRSA OX ≥ 4 µg/mL and Mueller Hinton agar (OX) 35 ± 2°C 0.5-1 Saline 24h S. aureus 43300TM XL ≥ 8 µg/mL (MHA) + 2% NaCl / (XL) Ambient air - Heavier inoculum improves detection of low level R - Phenotype interpretation: Oxacillin (OX) 35 ± 2°C BORSA MHA + 2% NaCl 0.5-1 Saline 24h S. aureus 29213TM OX ≥ 4 µg/mL and Amoxicillin/clavulanic acid (XL) Ambient air XL ≤ 4 µg/mL - Phenotype interpretation: OX ≥ 0.5 µg/mL Mueller Hinton agar 35 ± 2°C MRCNS 0.5-1 Saline Oxacillin (OX) 48h S. aureus 43300TM - Heavier inoculum (MHA) + 2% NaCl Ambient air improves detection of low Staphylococci level R - 0.1 mL inoculum/ 90mm plate - Phenotype interpretation: S. aureus VA ≥ 8µg/mL and 1) 24h. VISA/hVISA Brain Heart Infusion (VA) 35 ± 2°C QC ranges: TM TP ≥ 8µg/mL, 2 BHI broth Confirm at 29213 (macromethod) agar (BHI) 1) (TP) 1) Ambient air TP = 0.5-2 µg/mL OR 48h VA = 1-4 µg/mL TP ≥12 µg/mL (alone) = VISA/hVISA - Confirm VISA/hVISA by PAP 18-24h. S. aureus 29213TM Etest GRD2) (Vancomycin (VA) / 35 ± 2°C - Confirm VISA/hVISA by VISA/hVISA MHA + 5% blood 0.5 BHI broth Confirm at S. aureus 700698TM Teicoplanin (TP) 1)) Ambient air PAP 48h S. aureus 700699TM - Phenotype interpretation: VISA/VRSA 35 ± 2°C VA ≥ 4µg/mL = VISA (standard MHA 0.5 Saline Vancomycin (VA) 24h S. aureus 29213TM Ambient air method) VA ≥ 16µg/mL = VRSA (CLSI)

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RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere (CM) (DPC) MRSA (LZ) 16-20h. 35 ± 2°C Secondary MHA 0.5 Saline Vancomycin (VA) VA and TP S. aureus 29213TM Ambient air panel Teicoplanin (TP) 1) full 24h Quinupristin/ (QDA) Rifampicin (RI) 1) Staphylococci (PG) Erythromycin (EM) Non-MRSA Linezolid (LZ) 16-20h. 35 ± 2°C secondary MHA 0.5 Saline Trimethoprim/sulfamethoxazole (TS) VA and TP S. aureus 29213TM Ambient air panel Vancomycin (VA) full 24h Teicoplanin (TP) 1) Ciprofloxacin (CI)

Ampicillin (AM) Daptomycin (DPC) 16-20h. Linezolid (LZ) Confirm VA 35 ± 2°C MHA 0.5 Saline Quinupristin/dalfopristin (QDA) E. faecalis 29212TM and TP at Ambient air Vancomycin (VA) 24h Teicoplanin (TP) 1) Minocycline (MC) Enterococci - Heavier inoculum preferable E. faecalis - SM > 1024 µg/mL or 24h. QC ranges: GM > 512 µg/mL = (GM) high range 35 ± 2°C HLAR MHA 0.5-1 Saline Confirm SM GM = 4-16 µg/mL 29212TM positive HLAR (CLSI) (SM) high range 1) Ambient air at 48h SM = 64–256 µg/mL - SM > 512 µg/mL or GM > 128 µg/mL = positive HLAR (EUCAST)

Gentamicin (GM) / (PTc) 0.5 (PM) 35 ± 2°C Escherichia coli 25922TM MHA (1 for mucoid Saline 16-20h Ciprofloxacin (CI) Ambient air E. coli 35218TM organisms) (IP) (PTc) (AT) /cefotaxime + clavulanic E. coli 35218TM acid (CT/CTL) Klebsiella 700603TM 0.5 /ceftazidime + clavulanic 35 ± 2°C pneumoniae - Investigate ND results by ESBL MHA (1 for mucoid Saline 16-20h acid (TZ/TZL) Ambient air (ESBL positive) an alternative method organisms) Cefepime/cefepime + clavulanic acid 27853TM (PM/PML) 1) aeruginosa

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RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere 0.5 MBL MP/MPI /Meropenem +EDTA 35 ± 2°C K. pneumoniae 700603TM - Investigate ND results by MHA (1 for mucoid Saline 16-20h. 1) (MP/MPI) Ambient air K. pneumoniae BAA-2146TM an alternative method organisms) Enterobacteriaceae Klebsiella 700603TM pneumoniae /Cefotetan + 35 ± 2°C - Investigate ND results by AmpC 2) MHA 0.5 Saline 16-20h (negative) (CN/CNI) Ambient air an alternative method K. pneumoniae BAA-1144TM (positive)

Ceftazidime (TZ) Gentamicin (GM) 0.5 16-20h. Aztreonam (AT) 35 ± 2°C P. aeruginosa 27853TM MHA (1 for mucoid Saline 48h for slow Ciprofloxacin (CI) Ambient air E. coli 35218TM organisms) growers Imipenem (IP) (PTc) Pseudomonas spp. Piperacillin/tazobactam (PTc) P. aeruginosa 27853TM 0.5 16-20h. 35 ± 2°C (negative) - Investigate ND results by MBL IP/IPI 1) MHA (1 for mucoid Saline Imipenem/imipenem + EDTA (IP/IPI) 48h for slow Ambient air Stenotrophomonas 13636TM an alternative method organisms) growers maltophilia (positive) Ceftazidime (TZ) Meropenem (MP) 0.5 20-24h. (AK) 35 ± 2°C P. aeruginosa 27853TM MHA (1 for mucoid Saline 48h for slow / (AB) Ambient air E. coli 35218TM (AB) organisms) growers Levofloxacin (LE) Acinetobacter spp. Minocycline (MC) P. aeruginosa 27853TM 0.5 16-20h. 35 ± 2°C (negative) - Investigate ND results by MBL IP/IPI 1) MHA (1 for mucoid Saline Imipenem/imipenem + EDTA (IP/IPI) 48h for slow Ambient air Stenotrophomonas 13636TM an alternative method organisms) growers maltophilia (positive) Trimethoprim/sulfamethoxazole (TS) 0.5 Ceftazidime (TZ) 20-24h. 35 ± 2°C Burkholderia cepacia MHA (1 for mucoid Saline Levofloxacin (LE) 48h for slow E. coli 25922TM Ambient air organisms) Meropenem (MP) growers Minocycline (MC) Trimethoprim/sulfamethoxazole (TS) 0.5 Ceftazidime (TZ) 20-24h. Stenotrophomonas 35 ± 2°C E. coli 25922TM MHA (1 for mucoid Saline Levofloxacin (LE) 48h for slow maltophilia Ambient air E. coli 35218TM (TLc) organisms) Minocycline (MC) growers /clavulanic acid (TLc)

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FASTIDIOUS ORGANISMS

RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

Meropenem (MP) Cefotaxime (CT) MHA + 5% blood 0.5 MH broth or Benzylpenicillin (PG) 35 ± 2°C Pneumococci (CLSI) (1 for mucoid 20-24h 49619TM BHI broth Clindamycin (CM) 1) 5% CO pneumoniae MHF (EUCAST) organisms) 2 Vancomycin (VA) Trimethoprim/sulfamethoxazole (TS)

Benzylpenicillin (PG) Cefotaxime (CT) MHA + 5% blood 0.5 Chloramphenicol (CL) MH broth or 35 ± 2°C Streptococci (CLSI) (1 for mucoid Ofloxacin (OF) 20-24h S. pneumoniae 49619TM BHI broth 5% CO MHF (EUCAST) organisms) Linezolid (LZ) 1) 2 Vancomycin (VA) Daptomycin (DPC)

MH chocolate agar + Broth Abiotrophia & 35 ± 2°C 0.001% pyridoxal HCl + 1 (MH broth or 20-24h 5) 5) Granulicatella spp. 1) 5% CO 0.01% cysteine BHI broth) 2

Amoxicillin/ clavulanic acid (XL) Haemophilus Test 0.5 MH broth or Cefotaxime (CT) Haemophilus 49247TM Haemophilus 35 ± 2°C Media (HTM) (CLSI) (1 for mucoid HTM broth Meropenem (MP) 20-24h influenzae influenzae 5% CO MHF (EUCAST) organisms) (or BHI broth) Trimethoprim/sulfamethoxazole (TS) 2 H. influenzae 49766TM Chloramphenicol (CL) (MP, XM)

MHA + 5% blood MH broth or 35 ± 2°C Moraxella catarrhalis 1) (CLSI) 0.5 20-24h 5) 5) BHI broth 5% CO MHF (EUCAST) 2

- For obligate anaerobes, ensure anaerobic (MZ) Brucella agar + 24-72h conditions are Brucella broth Clindamycin (CM) 5% blood + (48h for CM). 35 ± 2°C Bacteroides fragilis 25285TM maintained throughout or MH broth Cefoxitin (FX) Anaerobes vitamin K (1 µg/mL) + 1 Confirm all Anaerobic B. thetaiotaomicron 29741TM processing (or Schaedler Imipenem (IP) hemin (5 µg/mL) (BBA) S results at system Eubacterium lentum 43055TM - DO NOT VORTEX Broth + vit K3) Piperacillin/tazobactam (PTc) (CLSI) 48h. - Anaerobiosis must be Benzylpenicillin (PG) achieved within 1-2 hours for MZ

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RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

- 1 strip/90mm plate - Strip handle to be 35 ± 2°C placed at edge of plate. MH broth + Amoxicillin (AC) Micro- - H. pylori colonies are MHA + 5% blood 5% serum Clarithromycin (CH) aerophilic, Helicobacter pylori 1) 3 72h or longer Helicobacter pylori 43504TM pin-point, translucent (≥ 2 weeks old) (CLSI) or BHI broth + Metronidazole (MZ) except MZ - and difficult to see. 5% serum Tetracycline (TC) first 24h Tilt plate and use anaerobic oblique light to read endpoint.

Ciprofloxacin (CI) Supplemented GC agar Benzylpenicillin (PG) MH broth 35 ± 2°C - 3-4 strips max./150mm Gonococci (CLSI) 0.5 Tetracycline (TC) 20-24h 49226TM (or BHI broth) 5% CO gonorrhoeae plate to facilitate reading MH chocolate agar (TX) 2 Spectinomycin (SC) 1)

Ciprofloxacin (CI) MHA + 5% blood Benzylpenicillin (PG) Broth 35 ± 2°C S. pneumoniae 49619TM Meningococci 1) (CLSI) 0.5 Trimethoprim/sulfamethoxazole (TS) 24h - Work in BSC (BHI broth) 5% CO E. coli 25922TM MH chocolate agar Meropenem (MP) 2 Ceftriaxone (TX)

- 3-4 strips max./150mm plate to facilitate reading - Do not invert plate Ciprofloxacin (CI) MHA + 5% blood 35 ± 2°C - Campylobacter colonies Broth Gentamicin (GM) Campylobacter Campylobacter spp. 1) (CLSI) 1 48-72h Micro- 33560TM may be translucent and (BHI broth) Erythromycin (EM) jejuni MHF (EUCAST) aerophilic difficult to see. Doxycycline (DC) Tilt plate and use oblique light to read endpoint.

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FASTIDIOUS GRAM-POSITIVE ORGANISMS 1)

RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

Arcanobacterium spp., monocytogenes*, Erysipelothrix, , MHA + 5% blood Broth 20-24h (48h if 35 ± 2°C , (CLSI) 1 5) 5) (BHI broth) required) 5% CO Bacillus spp., * MHF (EUCAST) 2 Rothia, Pediococcus*, Leuconostoc*, and Gemella spp.*

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FASTIDIOUS GRAM-NEGATIVE ORGANISMS 1)

RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

Broth 35 ± 2°C Bartonella spp. MH chocolate agar 1 3-5 days 5) 5) (BHI broth) 5% CO2

35 ± 2°C Regan-Lowe Broth Ambient air Bordetella pertussis Bordet-Jengou 3 3-5 days 5) 5) (BHI broth) in bags, or MHA + 5% blood moist

Broth 35 ± 2°C Capnocytophaga spp. BBA 1 48h 5) 5) (BHI broth) 5% CO2

Buffered Charcoal Broth 35 ± 2°C Legionella spp. 1 3-5 days 5) 5) Yeast Extract (BCYE) (BHI broth) 5% CO2

MHA + 5% blood Broth 35 ± 2°C Pasteurella spp. (CLSI) 1 48h 5) 5) (BHI broth) 5% CO MHF (EUCAST) 2

- Testing should only be Cysteine Heart Agar + Suspend 48h performed in appropriate Clindamycin (CM) 10% blood 1) (or + colonies from reference laboratories. Tetracycline (TC) 37 ± 2°C Francisella tularensis 2% haemoglobin 3)) 1 chocolate 48-72h 5) 5) - Perform all work within Ciprofloxacin (CI) 5% CO alternatively, Glucose agar in broth 2 BSC Class IIA and Gentamicin (GM) Cysteine Blood Agar (BHI broth) minimum BSL 2 environment.

MHA + Levofloxacin (LE) 1% haemoglobin + Broth Imipenem (IP) 35 ± 2°C H. influenzae 49247TM HACEK group 1% IsoVitalex 1 24-72h (BHI broth) Trimethoprim/sulfamethoxazole (TS) 5% CO H. influenzae 49766TM (IP) or HTM 2 Ceftriaxone (TX) or BBA

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MYCOBACTERIA AND AEROBIC ACTINOMYCETES 1)

RECOMMENED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

M7H9 broth + 0.5% tween + 2% glycerol Vortex with - Pre-incubation 24h sterile glass Ethambutol (EB) Mycobacterium 27294TM - 1 strip/90mm plate, seal Mycobacterium Middlebrook 7H11 + beads Ethionamide (ET) 37 ± 2°C 3-4 5-10 days tuberculosis plates tuberculosis 10% OADC agar 3-5 mins, Isoniazide (IZ) 5-10% CO 2 M. tuberculosis AW388 - Perform all work within settle 20 mins Rifampicin (RI) (not ATCC) BSC class IIA & adjust supernatant to correct turbidity.

M7H9 broth Vortex with sterile glass MHA + 10% OADC + beads Amikacin (AK) 35 ± 2°C M. avium 700898TM - 1 strip/90mm plate, seal 1 Nontuberculous 5% blood 3-5 mins, Ciprofloxacin (CI) (M. marinum M. avium spp. avium 35713TM plates (3 for 5-10 days mycobacteria (M. kansasii use same settle 20 mins Clarithromycin (CH) at 30°C) M. marinum 927TM - Antibiogram is species M. kansasii) TM agar as for MTB) & adjust Rifampicin (RI) 5% CO2 M. kansasii 12478 specific supernatant to correct turbidity.

Cefoxitin (FX) - Subculture twice before 30-35°C Rapid growing Imipenem (IP) M. fortuitum 6841TM preparing inoculum MHA + 5% blood 1 Saline 48-72h Ambient air, mycobacteria Ciprofloxacin (CI) M. peregrinum 700686TM - 3-4 strips max./150mm moist Clarithromycin (CH) plate to facilitate reading

Amikacin (AK) Trimethoprim/sulfamethoxazole (TS) 48-72h Broth 35 ± 2°C Nocardia spp. MHA + 5% blood 1 Ciprofloxacin (CI) (dependent S. aureus 29213TM (BHI broth) Ambient air Clarithromycin (CH) on spp.) Imipenem (IP)

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FUNGI

RECOMMENDED INOCULUM INCUBATION SPECIFIC QUALITY CONTROL ORGANISM MEDIA 6) SUGGESTED MIC PANEL 3) COMMENTS 4) PHENOTYPE Suspension Temperature/ McF equivalent Time (h) Strain ATCC® medium Atmosphere

Fluconazole (FL) RPMI 1640 + 0.5 Itraconazole (IT) 35 ± 2°C 24-48h. Candida albicans 90028TM - Once plate inoculated, 2% glucose + (1 for (AP) 1) Ambient air Yeast Saline 48-72h for C. krusei 6258TM re-dip swab and streak MOPS + Cryptococcus Flucytosine (FC) in bags, C. neoformans C. parapsilosis 22019TM again. 1.5% Bacto agar neoformans) Voriconazole (VO) moist Caspofungin (CS) 1)

Amphotericin B (AP) 1) RPMI 1640 + 0.5 Aspergillus 35 ± 2°C Itraconazole (IT) 16-72h C. parapsilosis 22019TM 2% glucose + spp. Saline + Ambient air Mould 1) Voriconazole (VO) 1) (Dependent Aspergillus flavus 204304TM MOPS + (1 for Fusarium, Tween 20 in bags, Posaconazole (POS) 1) on genus) A. fumigatus 204305TM 1.5% Bacto agar Rhizopus spp.) moist Caspofungin (CS) 1)

1) In the USA, For Research Use Only (RUO). The contents of this document do not in any way indicate or imply new in vitro diagnostic uses of Etest, outside those which are FDA-cleared for certain and organism groups. 2) Worldwide, For Research Use Only (RUO). 3) Example based on CLSI®, Performance Standards for Antimicrobial Susceptibility Testing supplement M100-S. Please use your own selection of MIC panels. 4) Additional Etest information is available at www.biomerieux.com/techlib. 5) For QC of fastidious organisms, please refer to local recommendations and/or CLSI M45-A Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious . 6) For media available in the bioMérieux range, see page 10 (consult www.biomerieux.com/techlib for availability of certificates of compatibility).

KEY:

BORSA Borderline ORSA (non-mec A resistance) due to type A macro-inducible ß-lactamase inhibited by clavulanic acid BSC Biological Safety Cabinet ESBL Extended Spectrum ß-Lactamase HLAR High-Level Resistance MBL Metallo ß-Lactamase MRCNS -Resistant -Negative Staphylococci (mec A+) MRSA Methicillin-Resistant aureus (mec A+) ND Non-Determinable OADC Oleic acid, Albumin, Dextrose Complex PAP Population Analysis Profile VISA/hVISA Vancomycin Intermediate/ hetero-Intermediate

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MEDIA AVAILABLE IN THE BIOMERIEUX RANGE Consult the bioMerieux Technical Library (www.biomerieux.com\techlib) for availability of certificates of compatibility

For non-fastidious bacteria: For fastidious bacteria: • Mueller Hinton 2 agar EUCAST recommendation: bioMérieux Ref. 43301/43309 - 20 / 100 plates 90 mm • Mueller Hinton 2 horse blood NAD agar bioMérieux Ref. 43302 - 20 plates 145 mm bioMérieux Ref. 43901/43919 - 20 / 100 plates 90 mm bioMérieux Ref. 43511 - 20 plates 120 x 120 mm bioMérieux Ref. 43904 - 20 plates 120 x 120 mm bioMérieux Ref. 41864 - 6 x 200 ml CLSI recommendation: bioMérieux Ref. 51075 - 500 g • Mueller Hinton 2 sheep blood agar bioMérieux Ref. 43321/43329 - 20 / 100 plates 90 mm bioMérieux Ref. 43324 - 20 plates 120 x 120 mm

For anaerobes: For fungi and yeasts: • Brucella blood agar (with Vitamin K1 + Hemin) • RPMI agar bioMérieux Ref. 411 968 - 20 plates 90 mm bioMérieux Ref. AEB 122 180 - 10 plates 90 mm bioMérieux Ref. AEB 122 182 - 10 plates 140 mm

SUSPENSION MEDIA AVAILABLE IN THE BIOMERIEUX RANGE

• Brain-Heart Infusion broth • API® NaCl 0.85 % Medium (saline) • Schaedler Broth + vit. K3 bioMérieux Ref. 42081 - 20 x 9 ml bioMérieux Ref. 20070 – 100 x 2ml bioMérieux Ref. 42106 - 20 x 13 ml bioMérieux Ref. 20040 – 100 x 3ml bioMérieux Ref. 20230 – 100 x 5ml

BIOMERIEUX, the blue logo, API and Etest are used pending and/or registered trademarks belonging to bioMérieux, or one of its subsidiaries, or one of its companies. CLSI is a registered trademark belonging to Clinical and Laboratory Standards Institute, Inc. The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection. Any other name or trademark is the property of its respective owner.

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