Microsoft word - rgmyco_us_gb_v1.19 _cim5759_.doc

SELECTION OF SUSCEPTIBILITY TEST BROTH
Use Sensititre CAMHBT for rapid growing mycobacteria,
Nocardia and other aerobic Actinomycetes or Mueller Hinton
broth with OADC for slow growing mycobacteria.
Sensititre broths are performance tested for use with SENSITITRE®
Broth Microdilution (MIC) Method:
INOCULATION AND INCUBATION
For Rapidly Growing Mycobacteria (RGM), Slowly
Rapid growing mycobacteria, Nocardia spp and other
Growing Nontuberculosis Mycobacteria, Nocardia and
aerobic actinomycetes
other Aerobic Actinomycetes
1.Sweep the confluent portion of growth from growth on an
agar plate with a swab. Emulsify in sterile water and adjust to a 0.5 McFarland Standard visually or using the Sensititre nephelometer. If particles are visible, vortex well. Actinomycetes typically have very hard crusty colonies. It may be necessary to vortex with glass beads to make a homogeneous suspension. If large clumps remain after INTENDED USE
vortexing, they should be allowed to settle and the Susceptibility testing of rapidly growing mycobacteria including supernatant used for the inoculum suspension. Mycobacterium fortuitum group (M. fortuitum, M. peregrinum, M. fortuitum third biovariant complex), M. chelonae, M. Moistening the swab with sterile water may facilitate a more abscessus, M. mucogenicum) and M. smegmatis group (M. smegmatis, M. goodii, M. wolinskyi). Nocardia spp and other aerobic actinomycetes. Slowly growing nontuberculosis Warning- the use of water supplemented with Tween may
mycobacteria (NTM), i.e. Mycobacterium avium complex, affect MICs
Mycobacterium kansasii and Mycobacterium marinum. 2.Transfer 50µl of the suspension into a tube of cation
Please refer to CLSI (NCCLS) (1) for details of testing M. adjusted Mueller-Hinton broth with TES buffer to give an inoculum of 5x105 cfu/mL (range 1x105 to 1x106 cfu/mL). . Mix PRINCIPALS OF USE
Each plate is dosed with antimicrobial agents at appropriate Steps 1 and 2 must be completed within 30 minutes. dilutions. Results can be read manually by visual reading of 3. Plates containing ≥ 32µg/mL doxycycline or minocycline
may show a precipitate after incubation. This can be PRECAUTIONS
prevented by reconstituting these well with 5µL sterile distilled Only personnel trained and qualified in susceptibility testing techniques should use the system. The laboratory should
have established biosafety guidelines for handling
4. Transfer 100µL to each well by either:
mycobacteria.
a. Sensititre AutoInoculator. Replace the tube cap with a
Sensititre single-use dosehead and inoculate the plate STORAGE AND SHELF LIFE
according to the AutoInoculator instructions. The plates should be stored at room temperature (15-25°C) b. Manual pipette. Pour the broth into a sterile seed trough
away from direct sunlight and direct heat. Each plate is and inoculate the plate using an appropriate pipette. Dose the packaged in foil with a silica gel desiccant. Do not use the panel with the Sensititre label facing the user. plate if past its expiration date, or the desiccant colour is not Pipettes should be periodically serviced and checked for Inoculate plate within 5 hours of removal from pouch. Inoculate broth into a plate within 30 minutes. PROCEDURE
5. A periodic check of the colony count of the positive control
well should be done. (See Appendix 1). Isolates should have an inoculum of 5x105 CFU/mL, (range 1x105 – 1x106) Materials required and not provided [TREK Product Code] 6. Cover all wells with the adhesive seal. Press all wells firmly
to assure adequate sealing. Avoid creases as these can lead Sensititre cation adjusted Mueller-Hinton broth with TES buffer 7. Incubate rapid growing mycobacteria aerobically at 300C in
a non-CO2 incubator for 72 hours. Check for growth. If poor, Sensititre cation adjusted Mueller-Hinton broth with OADC reincubate for up to a further 48 hours. Incubate Nocardia and other aerobic Actinomycetes at 350C in a non-CO2 incubator Sensititre doseheads (for use with AutoInoculator) [E3010] Plates can be stacked up to three high. Incubation of 4-5 days may be required for isolates of M. chelonae and M. abscessus 0.5 McFarland turbidity polymer standard [E1041] 50µL or 100µL pipettor and disposable tips Slow Growing Nontuberculous Mycobacteria (NTM
including MAC)
Same procedure as above except transfer 50µL of the organism suspension into 10mL of Sensititre Mueller-Hinton broth with 5% v/v OADC growth supplement. Invert the tube 8- Incubate at 350C in a non-CO2 incubator and read after 7 days. If growth is good in the positive control , read results. SPECIMEN COLLECTION AND PREPARATION
Otherwise re-incubate for up to 14 days. Prolonged incubation Specimens should be collected, transported, stored and then may require taking steps to prevent loss of well contents plated on to primary isolation medium to give isolated colonies through evaporation. Place the plates in a plastic container with the top ajar to facilitate gas exchange. READING TEST RESULTS
Results can be read using the Sensititre manual viewer or the Tentative Quality Control Ranges for mycobacteria QC SensiTouch. See Sensitouch User Manual. It is not necessary to remove the adhesive seal. Place the plate with the label facing the user. Growth appears as turbidity or as a deposit of cells at the bottom of a well. The MIC is recorded as the lowest concentration of antimicrobic that inhibits visible growth. Please refer to CLSI (NCCLS) M24-A (1) for guidance on reading endpoints. Reading faint growth on SensiTouch can be improved by use of bright indirect lighting against a The positive growth control wells should be read first. If any Mycobacteria end points can be difficult to interpret. CLSI (NCCLS) M24-A (1) provides reading guidelines and illustrations of various growth patterns. Negative wells can show a slight precipitate related to the inoculum. Reading QC strains with known MICs should be used for training Growth can range from a few colonies with no turbidity to heavy growth comparable to positive growth control. The MIC is the lowest concentration that completely inhibits growth except for sulphonamides, where the MIC is read as the lowest concentration that inhibits 80% growth compared to the a. Contamination
1 Ranges based on Sensititre in-house testing Contamination may result in growth in a well bordered by 2 The MIC range listed is for sulfizoxazole for wells showing no growth. Such a single well contamination can be ignored, but if multiple well contaminants are *** Range based on data from Reference 4 Occasionally a “skip” may be seen - a well showing no growth **** Incubation time dependent. Range based on 72 bordered by wells showing growth. There are variety of explanations including contamination, mutation, creased seal and misaligned dosing. A single skip can be ignored. Contact Sensititre Distributor or TREK Diagnostic Systems in However, in order to ensure effective antimicrobic therapy the event that quality control discrepancies cannot be NEVER read the skipped well as the MIC; always read the lowest well concentration above which there is consistently no PERFORMANCE
c. Mixed Cultures
Panels are designed to give comparable performance to CLSI Except as referred to in (a) above, if two end points are seen (NCCLS) reference micro-broth procedures (1). Performance as a distinct “button” of cells followed by several wells of has been independently investigated (references 5-7) diffuse growth with the “button” no longer visible (or seen as For further information contact TREK Diagnostic systems or smaller buttons), there may be a mixed bacterial population. Purity should be checked by sub-culturing growth onto suitable agar. Test results are invalid if a mixed culture is LIMITATIONS
1. Imipenem results for M.chelonae and M.abscessus should
INTERPRETATIVE GUIDELINES
2. Tobramycin is the aminoglycoside of choice for M. chelonae
Refer to the MIC Interpretive guidelines as provided by the and should only be reported for this organism (1) CLSI (NCCLS) (1), EUCAST or your national reference group. APPENDIX 1: Colony Count Procedure
QUALITY CONTROL
1.Immediately following inoculation plate, using a 1µl loop,
Frequency of quality control testing should be according to sample from the positive growth control well and inoculate local guidelines. Inocula should be cultured on a suitable medium to check for purity. Test results are invalid if a mixed 2.Take another loop (1µl) and sample from the same growth
well and mix with 50µl sterile deionised water. Inoculate 1µl of All Sensititre plates include positive control wells. Tests are this dilution onto an appropriate agar plate to obtain countable invalid unless there is distinct growth in all positive control 3.Incubate both plates at 30 or 35 0C (depending on type of
A number of factors influence MIC’s including organism state, inoculum density, temperature and broth. In practice, replicate 4.Read as follows:
MIC’s form a normal distribution with the majority of results lying within one dilution of the modal value. The test procedure can be considered satisfactory if control organism MIC’s are within range. Results should not be reported if QC
Colony Count
0.001 plate
0.001of 1/50 dilution
Table 1 lists tentative QC ranges using mycobacteria strains. Until other data becomes available, S.aureus ATCC 29213 and other non-mycobacterial QC strains and ranges from CLSI (NCCLS) document M100 (2) can be used for panel QC. BIBLIOGRAPHY
1. NCCLS M24-A. Susceptibility Testing of Nocardia, and
CLSI (NCCLS) M100 QC strains should use the same Other Aerobic Actinomycetes; Approved Standard- Second inoculation method as for rapid growing mycobacteria except Edition (2003). National Committee for Clinical Laboratory that 50µL of inoculum should be added to Sensititre Mueller Hinton broth with TES. Do not use broth supplemented with OADC. Panels should be read after 18 to 24 hours incubation 2.NCCLS M100-S13 (M7). Performance Standards for
Antimicrobial Susceptibility Testing; Thirteenth Informational Supplement. (2003). National Committee for Clinical
Laboratory Standards, Wayne, PA

3.Brown-Elliott, B; Wallace, R; Crist, C; Mann, L; and Wilson,
R. (2002). Comparison of In Vitro Activities of Galtifloxacin
and Ciprofloxacin against Four Taxa of Rapidly Growing
Mycobacteria. Antimicrobial Agents and Chemotherapy 46:
3283-3285
4.Wallace. R; Brown-Elliott, B; Crist, C; Mann, L;and Wilson,
R. (2002). Comparison of In Vitro Activities of Gatifloxacin and
Ciprofloxacin against Four Taxa of Rapidly Growing
Mycobacteria. Interscience Conference on Antimicrobial
Agents and Chemotherapy Abstract E-541
5.Woods. G; et.al. (2003). Multisite Reproducibility of Results
Obtained by Two Broth Dilution Methods for Susceptibility
Testing of Mycobacterium avium Complex. Journal of Clinical
Microbiology 41: 627-631
6.Woods, G; et.al (1999). Multisite Reproducibility of results
Obtained by Two Broth Dilution Methods for Susceptibility
Testing of Mycobacterium abscessus, M. chelonae and
Mycobacterium fortuitum. Journal of Clinical Microbiology 37:
1676-1682
7.Brown, B; Wallace, R; and Onyi, G. (1996). Activities of the
Glycylcyclines N,N-Dimethylglycylamido-Minocycline and N,N-
Dimethylglycylamido-6-Demethyl-6-Deoxytetracycline against
Nocardia spp. And Tetracycline- Resistant Isolates of Rapidly
Growing Mycobacteria. Antimicrobialial Agents and
Chemotherapy 40: 874-878
8. Clinical Microbiology Procedures Handbook, 2nd Edition.
2004. Editor H.D. Isenberg. ASM Press.

DISCLAIMER
Read the instructions for use and references before using the
product.
Any change or modification of the instructions may affect
results. TREK Diagnostic Systems will not be liable for any
damages resulting from any changes in storage, precautions,
handling or testing procedures of the current version of
instructions.
The information provided in this technical insert is current at
the time of printing and may change without notice. Contact
the local TREK distributor for latest information
Manufacturer: TREK Diagnostic Systems Limited
Imberhorne Lane, East Grinstead,
West Sussex RH19 1QX UK
Tel: +44 1342 318777

Distributed in USA by:
TREK Diagnostic Systems,
982 Keynote Circle, Suite 6,
Cleveland, Ohio, 44131
Sensititre® and SensiTouch® are registered trademarks of
TREK Diagnostic Systems.

RGMYCO_US_GB_V1.19

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