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Mycobacterial Testing With BacT/ALERT® Systems and Media

1. Sodium hydroxide the most commonly used decontaminant also serves as a mucolytic agent but must be used cautiously because it is only somewhat less harmful to tubercle bacilli than to the contaminating organisms The stronger the alkali the higher its temperature during the time it acts on the specimen and the longer it is allowed to act the greater will be the killing action on both contaminants and mycobacteria TECHNICAL TIPS If you are experiencing higher than expected false positives or breakthrough contamination use no more than 5 mL of sample per tube To avoid discarding sample split larger samples into multiple aliquots and process separately Pellets may be combined at end of processing for inoculation into one BacT ALERT MP culture bottle If less than 5 mL of sample is available add sterile 0 067 M phosphate buffer pH 6 8 or sterile water to bring the volume up to the 5 mL mark Use individual pour tubes to prevent the addition of excess decontamination reagent and to avoid the possibility of cross contamination between specimens Most commonly a combination liquefaction decontamination mixture is used These agents have no direct inhibitory effect on bacterial cells however their use permits treatment with lower concentrations of sodium hydroxide thereby indirectly improving the recovery of mycobacteria Specimens submitted for the culture of mycobacteria should be processed as soon as possible Th
2. Inoculation It is generally accepted that the use of a liquid medium in combination with at least one solid medium is essential for good laboratory practice in the isolation of mycobacteria Addition of a solid medium is advantageous for the detection of strains which occasionally do not grow in liquid medium aids in the detection of mixed mycobacterial infections and can serve as a back up for broth cultures if contaminated All positive cultures even if identified directly from the broth must be subcultured to solid media to detect mixed cultures and to correlate direct identification results with colony morphology It is important to resuspend the concentrated pellet in as little liquid as possible to maintain the largest numbers of organisms to inoculate cultures Larger numbers of organisms lessen the time to detection in the BacT ALERT culture bottle and enhance smear results Pfyffer GE Brown Elliott BA Wallace RJ Mycobacterium General Characteristics Isolation and Staining Procedures in Murray PR Baron EJ Pfaller MA et al eds Manual of Clinical Microbiology ed 8 Washington DC American Society for Microbiology 2003 p 549 Global Customer Support K5 31JAN09 17 of 76 Mycobacterial Support Manual Best Practices in Sample Processing TECHNICAL TIPS 1 Use individual disposable alcohol prep pads to clean the tops of all reagent or culture bottles before each entry into the bottle Add no more than 0
3. Organism no of strains 10 CFU ml 10 CFU ml M avium 4 M intracellulare 4 M tuberculosis 4 M fortuitum M chelonae M xenopi M bovis 2 M gordonae M simiae 510 k data on file at bioM rieux Inc Global Customer Support K5 31JAN09 56 of 76 Mycobacterial Support Manual APPENDIX G BacT ALERT MP Instructions for Use 43 03064 July 2008 PERFORMANCE CHARACTERISTICS OF THE TEST Seeded studies were performed using the following organisms at levels of 10 CFU bottle and 104 CFU bottle Time to Detectlon days Inoculum BacT ALERT MP Microorganism CFU bottle Plastic TB Complex AV tuberculosis 4 strains 106 5 6 10 3 s 10 13 7 18 8 MV bovis 2 strains 106 71 768 10 14 4 174 Photochromogens Runyoun I M kansasi 2 strains 10 74 8 8 s 102 13 1 15 0 M simiae 10 4 1 102 3 9 Scotochromogens Runyoun II M gordonae 10 14 1 s 10 23 7 A xenopi 2 strains 10 10 3 13 8 s 10 19 9 25 97 MV scrofulaceum 2 strains 10 12 6 23 3 10 20 7 NG Nonchromogens Runyoun Hi M avium 4 strains s 10 4 2 6 8 10 10 2 15 9 M intracellufare 4 strains 106 5 6 7 1 1 10 8 16 2 i malimoense 10 15 4 s 10 21 9 Rapid Growers Runyoun IV MV chelonae 108 2 2 10 4 1 M fortuitum 2 strains s 10 3 5 4 1 102 3 9 8 9 1 Each organism was tested with MB BacT Antibiotic Supplement Testing was performe
4. 46 of 76 Mycobacterial Support Manual APPENDIX C Suggested Daily 1 Read and log temperature of incubator s Workflow 2 Perform cell calibration if indicated with error 60 3 Identify any anonymous bottles as indicated on the screen 4 Print an Unload Positive Report and then Unload Positive BacT ALERT MB or MP culture bottles Either scan each bottle or remove bottles in a batch from the cells without scanning Work up per the laboratory protocol 5 Print an Unload Negative Report and then Unload Negative BacT ALERT MB or MP culture bottles Either scan each bottle or remove bottles in a batch from the cells without scanning 6 Prepare new samples for entry into the system following media package insert instructions and the laboratory protocol Affix the small bottle barcode label on laboratory worksheet if desired 7 Enter new data into the Data Management computer as required and Load Bottles Print load report to check for data entry errors 8 Perform manual backup if desired on the BacT ALERT 3D 9 Print or view the problem log on the Data Management computer if desired Global Customer Support K5 31JAN09 47 of 76 Mycobacterial Support Manual APPENDIX D Reagent Working Phosphate Buffer with Phenol Red Indicator Preparation Procedure for inclusion of Phenol Red Indicator in Phosphate Buffer lt Note This allows the user to visualize the pH of the pellet without actual pH paper t
5. CLSI documentation is available to describe the proper management of control organisms Working suspensions of QC organisms may be frozen in small aliquots 1 mL to 2 mL in appropriate tubes vials at 70 C 10 C The frozen suspension may be used for six months to a year Once thawed the culture suspension should not be refrozen l eLSI Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A Wayne PA Clinical and Laboratory Standards Institute 2008 pg 31 i Biomerieux Inc BacT ALERT MP Bottle Package Insert July 2008 3 Biomerieux Inc BacT ALERT MB Bottle Package Insert July 2008 Global Customer Support K5 31JAN09 74 of 76 Mycobacterial Support Manual APPENDIX J Commercial media are divided into two additional categories exempt and nonexempt which are determined by quality control performance data collected by CAP Exempt media includes commercial media documented to maintain consistent user performance with minimum variation and requires minimum quality control Provided the manufacturer can certify acceptable performance using the QC isolates listed in the table below automated AFB broths for detection of mycobacteria are considered exempt The user is instructed to refer to the package insert for specific QC information Because bioMe rieux s current Certificate of Conformance does not include all organisms listed in the table below the
6. MP Culture Bottle Procedure Mycobacterial Support Manual 1 Remove the metal seal by pulling on the center tab across the top down through the rim of the seal Avoid ripping the center tab completely away from the seal FN 2 Continue to pull the tab around the bottle to remove the seal completely from the bottle 3 Once the aluminum seal is removed the rubber septum can be lifted off the bottle with e g sterile forceps The septum may be easier to remove if pried out of the bottle on either side of the septum slit Special considerations are required to avoid contamination of the bottle Global Customer Support K5 31JANO09 33 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure 4 Aseptically inoculate the specimen into the bottle via a needle less device such as a sterile disposable pipette 5 Aseptically place the septum back into the bottle ensuring that the septum fits fully into the opening Place a reseal cap BacT ALERT Reseal P N 259787 over the septum and bottle opening The reseal must catch under the bottle rim and the underside of the reseal must be in full contact with the top of the septum The reseal should fit tightly on the bottle with a snap sound 6 This plastic reseal cap may be removed later for bottle sampling First vortex and invert bottle to mix contents Remove reseal by pulling the tab and tearing it off T FOR PLASTIC CULTURE BOTTLES gt BacT
7. When adding the neutralization buffer to stop the action of the digestion agent pour from an individual pour tube to avoid the possibility of cross contamination between specimens NOTE Ideally the volume of decontamination reagent and neutralization buffer used will not vary from tube to tube Add buffer to the 50 mL mark on the centrifuge tube to maximize buffering capability of this step Mix by inversion to ensure all digestion agent is neutralized within the sample tube To control breakthrough contamination rates after first assuring that procedure vortexing and sterile technique are adequate make small incremental increases of 0 5 in the decontamination reagent NaOH concentration Verify that a pH of 6 8 to 7 5 is maintained in the final sample pellet with each change in NaOH concentration Kantor IN Kim SJ Frieden t et al 1998 Laboratory Services in Tuberculosis Control Culture Part Ill WHO TB 98 258 p 37 Della Latta P ed Mycobacteriology and Antimycobacterial Susceptibility Testing in Isenberg HD ed Clinical Microbiology Procedures Handbook vol 2 Washington DC ASM Press 2004 sect 7 1 2 2 Global Customer Support K5 31JAN09 15 of 76 Mycobacterial Support Manual Best Practices in Sample Processing Concentration Mycobacteria are often present in clinical specimens in very small numbers therefore it is essential to concentrate by centrifugation before inoculating to cultures High cent
8. depending upon the species Growth of M tuberculosis using traditional mycobacterial media can take two to eight weeks or longer CDC Centers for Disease Control recommendations suggest that a liquid culture medium be used along with solid media for primary culture It is recommended that whenever possible liquid medium should be used to assure better and more rapid results The addition of solid medium along with a liquid medium maximizes the recovery provides an opportunity to look at colony morphology that is not possible in liquid media allows for detection of mixed mycobacterial infections and serves as a backup when liquid culture is contaminated An increase of 4 to 6 for mycobacterial isolation has been reported by the addition of an LJ slant along with a liquid medium while adding a liquid medium along with an LJ medium is reported to increase the recovery rate significantly 15 to 30 The liquid culture method is needed to provide more rapid detection growth and susceptibility results for M tuberculosis Essential components of a tuberculosis program recommendations of the Advisory Council for the Elimination of Tuberculosis MMWR 1995 44 No RR 11 13 Global Customer Support K5 31JAN09 2 of 76 Mycobacterial Support Manual Mycobacteria in our World The MB BacT and the BacT ALERT 3D Mycobacteria Detection Systems used in conjunction with the BacT ALERT MP culture bottle provide both a micro
9. important for the laboratory to monitor the overall rate of specimen contamination The goal is not to reduce this rate to zero since that would indicate too many mycobacteria are being lost in the decontamination process instead it is expected that under normal circumstances between 2 and 5 of specimens will be overgrown by normal flora 1 Kantor IN Kim SJ Frieden t et al 1998 Laboratory Services in Tuberculosis Control Culture Part Ill WHO TB 98 258 p 37 11 Della Latta P ed Mycobacteriology and Antimycobacterial Susceptibility Testing in Isenberg HD ed Clinical Microbiology Procedures Handbook vol 2 Washington DC ASM Press 2004 sect 7 1 2 2 12 CLSI Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A Wayne PA Clinical and Laboratory Standards Institute 2008 Global Customer Support K5 31JAN09 20 of 76 Mycobacterial Support Manual Best Practices in Sample Processing Global Customer Support K5 31JANO09 21 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure TECHNICAL PROCEDURE MYCOBACTERIA SPECIMEN PROCESSING SPECIMEN The BacT ALERT MP System consists of the BacT ALERT MP culture bottle with a removable closure used in conjunction with the MB BacT Antibiotic Supplement and or the MB BacT Reconstitution Fluid The BacT ALERT MP System is designed for use with the MB BacT or the BacT ALERT 3D Mycobacteria Detection
10. 5658 Fax 805 346 2760 website www hardydiagnostics com DECONTAMINATION REAGENTS FOR THE RECOVERY OF MYCOBACTERIA FROM SPUTUM STOCK ATCC QC Strains ATCC is the largest biological resource center in the CULTURES world with the most comprehensive source of reference cultures h ae The recommended Seeded Study s e Protocol strains are available 3 wa directly from ATCC The current list price may be accessed at Genuine Cultures www atcc org You may also contact Ne ATCC directly at 800 638 6597 American Type Culture Collection ATCC P O Box 1549 Manassas VA USA 20108 Tel 703 365 2700 800 638 6597 U S and Puerto Rico Canadian customers contact CEDERLANE 800 268 5058 North America M tuberculosis ATCC 27294 BTA MP culture bottle M intracellulare ATCC 13950 BTA MB amp BTA MP culture bottles M kansasii ATCC 12478 BTA MP culture bottle M fortuitum ATCC 6841 BTA MP culture bottle M avium ATCC 25291 BTA MB culture bottle Global Customer Support K5 31JAN09 64 of 76 APPENDIX H Mycobacterial Support Manual OTHER Allied Monitor 660 248 2823 REAGENTS Ferric Mycobactin J Product 62 0002 2 mg Fisher Scientific Healthcare Products 800 640 0640 e Ottowa Sand 500 gm Product MSX00701 Sigma Aldrich Chemical Company Inc 800 325 3010 e Hemin Product H9039 Bovine TISSUE STERILE DISPOSABLE TISSUE GRINDER AVAILABLE GRINDERS FRO
11. ALERT Reseal 100 units box P N 259787 4 Global Customer Support K5 31JAN09 34 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual WHAT TO DO WITH A POSITIVE BacT ALERT MP CULTURE BOTTLE Test procedure adapted from Instructions for Use 1 Load inoculated BacT ALERT MP culture bottles into the MB BacT or BacT ALERT 3D instrument following the instructions provided in the appropriate User Manual After culture bottles are loaded into the instrument they should remain there for 42 days or until designated positive When the instrument indicates a positive bottle remove the bottle according to procedures stated in the appropriate BacT ALERT User Manual NOTE To reduce sampling error and enhance smear interpretation proper mixing of a positive BacT ALERT MP culture bottle may be achieved by vortexing up to 30 seconds to break up any clumping present within the bottle This must be done in a biological safety cabinet using personal protective equipment 4 All bottles designated positive should be smeared and subcultured for acid fast bacilli Subculturing may be performed by removing the sample with a needle and syringe from bottles without a Reseal or from bottles with a Reseal a Subculturing from positive bottles without a Reseal using a needle and syringe In a biological safety cabinet mix bottle contents disinfect the bottle stopper and remove specimen fo
12. APPENDIX H Mycobacterial Support Manual Remel Inc produces NAC Attack mucolytic agent is N acetyl L cysteine and Sputagest mucolytic agent is Dithiothreitol for AFB specimen preparation a 3 NAC100 Thermo Fisher Scientific reme Y NAC So Remel Products 12076 Santa Fe Drive MAc atack P O Box 14428 Oxalic Acid Lenexa KS USA 66215 Phosphate Buffer Codium Citrate USA Phone 800 255 6730 EE International Phone 913 888 Sputagest 100 0939 Sputaqest 50 E mail IB Base Digestant Customersupport remel com TE Digestant N acetyl L cysteine NAC Attack WAC Attack REF R210250 Mucoltic Agent For digestion and decontamination procedures for AFB cultures Contents 5 sealed ampules ophilized N acetytLcysteine 5 bottles TB Base Digestant WPK 50 mifBottle MAC Attack REF R210500 Mucoltic Agent For digestion and decontamination procedures for AFB cultures Contents 5 sealed ampules hrophilzed N acet L cysteine 5 bottles TH Base Digestant WPK 100 mifBottle Cleland s Reagent Dithiothreitol sputagest 50 Sputagest 50 REF R21096 WMucoltic Agent Lyophilized rehydrates to 50 mi For digestion and decontamination procedures for AFB and fungal cultures For use with 50 ml TB Hase Digestant or Sodium Citrate 6 VialsiPk 50 milvial Global Customer Support K5 31JAN09 59 of 76 Mycobacterial Support Manual APPENDIX H SALUBRIS Inc SSS SSSSSSSsSsSsSsSsSSSSSSOUTEWAY TO HE
13. Global Customer Support K5 31JANO09 29 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure 4 Under a biological safety cabinet withdraw approximately 0 75 to 1 0 ml of the processed resuspended specimen pellet using a sterile syringe with at least 172 inch needle long enough to reach the bottom of the sample tube This avoids contaminating the specimen by using smaller more difficult to handle syringes Use 18 to 22 gauge needles to prevent sampling errors due to small bore needles Alternatively if bottle crimp and stopper are removed for needle less inoculation use one sterile transfer pipet per sample See page 34 of this manual To avoid biohazard exposure to the operator or contamination of the sample use syringes with needles long enough to reach the bottom of the tube 5 First add 0 5 ml of the sample to the appropriately labeled BacT ALERT MP culture bottle using aseptic technique 1 Sample into MP culture bottle Global Customer Support K5 31JANO09 30 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual 6 Second using the same syringe or pipet inoculate appropriate solid media 2 Sample to solid media 7 Third place sample on slides non sterile before discarding the syringe into a sharps biohazard container Invert and gently swirl bottles to mix all reagents and sample 3 Sample onto non sterile slides Global Custom
14. Manuals and appropriate media Instructions for Use bioM rieux the blue logo Bacl ALERT FAN and MB BacT are used pending and or registered trademarks of bioM rieux SA or one of its subsidiaries Amphyl is a registered trademark of Reckitt Benckiser Inc Angel Wing is a registered trademark of Covidien AG CLSI is a registered trademark of Clinical and Laboratory Standards Institute Inc ZIP is a registered trademark of lomega Corporation ATCC is a registered trademark of American Type Culture Collection REFERENCES BacT ALERT User Manual Version B 25 PN 514277 2EN1 12 2005 BacT ALERT Instructions for Use July 2008 Footnote references as cited in document Mycobacterial Support Manual Mycobacteria in our World BACKGROUND INFORMATION Mycobacteria in our World OBJECTIVES To understand the importance of Mycobacterial testing DISCUSSION Tuberculosis remains a major global public health problem The World Health Organization estimates that 9 million new cases and 2 million deaths are directly attributable to the disease each year Tuberculosis has been cited as the leading cause of death in many resource poor and developing countries Infections caused by M tuberculosis complex organisms are classically pulmonary If control of tuberculosis is not further strengthened in the future the World Health Organization estimates that between 2000 and 2020 nearly one billion people will be newly infected 200 million peop
15. Techniques and Procedures in Clinical Microbiology Washington D C American Society of Microbiology 1994 p8 Global Customer Support K5 31JAN09 14 of 76 Mycobacterial Support Manual Best Practices in Sample Processing bacilli gt The acceptable range is 3 to 5 with media without antimicrobial agents A contamination rate significantly less that 3 in the non antimicrobial agent containing media suggests overly harsh decontamination Greater than 5 growth suggests inadequate decontamination or incomplete digestion Overly harsh decontamination may damage or destroy mycobacteria in samples resulting in lower detection rates or prolonged time to detection TECHNICAL TIPS 1 Use individual pour tubes containing precise gt volumes of decontamination reagent to prevent wrod es the addition of excess reagent that will require pH CONN neutralization later and to avoid the possibility of cross contamination between specimens Vortex for a minimum of 20 seconds up to a maximum of 30 seconds Use a timer Make sure to invert the tube a few times during the vortexing process to insure decontamination of all surfaces of the specimen tube Extremely mucoid or bloody specimens may require additional NALC powder for complete digestion if you are using the NALC NaOH method Observe timing of decontamination step Longer exposure times cause more destruction of the mycobacteria Do not process too many samples at one time gt 20
16. between samples gt Use of individual pour tubes for reagents being added to test gt Use of syringes or pipettes long enough to reach bottom of tube without touching sides of tube when sampling pellet gt Careful recapping of centrifuge tube to include eNever having more than one tube top open at any given time eNever allowing a tube cap to be placed sample side down on working surfaces Global Customer Support K5 31JAN09 71 of 76 Mycobacterial Support Manual Appendix Q and A by Chapter Chapter 6 BacT ALERT MB Culture Bottle Technical Procedure 1 What are three acceptable inoculation methods for the BacT ALERT MB culture bottle 1 Collect the blood using a butterfly set and the BacT ALERT Blood Collection Adapter Cap and inoculate directly into the BacT ALERT MB culture bottle at the patient s bedside 3 5 ml per bottle 2 Blood may be collected in a sterile SPS or heparinized tube and inoculated into the BacT ALERT MB culture bottle in the laboratory 3 Syringe draw 3 ml 5 ml of blood without anticoagulant from the patient and inoculate directly into the blood culture bottle 2 What is the purpose of adding the MB BacT Enrichment Fluid MB BacT Enrichment Fluid is provided separately and must be added to the blood culture bottle for growth of mycobacteria The enrichment fluid includes a lytic agent Saponin Sodium polyanetholesulfonate SPS and other media supplements which eliminate the processing step pr
17. in BACT ALERT MB culture bottles Product description Product number Black cap 29 ml Black 259877 5 5 ml 210361 210362 Case Size and Storage 25 bottles per Case Room Temperature 15 30 C in dark 25 tests per kit Room Temperature 15 30 C in dark 120 case 60 case Room Temp Media Contents 1 0 ml into MB culture bottle Middlebrook 7H9 broth Pancreatic Digest of Casein Glycerol SPS in purified water with an atmosphere of COs nitrogen and oxygen under vacuum Bovine Serum Albumin Sodium Chloride Oleic Acid Saponin in purified water Enrichment fluid must be added to the MB bottle within a 24 hour window of inoculation of specimen Kit contains 5 vials Cap for use with butterfly collection set for direct draw of blood Insert allows blood to be filled into vacuum collection tubes NOTE Prior to use the BacT ALERT MB culture bottles should be examined for evidence of damage or deterioration discoloration Bottles exhibiting evidence of damage leakage or deterioration should be discarded The media in undisturbed bottles should be clear Do not use a bottle containing media that exhibits turbidity a yellow sensor or excess gas pressure these are signs of possible contamination Also inspect all MB BacT Enrichment Fluid vials for evidence of damage or contamination Do not use MB BacT Enrichment Fluid vials exhibiting turbidity Global C
18. in purified water Bottles contain an atmosphere of carbon dioxide in oxygen and nitrogen under vacuum The composition of the media may be adjusted to meet specific performance requirements MB BacT Enrichment Fluid MB BacT Enrichment Fluid vials contain a total fill volume of 5 5 ml each MB BacT Enrichment Fluid consists of Bovine Serum Albumin 14 5 w v Sodium Chloride 2 5 w v Oleic Acid 0 174 w v Saponin 4 4 w v in purified water Additional materials required MB BacT Mycobacteria Detection System or BacT ALERT 3D Microbial Detection System Middlebrook 7H11 or other mycobacterial agar or egg base medium CO incubator 37 C 2 C Sterile syringes and needles Mycobactericidal disinfectant Blood drawing apparatus Biological safety cabinet Sterile disposable gloves Disposable gowns Disposable masks Appropriate biohazard waste containers for materials contaminated with infectious agents Quality Control organisms Global Customer Support K5 31JAN09 38 of 76 BacT ALERT MB Culture Bottle Procedure Mycobacterial Support Manual SPECIMEN COLLECTION AND PREPARATION Correct specimen collection is extremely important when obtaining blood culture specimens Refer to Manual of Clinical Microbiology for proper specimen collection and transport procedures Proper skin disinfection is an essential requirement to reduce the incidence of contamination Blood collected in EDTA and coagulated blood are not acce
19. intake E may have biologically contaminated positive pressure plenum E fO alr recirculated 30 exhausted from a common plenum to the room Class ll Type mE 100FF intake AJBS E biologically contaminated plenum Under negative pressure or surrounded by negative pressure A mE 0 air recirculated 30 exhausted from a common plenum to a facility exhaust system mE OUFPM intake E clase U Type B2 E biologically contaminated plenum under negative pressure or surrounded by negative pressure E 40 air recirculated 60 exhausted from cabinet E exhaust air pulled through dedicated exhaust duct into facility exhaust system B1 Class Il Type B1 mE 100FPM intake E all biological contaminated plenums are negative to the room or surrounded by negative pressure plenums Global Customer Support K5 31JAN09 52 of 76 Mycobacterial Support Manual APPENDIX E 0 air recirculated 100 exhausted from cabinet exhaust air pulled through dedicated exhaust duct into facility exhaust system 100F FM intake Class ll Type B2 all ducts and plenums are under negative pressure all contaminated ducts are under negative pressure or surrounded by directhy exhausted negative pressure ducts or plenums References Biosafety in Microbiological and Biomedical Laboratories U S Department of Health and Human Services Public Health Service Centers for Disease Control and Prevention and National Institutes of Health Fift
20. into the sample tube 7 Re suspend the pellet in 1 2 ml of sterile phosphate buffer using individual sterile transfer pipettes to deliver the volume of buffer Recap tube and mix pellet by vortexing Use individual sterile disposable transfer pipettes Global Customer Support K5 31JANO09 26 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual bX Note It is recommended that you periodically check the final pH of the pellet with pH paper The pH should be neutral between 6 8 and 7 5 A pH above 7 5 could be detrimental to the mycobacteria or possibly delay the time to detection A pH lower than 6 8 may cause false positives Alternately you may use reagents with phenol red added to the buffer so that neutral pH may be visualized when the resuspension buffer is added to the pellet after decantation ALPHACTEC NAC PAC Kee 5 AN INOCULATION OF BOTTLES SLIDES AND SOLID MEDIA Use sterile technique during the following steps to avoid contaminating the culture bottles MAS and reconstitution fluid with environmental bacteria The BacT ALERT MP culture bottles MAS and reconstitution fluid should be at room temperature prior to mixing and inoculation lt Note Use individual disposable alcohol prep pads to clean the tops of all reagent or culture bottles before each entry Global Customer Support K5 31JANO09 27 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Pr
21. mucogenicum for example has been cultured from almost half of the samples of drinking water and ice investigated in the United States fewer than 5 of the clinical isolations are considered medically significant Likewise M gordonae rarely is associated with clinical disease and then almost exclusively in the setting of severe Global tuberculosis control surveillance planning financing WHO report 2007 Geneva World Health Organization WHO HTM TB 2007 376 Pfyffer GE Brown Elliott BA and Wallace RJ Jr Mycobacterium in Murray PR Baron EJ Pfaller MA et al eds Manual of Clinical Microbiology ed 8 Washington DC American Society for Microbiology 2003 pp 532 559 Roberts GD Koneman EW Kim YK Mycobacterium in Balows A Hausler WJ Jr Herrmann KL et al eds Manual of Clinical Microbiology ed 5 Washington DC American Society for Microbiology 1991 pp 304 339 CLSI Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A Wayne PA Clinical and Laboratory Standards Institute 2008 Global Customer Support K5 31JAN09 1 of 76 Mycobacteria in our World Mycobacterial Support Manual immunosuppression Many new species including M botniense M cookin M chlorophenolicum M frederiksbergense M hodleri and M murale have recently been identified from environmental samples but not yet identified as human pathogens M celatum is another organism frequently isolated i
22. mycobacterial blood bottle algorithms These algorithms employ bacterial and mycobacterial algorithms for the first 4 days of incubation and thereafter use the very sensitive mycobacterial algorithm only In addition to detecting rapid growers the bacterial algorithms detect any breakthrough bacterial contamination during the first four days of incubation Each sample is monitored independently with its own established baseline bioM rieux recommends a test protocol of 42 days for all mycobacterial bottles TRAINING SESSION Theory of Operation Organisms grow and produce CO gt 2 The bottle sensor lightens in color Reflectance is measured and analyzed A graph of the growth curve is produced AUN Mycobacterium tuberculosis Global Customer Support K5 31JANO09 6 of 76 Mycobacterial Support Manual Theory of Operation EXERCISE SESSION See Appendix I for answers 1 What metabolites were chosen to enhance CO2 production during Mycobacterial metabolism 2 What technology is used to monitor the production of CO2 3 Why does the sensor change colors from dark to light TIPS to REMEMBER CO is produced by growing organisms and results in a lowered pH The sensor changes color to lighter green or yellow A representative graph for most bacteria reflects a typical growth curve with a lag phase a log phase and a stationary phase of growth Mathematical algorithms in the software a
23. production affects the bottle sensor use of MB PROCESS and MB BLOOD algorithms Describe the bottle preparation procedure for the BacT ALERT MP culture bottle culture bottle and MB BacT Antibiotic Supplement Kit storage conditions reconstitution of the MB BacT Antibiotic Supplement sample size 0 5 ml crimp removal and use of reseal cap for needle less inoculation loading BacT ALERT MP culture bottles only into BacT ALERT MB drawers racks Describe specimen NALC NaOH decontamination digestion procedure contaminated specimen transport storage and preparation sterile specimen preparation bottle and reagent inoculation procedures describe best practices regarding specimen processing set up digestion decontamination of samples working or final NaOH concentration should be at least 2 use timer for incubation and vortexing times final pellet pH should be neutral 6 8 to 7 5 quality control of decontamination method technique related sources of contamination false positives and cross contamination mycobacteria requiring special growth requirements additives or temperatures causes of delayed detection times false negatives Describe the bottle preparation procedure for the BacT ALERT MB culture bottle storage conditions room temperature bottle preparation and addition of MB BacT Enrichment Fluid describe 3 methods of bottle inoculation sample size and fill volume is 3 to 5 ml how to handle bacterial growth in MB bottle never load anonym
24. some point prior to discarding as negative 7 Do not reuse BacT ALERT culture bottles Dispose of inoculated BacT ALERT culture bottles according to your laboratory protocol Autoclaving and or incinerating inoculated BacT ALERT bottles is appropriate i Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition U S Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health Fifth Edition US Government Printing Office Washington Feb 2007 Global Customer Support K5 31JAN09 41 of 76 Mycobacterial Support Manual BacT ALERT MB Culture Bottle Procedure EXERCISE SESSION See Appendix I for answers 1 What are three acceptable inoculation methods for the BacT ALERT MB culture bottle 2 What is the purpose of adding the MB BacT Enrichment Fluid 3 How long before inoculation or after inoculation may the MB BacT Enrichment Fluid be added TIPS to REMEMBER You must add 1 0 ml of the MB BacT Enrichment fluid to each BacT ALERT MB Culture bottle because it contains saponin to lyse the cells and growth factors necessary for Mycobacterial growth Global Customer Support K5 31JAN09 42 of 76 Mycobacterial Support Manual Appendix A Mycobacteria Support Manual Objectives After completion of this support manual the participant should be able to Describe theory and operation of the BacT ALERT 3D or MB BacT System how CO
25. 010 Becton Dickinson and Company www BD com e BD SAFETY LOK Syringes Global Customer Support K5 31JANO09 66 of 76 APPENDIX H Mycobacterial Support Manual e BD SAFETY LOK Syringes cont 309593 3mLBD Safety Lok syringe with 22 G x 1 1 2 in detachable needle regular bevel regular wall 309595 3 mL BD Safety Lok syringe with 21 G x 1 1 2 in detachable needle regular bevel regular wall BD Integra Syringe with Retracting BD PrecisionGlide Needle 305272 22Gx1 vin 305274 21Gx1 in QC SLIDES p W pm i ia N n 7 al p i a ka Ga KWIK QC Slides provide the performance of QC and sample staining in a single step Each slide has a positive and negative inoculated well and space for concurrent staining of culture isolates or clinical samples KVWIK OC Mycobacterium Slide SL43 10 Global Customer Support K5 31JANO09 67 of 76 Mycobacterial Support Manual APPENDIX H NEBHACTEC AFB QUALITY CONTROL SLIDES AFB Stain Control Slides Each slide contains an 4 7 6 positive staining control of Mycobacteniwim scoroiiac um and an 4 7 8 negative staining control of Escherichia cov AFE Stain Control Slides 10 slides pk 032 43 40 slides pk 032 40 Global Customer Support K5 31JANO09 68 of 76 APPENDIX H Mycobacterial Support Manual Global Customer Support K5 31JANO09 69 of 76 Mycobacterial Support Manual Appendix Q and A by Chapter Quest
26. 5 mL of MAS Mycobacterial Antibiotic Supplement to each BacT ALERT MP culture bottle Add 0 5 mL of MB BacT Reconstitution Fluid alone to the culture bottles for sterile body fluids Invert to mix MAS sample and broth Note Increasing the amount of MAS added to the bottle will increase false positives and or may increase time to detection of mycobacteria At neutral pH 0 5 mL of MAS should control most breakthrough contamination in properly digested decontaminated samples Re suspend lyophilized MAS with precisely 10 mL of reconstitution fluid When using syringes for obtaining sample from the tube for inoculation into the bottles use needles long enough to reach the bottom of the tube This aids in avoiding contamination and cross contamination of samples from gloved hands Do not use tuberculin syringes and needles for example because the needles are too short to reach the bottom of the tube Use larger bore needles to avoid sampling errors 18 22 gauge needles are OK while 23 gauge may be too small Decant supernatant immediately after centrifugation to avoid excessive contact time at alkaline pH and minimize the possibility of the pellet becoming loose Inoculate concentrates to media as soon as possible never leave overnight Although buffered the final concentrate may be slightly alkaline which will destroy mycobacteria in time The media in the BacT ALERT culture bottle serves to further neutralize the inoculum
27. ALTH WORLDWIDES MYCOPROSAFE MYCOPROSAFE MycoProSafe MPS is a Sample decontamination and concentration kit that permits the processing of samples for microscopy culture and molecular methods for identification and isolation of mycobacteria It is intended for in vitro diagnostic use MPS provides in individual sets all the materials needed for processing each sample It eliminates the problem of cross contamination while saving time and effort Stable pH 6 8 7 0 Cat MPS120 is available with 2 NaOH Headquarters and US office Baldwin Park 12 Alfred Street Suite 300 Woburn Massachusetts 01801 U S A Phone Toll Free 800 326 1484 Fax 617 249 0803 Europe 16 avenue de Beauval 92380 Garches France Phone 33 1 47 41 28 88 E mail info salubrisinc com Global Customer Support K5 31JANO09 60 of 76 APPENDIX H Mycobacterial Support Manual SDL Snap N Digest NALC is supplied sealed in a glass ampule within a plastic bottle of NaOH Sodium Citrate solution Squeeze the bottle to break the ampule when ready to use Reagent stable for 24 hours after ampule broken Product available in two sizes Kits also include packets of powder to make up sterile Phosphate Buffer pH 6 8 Cat No 667 Snap N Digest 75 ml 10 bottles Cat No 668 Snap N Digest 150 ml 10 bottles Cat No 6671 Phosphate Buffer powder 5 packages of 4 2 gm each Cat 669 100 Sterile TB Buffer pre mad
28. BIOM EIRIE UU X Mycobacterial Testing With BacT ALERT Systems and Media LCR 09 0112 PN 60 00467 0 Global Customer Support GCS K5 GCS TM 0383 31JANO9 Summar BIOMELRIEU xX MYCOBACTERIAL TESTING WITH BACT ALERT SYSTEMS AND MEDIA TABLE OF CONTENTS Chapter 1 Mycobacteria in our World Chapter 2 Theory of Operation Chapter 3 Media Overview Chapter 4 Best Practices in Sample Processing Chapter 5 MP Technical Procedure Chapter 6 MB Technical Procedure Appendix A Objectives Checklist Appendix B Additional References Appendix C Suggested Daily Workflow Appendix D Phosphate Buffer with Phenol Red Appendix E Biosafety Levels Appendix F Nomogram for proper centrifugation Appendix G Clinical Study and Performance Data Appendix H Miscellaneous Reagents Appendix Questions and Answers by Chapter Appendix J Quality Control Testing Appendix K How to Prepare Proper Quality Control Dilutions PURPOSE This manual contains theory troubleshooting Best Practices and tips to remember It is intended for use as a Support and instructional guide for customers and bioMerieux Applications Specialists in performing mycobacterial testing with BacT ALERT Systems and Media After completing this manual one should be competent in basic theory of sample processing and daily workflow of the BacT ALERT MP and MB culture bottles This manual should be used in conjuction with the referenced User
29. BacT ALERT MB culture bottle top with an individual disposable alcohol prep pad Allow to air dry 2 Using aseptic technique draw 3 ml 5 ml of blood without anticoagulant from the patient and inoculate directly into the culture bottle 3 After inoculation swab bottle tops with gauze soaked in 2 Amphyl or other tuberculocidal agent and allow to air dry CAUTION Cleaning agent must be compatible with polycarbonate plastic Global Customer Support K5 31JAN09 40 of 76 BacT ALERT MB Culture Bottle Procedure Mycobacterial Support Manual WHAT TO DO WITH A POSITIVE BacT ALERT MB CULTURE BOTTLE Adapted from the BacT ALERT MB Culture Bottle Instructions for Use CAUTION General caution should be taken when subculturing positive culture bottles as they could have been overfilled or contain high gas producing organisms Positive culture bottles contents may be under increased internal pressure Positive culture bottles should be transiently vented before staining or disposal to release any gas produced during microbial metabolism 1 Load inoculated BacT ALERT MB culture bottles into the MB BacT Mycobacteria Detection System non shaking or the BacT ALERT Microbial Detection System shaking following the instructions provided in the User Manual 2 After BacT ALERT MB culture bottles are loaded into the instrument they should remain there for at least 42 days or until designated positive The following procedures
30. Before removing bottles from the biological Safety Cabinet clean the tops of the bottles with a tuberculocidal agent CAUTION Be sure this agent is compatible with the polycarbonate BacT ALERT culture bottles Global Customer Support K5 31JAN09 18 of 76 Mycobacterial Support Manual Best Practices in Sample Processing Although N Acetyl L Cysteine Sodium Hydroxide is the digestion decontamination reagent recommended by bioM rieux there are many digestion decontamination methods in use for Mycobacterial testing around the world Most of these methods are successfully employed by BacT ALERT customers However Zephiran trisodium phosphate and _Cetylpyridinium chloride possess mycobacteriostatic properties and should not be used with any liquid broth system because the chemicals cannot be eliminated by washing or by buffering steps 2 Regardless of the method used strict adherence to the recommended procedure assures consistent and accurate results Regardless of the method used the most critical steps are Decant liquid completely after centrifugation Re suspend the pellet in sterile 0 067M phosphate buffer DH 6 8 Establish a processing method that consistently results in a sample with a neutral pH Use no more than the recommended 0 5 mL MAS per bottle These steps in combination with the other technical tips listed in this document will help to standardize the specimen processing procedure across samples provide be
31. H should be between 6 8 and 7 5 Global Customer Support K5 31JAN09 48 of 76 Mycobacterial Support Manual APPENDIX E Laboratory Biosafety Level Criteria The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Table 1 of this section and discussed in Section 2 The levels are designated in ascending order by degree of protection provided to personnel the environment and the community Standard microbiological practices are common to all laboratories Special microbiological practices enhance worker safety environmental protection and address the risk of handling agents requiring increasing levels of containment Biosafety Level 1 is suitable for work involving well characterized agents not known to consistently cause disease in immunocompetent adult humans and present minimal potential hazard to laboratory personnel and the environment BSL 1 laboratories are not necessarily separated from the general traffic patterns in the building Work is typically conducted on open bench tops using standard microbiological practices Special containment equipment or facility design is not required but may be used as determined by appropriate risk assessment Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science Biosafety Lev
32. M MANY SOURCES tyca Heming Is Kendali Kendall PRECISION Disposable Tissue Grinder Systems Features a Ready to use in sterile pouch Completely disposable after grinding no clean up or sterilization required Specimen can be cooled during grinding a srinder tube doubles as a specimen container for transport and storage a Protective sleeve helps to contain any aerosolization created during grinding issue can be ground quickly due to fresh grinding surface Grinders pictured here available from LifeLine Medical Inc 22 Shelter Rock Lane Danbury CT 06810 800 452 4566 Toll Free 203 748 3806 Tel DIRECT BacT ALERT Blood Collection Adapter for BTA MB culture bottle DRAW BLOOD ADAPTOR cap 210361 insert 210362 Global Customer Support K5 31JANO09 65 of 76 Mycobacterial Support Manual APPENDIX H Kendall Healthcare 1 800 962 9888 www kendallhg com e Angel Wing Blood Collection Device Dome holder with Male Adapter and insert for BacT ALERT blood culture bottles Product 8881225245 e Angel Wing Blood Transfer Device Tube holder with Female Adapter and insert for BacT ALERT blood culture bottles Product 8881225241 SAFETY Vanishpoint Safety Syringes SYRINGES vy VANISH POmTNELt 3ml VanishPoint Syringe Retractable Technologies Inc 511 Lobo Lane P O Box 9 Little Elm TX 75068 0009 Toll free 888 806 2626 Phone 972 294 1
33. MB culture bottles may incubate in shaking or non shaking cabinets and drawers However these bottles may NEVER be loaded anonymously Scanning the bottle barcode directs the software to assign a specific mycobacteria blood bottle algorithm to analyze the bottle readings in which the Delta portion of the mycobacterial algorithm is disabled Global Customer Support K5 31JAN09 12 of 76 Mycobacterial Support Manual Best Practices in Sample Processing INTRODUCTION TO PROCESSING PROCEDURAL STEPS AND BEST PRACTICES OBJECTIVES To understand the processing procedure and to know how to obtain the best sample possible for patient testing DISCUSSION BACT ALERT MP CULTURE BOTTLE PROCEDURE The BacT ALERT MP System includes the BacT ALERT MP culture bottles and the MB BacT Antibiotic Supplement Kit MAS The MB BacT Antibiotic Supplement Kit includes MB BacT Reconstitution Fluid and MB BacT Antibiotic Supplement The reconstituted antibiotic supplement is intended to reduce the incidence of breakthrough contamination due to bacteria that may survive the decontamination digestion process The MB BacT Reconstitution Fluid contains components necessary to ensure optimal growth of mycobacteria present in the patient sample and must be added to all bottles to ensure growth of mycobacteria The red color of the reagent allows the user to know that this reagent has been added to the BacT ALERT MP culture bottle Inoculation of soli
34. Systems for recovery and detection of mycobacteria from sterile body specimens other than blood and from digested decontaminated clinical specimens REAGENTS BacT ALERT MP color coded red BacT ALERT MP disposable culture bottles with a removable closure contain 10 ml of media and an internal sensor that detects carbon dioxide as an indicator of microbial growth The BacT ALERT MP culture bottle may be inoculated via a sterile syringe with either an attached locking needle or a sheathed needle or by removing the closure and using a needleless inoculation device The media formulation consists of Middlebrook 7H9 Broth 0 47 w v Pancreatic Digest of Casein 0 1 w v Bovine Serum Albumin 1 0 w v Catalase 48 u ml in purified water Bottles contain 10 ml of media and are prepared with an atmosphere of carbon dioxide nitrogen and oxygen under vacuum The composition of the media may be adjusted to meet specific performance requirements MB BacT Antibiotic Supplement Lyophilized supplement formulated to contain Amphotericin B 0 0180 w v Azlocillin 0 0034 w v Nalidixic Acid 0 0400 w v Polymyxin B 10 000 units Trimethoprim 0 00105 w v Vancomycin 0 0005 w v and a bulking agent prior to processing The composition of the supplement may be adjusted to meet specific performance requirements Reconstitute with 10 ml of MB BacT Reconstitution Fluid NOTES e One vial of reconstituted MB BacT Antibioti
35. T ALERT MB culture bottles are in one case 25 bottles 2 What are the storage conditions for the MB Blood bottles and the MB BacT Enrichment Fluid Room Temperature 15 30 C in dark 3 How many bottles can be prepared with one box of MB BacT Enrichment Fluid 25 bottles Chapter 4 Best Practices in Sample Processing Chapter 5 BacT ALERT MP Culture Bottle Technical Procedure The following techniques enhance Time to Detection by increasing the numbers of viable mycobacteria recovered from a sample gt Proper centrifugation at 3000 xg Use the nomogram gt Immediate decantation after centrifugation gt Resuspension of centrifuged pellet with lt 2 ml of phosphate buffer gt Final pH of pellet between 6 8 and 7 5 gt Use of ONLY 0 5 mL of MAS gt DO NOT attempt to concentrate MAS by using lt 10 mL of reconstitution fluid The following techniques help to control breakthrough contamination gt Transport specimens to lab promptly and refrigerate immediately gt Use of proper sample volume gt Use of NACL NaOH working concentration 2 processing reagent gt Complete vortexing 20 30 seconds with inversion and use of a timer gt Incubating 15 minutes at room temperature with use of a timer gt Inoculation of tests in correct order MP culture bottle solid media slide gt DO NOT attempt to concentrate MAS by using lt 10 mL of reconstitution fluid The following techniques help prevent crossover contamination
36. acteria spp other than M tuberculosis complex Clinical specimens may also contain M tuberculosis and care must be exercised to ensure the correct identification of cultures Special caution should be exercised in handling M ulcerans to avoid skin exposure NSF International The National Sanitation Foundation conducts tests on biological safety cabinets to ensure the products meet minimum standards for cabinet classifications devised by NSF 7 NSF Standards are reviewed every 5 years n Tests are conducted on cabinets submitted to NSF by the manufacturers 5 Products which meet these standards are certified by NSF z Tests on cabinets are repeated every 5 years New Standard for Biological Safety Cabinets NSF ANSI National Sanitation Foundation American National Standards Institute NSF ANSI 49 2002 pertains to all models of Class II cabinets Type A1 A2 B1 B2 and provides a series of specifications regarding e Design construction e Performance e Installation recommendations e Recommended microbiological decontamination procedure e References and specifications pertinent to Class II Biosafety Cabinetry Global Customer Support K5 31JAN09 51 of 76 APPENDIX E Mycobacterial Support Manual Class Ill Cabinet Types New NSF Classification Adopted 2002 Previous NSF Classification General Description E 70 air recirculated 30 exhausted from a common plenum to the room Ad Class ll Type A E YSFPM
37. agents requiring BSL 4 containment must be handled at this level until sufficient data are obtained either to confirm continued work at this level or re designate the level Laboratory staff must have specific and thorough training in handling extremely hazardous infectious agents Laboratory staff must understand the primary and secondary containment functions of standard and special practices containment equipment and laboratory design characteristics All laboratory staff and supervisors must be competent in handling agents and procedures requiring BSL 4 containment Access to the laboratory is controlled by the laboratory supervisor in accordance with institutional policies Global Customer Support K5 31JAN09 49 of 76 APPENDIX E Mycobacterial Support Manual Not known to consistently cause discases in health adults Agents associated with human disease Routes of transmission include percutaneous injury ingestion mucous membrane exposure Indigenous or exobe agents with potential for aerosol transmission Disease may have serious of lethal consequences Standard Microbiological Practices TABLE I PRACTICES BSL 1 practice plus Limited access Biohazard warming signs Sharps precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies BSL 2 practice plus Controlled access Decontamination of all waste Decontamination of laboratory clothing befo
38. ain a new specimen for culture Signal negative cultures at the maximum test time should be visually examined for turbidity If contents of the bottle are turbid aseptically obtain a sample for acid fast staining and subculture according to your laboratory s protocol Bottles not exhibiting turbidity may be discarded Decontaminate all bottles prior to disposal Global Customer Support K5 31JAN09 35 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure EXERCISE SESSION 1 What specimens may be added to the BacT ALERT MP culture bottle 2 What is the bioM rieux recommended processing decontaminating reagent 3 What is the optimal pH of the centrifuged pellet prior to adding to the BacT ALERT MP Culture Bottle 4 What is the recommended time to test for BacT ALERT MP culture bottles TIPS to REMEMBER 1 The following techniques enhance Time to Detection by increasing the numbers of viable mycobacteria recovered from a sample Proper centrifugation at 3000 xg Immediate decantation after centrifugation Resuspension of centrifuged pellet with lt 2 ml of phosphate buffer Final pH of pellet between 6 8 and 7 5 Use of ONLY 0 5 mL of MAS 0AO0O0 2 The following techniques help to control breakthrough contamination Use of proper sample volume Use of NACL NaOH working concentration 2 processing reagent Complete vortexing 20 30 seconds with inversion and use of a timer Incubating 15 minute
39. al to remaning 45 mi of TE Base Digestant Pelle Use as needed for up io 24 Manual NALC NaOH Method Weigh 29 g of sodium citrate Add to l Iter water This Is Suton A Weigh 40 q of NaOH pallets Rapidly add to water keeping Water CoN to 1 iter This E salution E Combine equal volumes of sakin A and B Senize 121 C for 15 min ThE E NaOH Sodium cfrate sahin Dwd and store in appropriate warking volumes JUST BERORE LSE wean MALZ to Gesined armour Add MALZ to NaH sodium working volume ti Mix z 11 e eded Tor up bo 24 Maurs 62 of 76 APPENDIX H Mycobacterial Support Manual NALC is supplied in an ampule within a plastic bottle of NaOQH Sodium Citrate solution Squeeze the bottle to break the NALC ampule Use within 24 hours One packet of Phosphate Buffer makes 500 ml Product is available in two sizes Product Unit Catalog No BEL MycoPrep Specimen 10 75 mL bottles of NALC NaQH Solution and 240862 Digestion Decontamination Kit 5 packages of Phosphate Buffer ipH 6 8 10 150 77L bottles of NALT NaOH Solution and 240863 10 packages of Phosphate Buffer pH 6 8 Becton Dickinson 1 Becton Drive Franklin Lakes NJ USA 07417 201 847 6800 www BD com Global Customer Support K5 31JANO09 63 of 76 Mycobacterial Support Manual APPENDIX H Hardy Diagnostics DIAGNOSTICS HARDY DIAGNOSTICS 1430 W McCoy Lane Santa Maria CA 93455 Phone 805 346 2766 ext
40. al Theory of Operation THEORY OF OPERATION OBJECTIVES To understand how the BacT ALERT Systems operate DISCUSSION Theory of Operation The MB BacT Classic and BacT ALERT 3D Mycobacteria Detection System are totally automated test systems for incubating and monitoring culture bottles for microbial growth Both systems use the same technology to read and determine positive and negative cultures The system consists of incubators data management computers growth media bottles and reagents Mycobacteria behave like most other bacteria with respect to carbohydrate metabolism energy production and the biosynthesis of low weight metabolites Glycerol and oleic acid were selected as primary carbon sources in the BacT ALERT MP and BacT ALERT MB culture bottles because of their ability to maximize the amount of carbon dioxide CO2 generated by mycobacteria Once ingested glycerol and oleic acid are converted to Acetyl CoA and are oxidized through the Krebs or Tricarboxylic Acid Cycle TCA Carbon Dioxide CO2 and free electrons are the major metabolic byproducts of oxidation Although mycobacteria divide slowly the level of CO2 generated is similar to the levels seen in most common bacteria The MB BacT and BacT ALERT 3D Mycobacteria Detection Systems utilize a colorimetric sensor and reflected light to monitor the amount of COz dissolved in the culture medium If microorganisms are present in the test sample they produce CO
41. aminated on exit from facility Agent Summary Statements Agent Mycobacterium tuberculosis complex The Mycobacterium tuberculosis complex includes M tuberculosis M bovis M africanum and M microti that cause tuberculosis in humans and more recently recognized M caprae and M pinnipedii that have been isolated from animals M tuberculosis grows slowly requiring three weeks for formation of colonies on solid media The organism has a thick lipid rich cell wall that renders bacilli resistant to harsh treatments including alkali and detergents and allows them to stain acid fast Containment Recommendations BSL 2 practices and procedures containment equipment and facilities are required for non aerosol producing manipulations of clinical specimens such as preparation of acid fast smears All aerosol generating activities must be conducted in a BSC BSL 3 practices containment equipment and facilities are required for laboratory activities in the propagation and manipulation of cultures of any of the subspecies of the M tuberculosis complex Agent Mycobacterium spp other than M tuberculosis complex and M Leprae More than 100 species of mycobacteria are recognized These include both slowly growing and rapidly growing species In the past mycobacterial isolates that were not identified as M tuberculosis complex were often called atypical mycobacteria but these are now more commonly referred to as nontuberculous myc
42. and should be kept on file QC should be carried out on culture media and reagents Quality of laboratory procedures may also be evaluated by establishing normal averages of different parameters of results and then monitoring these parameters on specified time intervals Laboratory prepared media should be thoroughly quality controlled Inoculate 1 to 3 of each batch of newly prepared medium with a standardized suspension of at least three mycobacterial species see below Identify a previously prepared good lot of medium or a commercially prepared medium as a positive control Using this medium establish criteria for optimal growth to be obtained with a standardized inoculum and incubation period The number and size of colonies within a specified time of incubation should be critically evaluated If a newly prepared batch lot of medium when tested yields results below the established range it should be considered unsatisfactory colony counts 20 above or below the established range may be considered as acceptable or laboratories may establish their own criteria If the performance of the test medium is better than the control medium this medium should be saved for the future QC control medium only after confirmation of its performance with repeat testing This QC testing should be carried out with each newly prepared batch of medium For commercially prepared media follow the recommended procedures and criteria of performance The followi
43. bial detection system and a culture media with suitable nutritional and environmental conditions to recover mycobacterial species commonly isolated from patient specimens other than blood The MB BacT Antibiotic Supplement is intended to reduce the incidence of break through contamination due to bacteria that may survive the decontamination concentration process MB BacT Antibiotic Supplement must be added to BacT ALERT MP culture bottles prior to inoculation of all non sterile specimens For recovery of mycobacteria present in sterile specimens only the MB BacT Reconstitution Fluid should be added to the BacT ALERT MP culture bottles MB BacT Reconstitution Fluid contains components that are necessary to ensure optimal growth of mycobacteria present in the patient sample Inoculated bottles are placed into the instrument where they are incubated and continuously monitored for the presence of mycobacteria that will grow in the BacT ALERT MP culture bottle At the time of detection approximate colony forming units CFUs per ml are 10 10 The MB BacT and the BacT ALERT 3D Mycobacteria Detection Systems used in conjunction with the BacT ALERT MB culture bottle provides both a microbial detection system and a culture media with suitable nutritional and environmental conditions to recover mycobacterial species commonly isolated from patient blood MB BacT Enrichment Fluid must be added to BacT ALERT MB culture bottles withi
44. c Supplement is sufficient for 20 BacT ALERT MP culture bottles e Once reconstituted the MB BacT Antibiotic Supplement has a shelf life of 7 days when stored at 2 8 C The expiration date for the reconstituted supplement should be recorded on the vial label e Remaining MB BacT Reconstitution Fluid may be stored at 2 8 C for later use with sterile specimens MB BacT Reconstitution Fluid Oleic acid 0 05 w v Glycerol 5 w v Amaranth 0 004 w v and Bovine Serum Albumin 1 w v in purified water Each bottle contains a total fill volume of 15 ml The composition of the reconstitution fluid may be adjusted to meet specific performance requirements EQUIPMENT AND MATERIALS Additional materials required Sterile distilled or deionized water Middlebrook 7H11 or other mycobacterial agar or egg base medium Autoclave or other safe disposal method N Acetyl L Cysteine powder decontamination reagents Sterile 0 067 M phosphate buffer pH 6 8 Centrifuge CO Incubator 37 C Global Customer Support K5 31JAN09 22 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual Sterile syringes with either attached locking needles or sheathed needles Sterile pipettes or other sterile needleless inoculation device for bottles with removed closures Mycobactericidal disinfectant Alcohol swabs Vortex mixer Sterile 50 ml conical polypropylene centrifuge tubes Biological safety cabinet Sterile disposabl
45. d Procedure DISCUSSION BacT ALERT MB culture bottles with the addition of MB BacT Enrichment Fluid when used with the MB BacT Mycobacteria Detection System non shaking and the BacT ALERT Microbial Detection System shaking is a non selective culture medium for the qualitative culture and recovery of mycobacteria from blood specimens BacT ALERT MB culture bottles in combination with the MB BacT Enrichment Fluid are designed for the cultivation of Mycobacterium sp commonly isolated from blood The medium will support growth of other aerobic organisms including yeast fungi and bacteria This complete system includes a lytic agent Saponin Sodium polyanetholesulfonate SPS and other media supplements which eliminate the processing step prevent clotting of blood and enhance the growth of mycobacteria A 3 5 ml volume of blood can be inoculated directly into the BacT ALERT MB culture bottle Inoculated bottles are placed into the instrument where they are incubated 35 C 37 C and continuously monitored for microbial growth REAGENTS BacT ALERT MB BacT ALERT MB color coded black BacT ALERT MB sterile disposable culture bottles contain 29 ml of media and an internal sensor that detects carbon dioxide as an indicator of microbial growth The media formulation consists of Middlebrook 7H9 Broth 0 47 w v Pancreatic Digest of Casein 0 1 w v Glycerol 1 0 w v Sodium polyanetholesulfonate 0 025 w v
46. d for single bottles atf 1 6and in triplicate at 10 Data listed represent a range of values from multiple strains except for M simiae M gordonae M malmoense and M chelonge where data for individual organism is given 2 One of three bottles showed no growth after 42 days value given is average of two bottles One organism tested showed no growth for three of three bottles after 42 days Global Customer Support K5 31JANO09 57 of 76 Mycobacterial Support Manual APPENDIX H SPECIMEN SOURCES OF MISCELLANEOUS SUPPLIES PROCESS REAGENTS NOTE Many sources exist for the product types listed here This manual shows a representative listing available from various websites but is not a complete list MYCOBACTERIOLOGY WAC PAC EMS t Digestion Systems Neutralizing Buffers Pellet Resusp Buffer AFE Reagents l AFE Stains REBATE Abas ee H i TEP a ea EAPN A AA T Quality Control Slides Pe 1 mi i J a i TE a a i ry oi Mi bi CELL BOND Slides m aiia NAC PAC Systems NAC PAC Red Alpha Tec Systems Inc manufactures an array of reagents for effective specimen preparation for AFB testing that is available worldwide Alpha Tec Systems Inc P O Box 5435 Vancouver WA USA 98662 For product inquiry and pricing USA phone 800 221 6058 International phone 360 260 2779 E mail CustSvc AlphaTecSystems com Global Customer Support K5 31JAN09 58 of 76
47. d media and the BacT ALERT MP culture bottle is recommended for optimal recovery of mycobacterial organisms from specimens The recommended decontamination method is N Acetyl L Cysteine Sodium Hydroxide final or working concentration of 2 for non sterile specimens For sterile body fluids except whole blood 0 5 ml sample is inoculated directly into the BacT ALERT MP culture bottle with 0 5 ml of the MB BacT Reconstitution Fluid Non sterile specimens including fluids that contain ANY bacteria other than mycobacteria must be decontaminated and 0 5 ml of pellet pH neutral inoculated into the bottle with 0 5 ml of reconstituted antibiotic supplement The red colored MB BacT Reconstitution Fluid provides nutrients and growth factors and is required The MB BacT Antibiotic Supplement is rehydrated with MB BacT Reconstitution Fluid All BacT ALERT MP culture bottle media should have a light pink color prior to inoculation indicating that either reconstitution fluid or reconstituted antibiotic supplement has been added The reconstituted antibiotic supplement is not intended to overcome lack of good sterile technique or grossly contaminated specimens Proper specimen handling and good laboratory techniques must be practiced to avoid a high contamination rate Drug resistant strains of bacteria may not be inhibited THEORY AND BEST PRACTICES MYCOBACTERIA SPECIMEN PROCESSING Liquefaction Many specimens submitted for mycobacterial isola
48. dro Characterization of a Novel Rapidly Growing Mycobagqcterium species Associaed with Sepsis J Clin Microbiol 2003 p 5650 5653 3 Ana Paula S Louro Ken B Waites Ecaterina Georgescu and William H Benjamin Jr Direct Identification of Mycobacterium avium Complex and Mycobacterium gordonae from MB BacT bottles using AccuProbe J Clin Microbiol 2001 39 570 573 4 Claudio Scarparo Paola Piccoli Alessandra Rigon Giulana Ruggiero Domenico Nista and Claudio Piersimoni Direct Identification of Mycobacteria from MB BacT Alert 3D Bottles Comparative Evaluation of Two Commercial Probe Assays J Clin Microbiol 2001 39 3222 3227 5 Fernando Alcaide Miguel Angel Benitez Josep M Escribo and Rogelio Martin Evaluation of the Bactec MGIT 960 and the MB BacT Systems for Recovery of Mycobacteria from Clinical Specimens for Species Identification by DNA AccuProbe J Clin Microbiol 2000 38 1 398 401 6 William H Benjamin Jr Ken B Waites Stephen A Moser and Andreas Roggenkamp The MB BacT Is a Sensitive Method of Isolating Mycobacterium tuberculosis from Clinical Specimens in a Laboratory with a Low Rate of Isolation J Clin Microbiol 2000 38 3133 3134 7 Harris G Rayner A Blair J Watt B Comparison of three isolation systems for the culture of mycobacteria from respiratory and non respiratory samples J Clin Pathol 2000 Aug 53 8 615 8 8 Francesca Brunello Flavio Favari and Rob
49. e Phosphate Buffer 10 bottles 100 ml Cat 669 500 Sterile TB Buffer pre made Phosphate Buffer 1 bottle 500 ml Scientific Device Laboratory Inc 411 East Jarvis Avenue Des Plaines IL USA 60018 847 803 9495 for product inquiry and pricing or www scientificdevice com Global Customer Support K5 31JANO09 61 of 76 Mycobacterial Support Manual APPENDIX H BD BBL MycoPrep SBD Mycobacteria Testing Product Number 740862 240862 BD MecoPrep Specimen Digestion Decontamination Kit Consists of 10 75 mL bottles of H4LC NaOH Solution and 5 packages of Phosphate Buffer pH 6 8 1 ea Availability Product type Brand Key Product Features US Canada Mexico Australia New Zealand BO BBL Specimen Collection and Processing BO MycoPrep Mycobacteria Testing Product Number 740863 240863 BD MycoPrep Specimen Digestion Decontamination Kit Consists of 10 150 mL bottles of N4LC NHaGH Solution and 10 packages of Phosphate Buffer pH 6 3 1 eaj Maximum Time and Labor Savings When Compared with Other Reagent Preparation Protocols BD BBL MycoPrep Kit 1 Sgu bottle to snap the MALC amput 2 Mix by gentle shaking 3 Use as neededfor up ta 24 Maura Global Customer Support K5 31JAN09 Typkal TB Base Digestant and MAC 50 System Adi MAC DI val to come fo oam Temi perature Trara ferS mL of TE Base Digestant to MAZC 50wal Sar to dave Add contents of NAC 50 vi
50. e best yield of mycobacteria may be expected to result from the use of the mildest decontamination procedure that sufficiently controls contaminants Strict adherence to specimen processing procedures is mandatory to ensure survival of the maximal number of mycobacteria All currently available digesting decontaminating agents are to some extent toxic to tubercle bacilli therefore to ensure the survival of the maximum number of bacilli in the specimen the digestion decontamination procedure must be precisely followed In order for enough tubercle bacilli to survive to give a confirmatory diagnosis it is inevitable that a proportion of cultures will be contaminated by other organisms It is also important to note that a laboratory which experiences no contamination is probably using a method that kills too many of the tubercle Cernoch PL Enni RK Saubolle MA Wallace RJ Weissfeld AS ed Laboratory Diagnosis of the Mycobacterioses Cumulative Techniques and Procedures in Clinical Microbiology Washington D C American Society of Microbiology 1994 p8 i Pfyffer GE Brown Elliott BA Wallace RJ Mycobacterium General Characteristics Isolation and Staining Procedures in Murray PR Baron EJ Pfaller MA et al eds Manual of Clinical Microbiology ed 8 Washington DC American Society for Microbiology 2003 p 544 Cernoch PL Enni RK Saubolle MA Wallace RJ Weissfeld AS ed Laboratory Diagnosis of the Mycobacterioses Cumulative
51. e blood specimen then this step must be performed in a biological safety cabinet while wearing appropriate protective clothing to comply with safety standards set forth by CDC NIH for Biosafety Level 3 guidelines To avoid cross contamination use a new syringe for each bottle containing blood Clean tops of inoculated BacT ALERT MB culture bottles with a tuberculocidal agent CAUTION must be compatible with polycarbonate plastic after addition of blood and prior to removal from the biological safety cabinet This removes any possible mycobacteria left on the tops of the bottles 1 Pfyffer GE Brown Elliott BA and Wallace RJ Jr Mycobacterium in Murray PR Baron EJ Pfaller MA et al eds Manual of Clinical Microbiology ed 8 Washington DC American Society for Microbiology 2003 p 543 BioMerieux Package Insert Jul 2008 i Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition U S Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health Fifth Edition US Government Printing Office Washington Feb 2007 Global Customer Support K5 31JAN09 39 of 76 Mycobacterial Support Manual BacT ALERT MB Culture Bottle Procedure CD ee a ii DIRECT DRAW INOCULATION PROCEDURE 1 Prior to inoculation disinfect the culture bottle top with an alcohol swab or equivalent Allow to air dry 2 Collect the blood using a butterfly set and the BacT ALERT B
52. e gloves Disposable gowns Disposable masks Microscope Materials for staining slides Quality Control organisms Materials available from bioMerieux BacT ALERT 3D Mycobacteria Detection Systems BacT ALERT MP culture bottles PN 259797 MB BacT Antibiotic Supplement Kit PN 259760 BacT ALERT Reseals PN 259787 to use removable closure system PROCEDURE 1 Use no more than 10 ml of the specimen in a 50 ml conical centrifuge tube Split larger samples into two or more aliquots and process the aliquots Pellets may be combined at the end of the decontamination procedure for inoculation into one BacT ALERT MP Culture bottle Assure that sample volumes used are less than or equal to 10 ml Split larger volumes into two or more tubes and process Add sterile saline to small volumes to standardize amount of NaOH added for decontamination and control final pH b lt INote Splitting very mucoid samples may be accomplished more easily after adding an equal amount of decontamination digestion reagent vortexing and inverting 30 seconds Pour equal amounts into two or more 50 ml conical centrifuge tubes Process and combine same sample sediments for inoculation of culture bottle solid media and slide Global Customer Support K5 31JANO09 23 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure 2 Add an equal volume of N acetyl L cysteine NALC Sodium Hydroxide NaOH solution to t
53. el 2 builds upon BSL 1 BSL 2 is suitable for work involving agents that pose moderate hazards to personnel and the environment It differs from BSL 1 in that 1 laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures 2 access to the laboratory is restricted when work is being conducted and 3 all procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment Biosafety Level 3 is applicable to clinical diagnostic teaching research or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through inhalation route exposure Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents and must be supervised by scientists competent in handling infectious agents and associated procedures All procedures involving the manipulation of infectious materials must be conducted within BSCs other physical containment devices or by personnel wearing appropriate personal protective equipment Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of life threatening disease aerosol transmission or related agent with unknown risk of transmission Agents with a close or identical antigenic relationship to
54. er Support K5 31JANO09 31 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure 8 Clean tops of inoculated BacT ALERT MP culture bottles with a tuberculocidal agent CAUTION Cleaning agent must be compatible with polycarbonate plastic prior to removal from the biological safety cabinet if they were inoculated with a needle This removes any possible mycobacteria left on the tops of the bottles 9 Load bottles into the red handled MB drawers for BacT ALERT 3D instrument or into the MB BacT instrument Refer to User Manual for assistance in loading Default test day protocol holds bottles 42 days before reporting final negative result Bottles should remain loaded for 42 days unless the instrument signals a positive NEEDLE LESS INOCULATION OF BOTTLES SLIDES AND SOLID MEDIA Perform this procedure under the biological safety cabinet Take care to prevent contamination during both bottle preparation and inoculation of the patient sample If aseptic needle less inoculation is chosen follow the instructions below after aseptically adding either the MAS or reconstitution fluid as described in this procedure Forceps lt Caution Use care when removing the metal crimp seal The use of forceps or other mechanical device is recommended If the pull tab breaks free from the seal resulting in sharp edges never attempt to remove the seal by hand Global Customer Support K5 31JANO09 32 of 76 BacT ALERT
55. erta Fontana Comparison of the MB BacT and Bactec 460 TB Systems for Recovery of Mycobacteria from Various Clinical Specimens J Clin Microbiol 1999 37 1206 1209 9 F Zuhre Badak Servet Goksel Ruchan Sertoz Asuman Guzelant Ahmet Kizirgil and Altinay Bilgic Cord Formation in MB BacT Medium is a Reliable Criterion for Presumptive Identification of Mycobacterium tuberculosis Complex in Laboratories with High Prevalence of M tuberculosis J Clin Microbiol 1999 37 4189 4191 10 Carmen Nogales Samuel Bernal Monica Chavez and Francesca Brunello Comparison of the MB BacT and Bactec 460 TB Systems J Clin Microbiol 1999 37 3432 3433 11 W H Benjamin Jr K B Waites A Beverly L Gibbs M Waller S Nix S A Moser and M Willert Comparison of the MB BacT System with a Revised Antibiotic Supplement Kit to the Bactec 460 System for Detection of Mycobacteria in Clinical Specimens J Clin Microbiol 1998 36 11 3235 3238 Global Customer Support K5 31JAN09 45 of 76 Mycobacterial Support Manual APPENDIX B 12 P Rohner B Ninet C Metral S Elmer and R Auckenthaler Evaluation of the MB BacT system and comparison to the Bactec 460 system and solid media for isolation of mycobacteria from clinical specimens J Clin Microbiol 1997 35 12 3127 3131 13 J G Magee R Freeman A Barrett Enhanced speed and sensitivity in the cultural diagnosis of pulmonary tuberculosis with a continuou
56. esting If the pH of the resuspended pellet is out of range it will appear pink and should be adjusted to neutral pH and become colorless 1 0 067 M M 15 phosphate buffer pH 6 8 a Stock alkaline buffer NazHPO anhydrous 9 47 g distilled water to 1 000 ml b Stock acid buffer distilled water to 1 000 ml Add dry buffer powders to separate 1 000 ml volumetric flasks Add distilled water to the 1 000 ml mark in each flask Combine equal volumes of stock alkaline and acid buffers Check the pH of the working solution with a pH meter Add small amounts of alkaline buffer to raise the pH or small amounts of acid buffer to lower it 2 Phenol Red indicator Phenol Red 1 0g or 0 2g distilled water to 500 ml to 100 ml Add dry powder to volumetric flask and add distilled water to the volume mark Mix well 3 Working Phosphate Buffer pH 6 8 with Phenol Red Indicator Add 14 ml of the Phenol Red indicator to 4000 ml Working Phosphate Buffer Sterilize at 121 C for 20 minutes The solution may be stored at room temperature but refrigeration is recommended This is really a color intensity judgment Add more or less as desired lt Note Use the above solution to buffer the NALC NaOH specimen mixture and to re suspend the processed centrifuged MB BacT pellet Further neutralization of the re suspended pellet if required is accomplished by the drop wise addition of sterile 2N HCL until the indicator becomes clear The p
57. event clotting of blood and enhance the growth of mycobacteria 3 How long before inoculation or after inoculation may the MB BacT Enrichment Fluid be added Enrichment Fluid is provided separately and must be added to the blood culture bottle for growth of mycobacteria This addition can be done within 24 hours of specimen inoculation or if specimen inoculation is carried out at bedside enrichment fluid can be added after receiving the inoculated vials in the laboratory Global Customer Support K5 31JAN09 72 of 76 Mycobacterial Support Manual BacT ALERT Media Quality Control APPENDIX J The following information is provided from Clinical Laboratory and Standards Institute CLSI documentation as listed in appropriate footnotes QC is an important integral part of laboratory testing Laboratories should establish a protocol of all the aspects of QC testing some of which are described below The frequency of the QC testing depends upon the workload in a laboratory type of media used and laboratory established policies All QC procedures should be written and results of QC testing documented in the laboratory records In case there is a situation of unsatisfactory performance corrective measures should be taken and their outcome should be documented Records of batch numbers and expiration dates of all media and reagents used should be documented QC records of commercial media may be obtained from the manufacturer
58. f supernatant completely into a splash proof discard container This allows for better pH control after resuspension of the pellet Carefully wipe the lip of the tubes with individual gauze pad dampened with disinfectant avoid getting the disinfectant into the sample tube Re suspend the pellet in 1 2 mL of sterile 0 067 M phosphate buffer pH 6 8 using individual sterile transfer pipettes Lower resuspension volumes less dilution increase the numbers of organisms in the 0 5 mL sample to be inoculated into the bottle Do not put other additives such as albumin into the sample pellet prior to inoculation Periodically check the final pH of the re suspended pellet It should be between pH 6 8 and 7 5 Higher pH may cause false positives and be detrimental to the mycobacteria delaying the time to detection Lower pH may cause false positives Kantor IN Kim SJ Frieden t et al 1998 Laboratory Services in Tuberculosis Control Culture Part Ill WHO TB 98 258 p 38 Global Customer Support K5 31JAN09 16 of 76 Mycobacterial Support Manual Best Practices in Sample Processing Relative Centrifugal Force Nomogram 350 13000 6 000 10000 5 000 300 i 5000 4005 Foo ee 3000 z g 1000 2000 180 500 160 140 200 ay 000 ji i DEW 50 J 500 BO 2 ro j 60 5 50 n 0 RATUS Relative Centrifugal SPEED rem Fiki irm xg Diagram reprinted with permission from Bockman Insiremenis Inc Palo Allo CA
59. gt 2 as they metabolize the substrates in the culture medium The liquid emulsion sensor LES is impermeable to most ions including hydrogen ions and to components of media and whole as well as degraded blood It is freely permeable to CO2 As CO diffuses across the membrane and dissolves in the water contained in the sensor free hydrogen ions are generated COs H20 lt HCO Ht HCO3 Free hydrogen ions interact with the indicator in the sensor As COz is produced the concentration of hydrogen ions increases and the pH falls causing the sensor to change to lighter green or yellow A Light Emitting Diode LED projects light onto the sensor The light reflected by the sensor is measured by a photodetector and as more CO is generated more red light is reflected lighter colors reflect more light than darker colors The photodiode converts the reflected light into an electrical signal that is measured every 10 minutes and plotted every hour as a point on a graph Mathematical algorithms analyze the readings and slope of the curve over time to determine Global Customer Support K5 31JAN09 5 of 76 Theory of Operation Mycobacterial Support Manual positives and negatives A representative graph for most bacteria reflects a typical growth curve with a lag phase log phase and stationary phase of growth The MB BacT and BacT ALERT 3D Mycobacteria Detection Systems monitor bottles using specific mycobacterial process bottle and
60. h Edition 2007 U S Government Printing Office Washington DC The Baker Company P O Drawer E 161 Gatehouse Road Sanford Maine 04073 USA 800 992 2537 E mail bakercoATbakercoDOTcom Global Customer Support K5 31JAN09 53 of 76 Mycobacterial Support Manual APPENDIX F Nomogram to Define Relative Centrifugal Force RCF for a Centrifuge The centrifugal forces at a given radius are a function of rpm s This nomogram is used for determining the relative centrifugal force RCF the revolutions per minute rpm or the radius when only two of these three variables are known To find the unknown value draw a straight line using a ruler through the two known values Read the unknown value from the point of intersection on the column corresponding to that variable 350 15 000 6 000 10 000 5 000 300 5 000 4000 250 _ 3000 200 1000 A 180 aia 500 160 140 200 1000 100 100 50 90 5 80 20 o 70 10 60 5 50 n 200 RADIUS Relative Centrifugal SPEED mm Field rpm xg Diagram reprinted with permission from Beckman Instruments Inc Palo Alto CA Global Customer Support K5 31JANO09 54 of 76 Mycobacterial Support Manual APPENDIX G Clinical Study Data Comparison of BacT ALERT MP Culture Bottles and Conventional Methods for Recovery of all Mycobacteria Percent Sensitivity MAI TB chelonae fortuitum gordonae Kansasil simiae terrae lt xs Ss S S S amp S S
61. he specimen from an individual pour tube Do not exceed 10 ml of NALC NaOH per tube Individual pour tubes prevent the addition of excess NaOH that will require pH neutralization later and avoid the possibility of cross contamination between specimens Use individual pour tubes for NALC NaOH 3 Vortex for 20 seconds up to a maximum of 30 seconds Use a timer This breaks down the mucus Be sure to invert the tube a few times during this process to insure that all areas of the specimen tube are decontaminated Vortex gently because over foaming will cause NALC to oxidize and become inactive Vortexing over 30 seconds may inactivate NALC It may be necessary to add an extra pinch of NALC powder to very mucoid or bloody specimens to digest the specimen completely If liquefaction is not complete during the initial vortex period you may agitate the solution at intervals during step 4 to follow Use a timer for 20 30 seconds of vortexing Global Customer Support K5 31JANO09 24 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual Incubate at room temperature for 15 minutes to decontaminate Use a timer All specimens first to last must incubate for 15 minutes Sample batches that are too large gt 20 samples may cause incubation time to exceed 15 minutes and possibly delay time to detection You may choose to invert and gently shake by hand at intervals during this incubation period 4 a Usea time
62. ion for MB culture bottle Mycobacterium avium and M intracellulare 3 For Mycobacterium tuberculosis and kansasii isolates Transfer the supernatant into sterile 7H9 broth and adjust to a McFarland 0 5 Standard 1 x 10 CFU ml Dilute this stock as follows 1 0 ml of the above in 9 0 ml 7H9 broth 10 1 0 ml of the above in 9 0 ml 7H9 broth 10 1 0 ml of the above in 9 0 ml 7H9 broth 10 working dilution for MP culture bottle Mycobacterium tuberculosis and M kansasii Global Customer Support K5 31JAN09 76 of 76
63. ions and Answers by Chapter Chapter 1 Mycobacteria in our World 1 With 2 million deaths a year what is the leading cause of death in many resource poor and developing countries The World Health Organization estimates that 9 million new cases and 2 million deaths are directly attributable to Tuberculosis and it has been cited as the leading cause of death in many resource poor and developing countries 2 What are MOTT Mycobacteria other than the tubercle bacillus MOTT are also referred to as nontuberculosis mycobacteria NTM 3 Why is a solid media recommended when using liquid media culture systems It is recommended that whenever possible liquid medium should be used to assure better and more rapid results The addition of solid medium along with a liquid medium maximizes the recovery provides an opportunity to look at colony morphology that is not possible in liquid media allows for detection of mixed mycobacterial infections and serves as a backup when liquid culture is contaminated An increase of 4 to 6 for mycobacterial isolation has been reported by the addition of an LJ slant along with a liquid medium while adding a liquid medium along with an LJ medium is reported to increase the recovery rate significantly 15 to 30 The liquid culture method is needed to provide more rapid detection growth and susceptibility results for M tuberculosis Chapter 2 Theory of Operation 1 What metabolites were chosen to enhance CO2
64. le will become sick and another 35 million people will die from tuberculosis Mycobacteria other than the tubercle bacillus MOTT are referred to here as nontuberculosis mycobacteria NTM The majority of these isolates require incubation at 35 to 37 C except where noted These organisms are most commonly found in soil and water and have been implicated as opportunistic pathogens in patients with underlying lung disease immunosuppression or percutaneous trauma NTM associated with pulmonary disease are M avium complex MAC M kansasil M asiaticum M fortuitum complex rarely pulmonary M szulgai M malmoense M shimoidei grows well at 45 C M celatum and M xenopi optimum temperature of 40 42 C M marinum and M ulcerans which require incubation at 30 C are NTM associated with cutaneous infections NTM producing other types of infection e g disseminated disease lymphadenitis osteomyelitis etc are MAC M kansasii M scrofulaceum M fortuitum complex M chelonae isolates generally require incubation at 30 C M szulgai M simiae M genavense requires Mycobactin J and 8 to 12 weeks incubation M celatum and M haemophilum requires addition of hemin and incubation at 30 C Classic examples of NTM contamination include the isolation of M gordonae M mucogenicum or M terrae complex from sputum on culture These species are common in tap water and almost never cause chronic lung disease M
65. lood Collection Adapter Cap and inoculate directly into the BacT ALERT MB culture bottle at the patients bedside 3 5 ml per bottle To prevent overinoculation monitor the blood volume intake into the Culture Bottle using the 5 ml incremental markings on the bottle label 3 After inoculation swab bottle tops with gauze soaked in 2 Amphyl or other mycobacteriocidal agent and allow to air dry 4 Transfer the inoculated culture bottle promptly to the testing laboratory BLOOD COLLECTION TUBE INOCULATION PROCEDURE 1 Blood may also be collected in a sterile SPS Sodium polyanetholesulfonate or heparinized tube and inoculated into the BacT ALERT MB culture bottle in the laboratory No prior processing of the specimen is required Blood collected in EDTA is unacceptable since EDTA inhibits mycobacterial growth even in trace amounts 2 Disinfect the blood collection tube top prior to blood collection with an alcohol swab or equivalent Allow to air dry Disinfect the BacT ALERT MB culture bottle top with an alcohol swab or equivalent Allow the septa to air dry prior to inoculation of the blood specimen 3 Using aseptic technique remove 3 5 mls from the collection tube with a sterile syringe and inoculate into the culture bottle 4 After inoculation swab bottle tops with gauze soaked in 2 Amphyl or other mycobacteriocidal agent and allow to air dry SYRINGE INOCULATION PROCEDURE 1 Prior to inoculation disinfect the
66. n 24 hours of inoculation MB BacT Enrichment Fluid contains components that are necessary to ensure optimal growth of mycobacteria present in the patient sample Tenover FC Crawford JT Huebner RE et al The resurgence of tuberculosis is your laboratory ready J Clin Micro 31 4 767 770 1993 bioM rieux BacT ALERT MP Instructions for Use July 2008 Global Customer Support K5 31JANO09 3 of 76 Mycobacteria in our World Mycobacterial Support Manual EXERCISE SESSION See Appendix I for answers 1 With 2 million deaths a year what is the leading cause of death in many resource poor and developing countries What are MOTT Why is a solid media recommended when using liquid media culture systems TIPS to REMEMBER Mycobacteria are aerobic nonsporeforming nonmotile acid fast bacilli which have slow to very slow growth rates generation times of species vary from 2 to gt 20 hours and growth of M tuberculosis using traditional mycobacterial media can take two to eight weeks or longer Liquid medium should be used to assure better and more rapid results The addition of solid medium along with a liquid medium maximizes the recovery provides an opportunity to look at colony morphology that is not possible in liquid media allows for detection of mixed mycobacterial infections and serves as a backup when liquid culture is contaminated Global Customer Support K5 31JANO09 4 of 76 Mycobacterial Support Manu
67. n settings in which it is not considered clinically significant though the pathogenicity seems higher than many species of NIM especially in patients with acquired immune deficiency syndrome AIDS some species are associated with specific disease syndromes and their isolation in that setting is often highly significant Table 1 below shows examples of mycobacterial species and clinical conditions in which they often play a significant role Table l Examples of Mycobacterial Species and Clinical Conditions Clinical setting M a abscessus or M Cystic fibrosis CF or bronchiectasis in Respiratory avium complex older Caucasian women M fortuitum Fsophageal achalasia and chromic Respiratory regurgitation or history of mineral oil ingestion M marinum Extremity lesion after fish tank or other Biopsy aspirate marine exposure M kansasii or MAC Hairy cell leukemia advanced HIV disease Blood bone marrow M chelonae M Disseminated skin lesions in patients on Biopsy aspirate abscessus or Md chronic immunosuppressive drugs eg haemophilum corticosteroids M chelonae M Central venous catheter prosthetic device Biopsy aspirate mucogenicum surgical wound injection site or other local M fortuitum or trauma M abseessus Table is from CLSI M48 A Mycobacteria are aerobic nonsporeforming nonmotile acid fast bacilli which have slow to very slow growth rates generation times of species vary from 2 to gt 20 hr
68. nalyze the readings and slope of the curve over time to detect log phase growth and determine positive and negative results Global Customer Support K5 31JAN09 7 of 76 Mycobacterial Support Manual BacT ALERT MYCOBACTERIA PRODUCTS OVERVIEW MEDIA OVERVIEW OBJECTIVES To be familiar with BacT ALERT Mycobacterial Products DISCUSSION BacT ALERT MP System Reagent Product Product description number B BACT ALER Red cap 259797 10 ml MP culture bottle Plastic 259760 MB Bac Antibiotic Supplement kit contains 2 reagents for MP culture bottle Lyophilized antibiotic cake Reagent 1 is MB BacT Antibiotic Supplement Reagent 2 is Red fluid MB BacT Reconstituiton Fluid 15 ml i Global Customer Support K5 31JAN09 BacT ALER Reseal Case Size and Storage per case 2 8 C in dark 100 tests per kit 5 vials per kit 2 8 C in dark 5 vials per kit 2 8 C in dark 100 box Room Temp 100 bottles 0 5 ml 0 5 ml for non sterile specimens 0 5 ml into MP culture bottle for non sterile specimens rehydrate reagent 1 with 10 ml of reagent 2 0 5 ml into MP culture bottle for sterile specimens Caan Media Contents Middlebrook 7H9 broth Pancreatic Digest of Casein Bovine Serum Albumin Catalase in purified water with an atmosphere of COs nitrogen and oxygen under vacuum Kit contains 5 vials of lyophilized antibi
69. ng three mycobacterial cultures are recommended for QC testing M tuberculosis H37Ra ATCC 25177 M kansasii ATCC 12478 M fortuitum ATCC 6841 Commercially prepared media are thoroughly QC tested by the manufacturer However users should refer to CLSI NCCLS document M22 for media that are exempt nonexempt from user QC It is also important to visually examine commercially prepared media before use to make sure the tubes are not damaged cracked and that the medium has not been contaminated or changed in its appearance during transport or storage For high volume and reference laboratories it is permissible to carry out QC testing of a new batch of commercial medium or reagents and document results Follow the QC Global Customer Support K5 31JAN09 73 of 76 Mycobacterial Support Manual APPENDIX J procedures and criteria recommended by the manufacturers The following information is from bioM rieux BacT ALERT MP and MB Culture Bottle Instructions for Use BacT ALERT MP culture bottles A Certificate of Conformance is provided with each case of BacT ALERT MP culture bottles indicating satisfactory growth performance of M tuberculosis ATCC 25177 and M intracellulare ATCC 13950 Upon receipt new lots or shipments of BacT ALERT MP culture bottles or reagents may be tested for quality control Refer to CLSI NCCLS M22 A3 for appropriate Quality Control organisms Follow the Bottle preparation and inoculati
70. obacteria or mycobacteria other than Global Customer Support K5 31JANO09 50 of 76 Mycobacterial Support Manual APPENDIX E tuberculosis Many of the species are common environmental organisms and approximately 25 of them are associated with infections in humans A number of additional species are associated with infections in immunocompromised persons especially HIV infected individuals All of these species are considered opportunistic pathogens in humans and none are considered communicable Mycobacteria are frequently isolated from clinical samples but may not be associated with disease The most common types of infections and causes are 1 pulmonary disease with a clinical presentation resembling tuberculosis caused by M kansasili M avium and M intracellulare 2 lymphadenitis associated with M avium and M scrofulaceum 3 disseminated infections in immunocompromised individuals caused by M avium 4 skin ulcers and soft tissue wound infections including Buruli ulcer caused by M ulcerans swimming pool granuloma caused by M marinum associated with exposure to organisms in fresh and salt water and fish tanks and tissue infections resulting from trauma surgical procedures or injection of contaminated materials caused by M fortuitum M chelonei and M abscesens Containment Recommendations BSL 2 practices containment equipment and facilities are recommended for activities with clinical materials and cultures of Mycob
71. ocedure DJ Note MAS may be pre added to the BacT ALERT MP culture bottles and left in the refrigerator up to 24 hrs within the limits of the expiration date of the MAS before inoculation Allow bottles to come to room temperature before using 1 Label BacT ALERT MP culture bottles solid media and slides appropriately Do not cover bottle s barcode 2 Reconstitute MB BacT Antibiotic Supplement with 10 ml of MB BacT Reconstitution Fluid using aseptic technique Never re use syringes Aseptically withdraw 10 mL of Reconstitution Fluid Aseptically add 10 mL of Reconstitution Fluid to Antibiotic Supplement vial and swirl to mix contents Global Customer Support K5 31JANO09 28 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual 3 Add 0 5 ml reconstituted antibiotic supplement to each BacT ALERT MP culture bottle using aseptic technique for non sterile samples For sterile body fluids you may add 0 5 ml reconstitution fluid alone to the culture bottles Antibiotics are added to lower breakthrough contamination of non sterile specimens Invert and gently swirl bottles to mix all contents A Add 0 5 mL reconstituted antibiotic supplement to each bottle for non sterile specimens and 0 5 mL reconstitution fluid alone to bottles for sterile specimens Do a PINK CHECK The addition of this reagent is easily confirmed by visualizing a pink color in inoculated bottles
72. on procedure 1 Add 0 5 ml of rehydrated MB BacT Antibiotic Supplement to each BacT ALERT MP culture bottle required for testing 2 Inoculate representative bottles with 0 5 ml of the control organisms diluted to 10 CFU ml in sterile physiological saline or sterile unsupplemented Middlebrook 7H9 Broth approved lot 3 After obtaining anticipated results use remaining bottles for testing clinical specimens If expected results are not obtained contact bioM rieux Customer Service BacT ALERT MB culture bottles A Certificate of Conformance is provided with each case of BacT ALERT MB culture bottles indicating satisfactory growth performance of M avium ATCC 25291 and M intracellulare ATCC 13950 Upon receipt new lots or shipments of BacT ALERT MB culture bottles or reagents may be tested for quality control Refer to CLSI NCCLS M22 A3 for appropriate Quality Control organisms Follow the bottle preparation and inoculation procedure 1 Add 1 0 ml MB BacT Enrichment Fluid to each BacT ALERT MB culture bottle required for testing 2 Inoculate representative bottles with 5 0 ml of the control organisms diluted to 10 CFU ml in sterile defibrinated sheep blood Subculture on Middlebrook 7H11 plates to determine viability and count 3 After obtaining anticipated results use remaining bottles for testing clinical specimens If expected results are not obtained contact bioM rieux Customer Service
73. otic power and 5 vials of reconstitution fluid Lyophilized cake of 6 antibiotics Amphotericin B Azlocillin Nalidixic Acid Polymyxin B Trimethoprim and Vancomycin Contains oleic acid Glycerol Amaranth and Bovine Serum Albumin in purified water growth factors Use 10 ml to rehydrate one antibiotic vial Use remaining 5 ml for sterile body fluids in MP culture bottle Clear plastic snap cap used to reseal stopper after removing bottle crimp for needless inoculation of MP bottle 8 of 76 MEDIA OVERVIEW Mycobacterial Support Manual NOTE Prior to use the BacT ALERT MP culture bottles should be examined for evidence of damage or deterioration discoloration Bottles exhibiting evidence of damage leakage or deterioration should be discarded The media in undisturbed bottles should be clear Do not use a bottle containing media that exhibits turbidity a yellow sensor or excess gas pressure these are signs of possible contamination Also inspect all MB BacT Antibiotic Supplement Kit vials for evidence of damage or contamination Do not use MB BacT Reconstitution Fluid vials exhibiting turbidity TRAINING SESSION BacT ALERT MP culture bottles MB BacT Antibiotic Supplement Kit and BacT ALERT Reseals BacT ALERT MP culture bottles Plastic 100 bottles per case BacT ALERT Reseals PN 259787 Reagent 1 Lyophilized antibiotic cake Reagent 2 Reconstituiton fluid NOTE Rehydrate p
74. ously loading BacT ALERT MB culture bottles into either shaking BC or non shaking MB drawers racks Describe testing methods any additional QC setting up QC cultures in BacT ALERT MP and MB culture bottles setting up seeded cultures in BacT ALERT MP and MB culture bottles Review of appendix sections completed Technologist s Signature Date Global Customer Support K5 31JAN09 43 of 76 Mycobacterial Support Manual APPENDIX B 10 11 APPENDIX B Other References for Mycobacterial testing Instructions for Use bioM rieux Inc BacT ALERT MP culture bottle product 259797 also contains information for MB BacT Antibiotic Supplement Kit product 259760 BacT ALERT Reseal product 259787 Instructions for Use bioM rieux Inc BacT ALERT MB culture bottle product 251011 also contains information for MB BacT Enrichment Fluid product 259877 Manual of Clinical Microbiology 9 edition Murray Baron Jorgensen Landry Pfaller ASM Press Washington D C 2007 Public Health Mycobacteriology A Guide for the Level IIl Laboratory US Department of Health and Human Services Center for Disease Control Atlanta GA 1985 Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition U S Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health Fifth Edition US Government Printing Office Washington Feb 2007 Clinical and Laborato
75. owder with 10 ml reconstitution fluid and discard unused portion 7 days after rehydration EXERCISE SESSION See Appendix I for answers How many BacT ALERT MP culture bottles are in one case What are the storage conditions for the BacT ALERT MP culture bottles and the MB BacT Antibiotic Supplement Kit 3 How many bottles can be prepared with one box of the MB BacT Antibiotic Supplement Kit NO TIPS to REMEMBER MB BacT Reconstitution Fluid vials contain 15 ml The MB BacT Antibiotic Supplement vials are reconstituted with 10 ml of MB BacT Reconstitution Fluid leaving 5 ml for sterile body fluid bottles Either 0 5 ml of the reconstituted antibiotic Global Customer Support K5 31JANO09 9 of 76 Mycobacterial Support Manual MEDIA OVERVIEW cake non sterile decontaminated specimens or 0 5 ml of reconstitution fluid sterile specimens must be added to bottles to assure the presence of Mycobacterial growth factors BacT ALERT MP culture bottles must be loaded into cabinets BacT ALERT Classic or drawers BacT ALERT 3D that are designated MB cabinets or drawers This assures that the correct mycobacterial culture bottle algorithm is assigned to analyze the bottle readings DISCUSSION BacT ALERT MB System B BACT ALER MB Culture Bottle Glass MB Bac Enrichment Fluid for the MB bottle BACT ALER Blood Collection Adapter Cap Insert for filling of blood
76. production during Mycobacterial metabolism Once ingested glycerol and oleic acid are converted to Acetyl CoA and are oxidized through the Krebs or Tricarboxylic Acid Cycle TCA Carbon Dioxide CO2 and free electrons are the major metabolic byproducts of oxidation 2 What technology is used to monitor the production of CO2 The MB BacT and BacT ALERT 3D systems utilize a colorimetric sensor and reflected light to monitor the amount of CO2 dissolved in the culture medium 3 Why does the sensor change colors from dark to light As CObd diffuses across the membrane and dissolves in the water contained in the sensor free hydrogen ions are generated COs H2O e HCO H HCO3 Free hydrogen ions interact with the indicator in the sensor As COz is produced the concentration of hydrogen ions increases and the pH falls causing the sensor to change to lighter green or yellow Global Customer Support K5 31JAN09 70 of 76 Mycobacterial Support Manual Appendix Q and A by Chapter Chapter 3 Media Overview BacT ALERT MP Culture Bottle 1 How many BacT ALERT MP culture bottles are in one case 100 bottles 2 What are the storage conditions for the BacT ALERT MP culture bottles and the MB BacT Antibiotic Supplement Kit 2 8 C in dark 3 How many bottles can be prepared with one box of the MB BacT Antibiotic Supplement Kit 100 bottles Chapter 3 Media Overview BacT ALERT MB Culture Bottle 1 How many Bac
77. ptable Direct inoculation of blood onto a solid medium is not recommended Refer to the BacT ALERT MB Culture Bottle Instructions for Use for recommended collection procedures BioM rieux recommends that inoculated culture bottles be placed into the BacT ALERT Microbial Detection System as soon as possible after collection Inoculated culture bottles delayed in entry should be maintained at room temperature until they can be loaded into the incubator BOTTLE PREPARATION 1 Label the culture bottle with patient information The icons on the bottle label can be defined by the user 2 Remove plastic flip top from culture bottle and disinfect with an alcohol swab or equivalent Allow to air dry 3 Enrichment Fluid is provided separately and must be added to the blood culture bottle for growth of mycobacteria This addition can be done up to 24 hours before specimen inoculation or if specimen inoculation is carried out at bedside Enrichment Fluid can be added up to 24 hours after receiving the inoculated bottles in the laboratory e Remove the plastic flip top from the Enrichment Fluid vial and disinfect with an alcohol swab or equivalent Allow to air dry e Aseptically add 1 ml of Enrichment Fluid to each BacT ALERT MB culture bottle e Employ careful aseptic technique to avoid contamination of the Enrichment Fluid and medium in the culture bottle NOTE If the Enrichment Fluid is added following inoculation of th
78. r acid fast staining and subculturing Disinfect bottle stopper after subculture b Subculturing from positive bottles sealed with the Reseal Pull the center tab away from the Reseal and remove the seal completely from the bottle After the septum is removed aseptically the sample may be removed If it is desired to keep the bottle for further testing aseptically replace the stopper and place a new Reseal over the stopper and bottle If the acid fast smear is positive proceed with the Mycobacteria specific identification procedures employed by your institution If the concentrated smear is negative for acid fast bacilli a Gram stain should be performed If both the acid fast smear and the Gram stain are negative indicating a possible false positive the bottle should be loaded back into the instrument until growth of subculture or redesignation as positive or negative A positive bottle status will automatically revert to negative to date when reloaded into BacT ALERT detection systems Cultures which were initially determined false positive and were redesignated positive should be smeared and subcultured If non mycobacterial organisms are seen on the Gram stain reprocess entire bottle contents through another decontamination procedure and inoculate into a new BacT ALERT MP culture bottle or discard and obtain another specimen for culture If the new BacT ALERT MP culture bottle again grows non mycobacterial organisms discard and obt
79. r for 15 minutes RT incubation i D E e NR f 4 Dilute specimen to a volume of 50 ml with sterile phosphate buffer from an individual pour tube This reduces the action of the NaOH which could injure the mycobacteria Individual pour tubes avoid the possibility of cross contamination between specimens Use individual pour tubes for Phosphate Buffer 5 Mix by inverting to be sure that all surfaces are coated and that the NaOH is neutralized Refer to the nomogram provided on the next page and in Appendix F of this manual to determine the proper relative centrifugal force RCF using the centrifuge speed and radius Global Customer Support K5 31JANO09 25 of 76 Mycobacterial Support Manual BacT ALERT MP Culture Bottle Procedure Centrifuge for 15 minutes at 3000 x g RCF preferably at refrigerated temperatures to increase cell viability and optimal recovery TF ihai ir ji _ os o p ao H Fii prun 7 L ri r aI aT TITT TTA z E amp EEEE E T f a E al L i E yioo T it 4 i a Pe EJA e s Tages pimal ari prena nm Pe kea eee le Pis iha d4 6 Immediately after centrifuge has stopped pour off all supernatant completely into a splash proof discard container filled with an appropriate tuberculocidal agent Carefully wipe the lip of the tubes with individual gauze pads dampened with the disinfectant to clean up any drips avoid getting the disinfectant
80. rd Edition NCCLS document M22 A3 ISBN 1 56238 536 4 NCCLS 940 West Valley Road Suite 1400 Wayne Pennsylvania 19087 1898 USA 2004 pg 5 NCCLS Quality Control for Commercially Prepared Microbiological Culture Media Approved Standard Third Edition NCCLS document M22 A3 ISBN 1 56238 536 4 NCCLS 940 West Valley Road Suite 1400 Wayne Pennsylvania 19087 1898 USA 2004 pg 14 17 Global Customer Support K5 31JAN09 75 of 76 Mycobacterial Support Manual APPENDIX K Procedure for 1 Prepare a culture grown on Lowenstein Jensen or Middlebrook 7H11 preparation of media in a sterile screw cap tube containing Middlebrook 7H9 broth with appropriate sterile Ottawa Sand or glass beads Place colonies in a screw cap tube QC dilutions tighten the cap and vortex 20 30 seconds or until well dispersed Allow 10 and 10 the suspension to stand for 15 minutes to allow large particles to settle 2 For Mycobacterium fortuitum intracellulare and avium isolates Transfer the supernatant into sterile 7H9 broth and adjust to a McFarland 0 5 Standard 1 x 10 CFU ml Dilute this stock as follows 1 0 ml of the above in 9 0 ml 7H9 broth 107 1 0 ml of the above in 9 0 ml 7H9 broth 10 1 0 ml of the above in 9 0 ml 7H9 broth 10 1 0 ml of the above in 9 0 ml 7H9 broth 10 working dilution for MP culture bottle Mycobacterium fortuitum 1 0 ml of the above in 9 0 ml sterile defibrinated sheep blood 10 working dilut
81. re laundering Baseline serum PRIMARY BARRIERS AND SAFETY EQUIPMENT Primary barriers Class I or Il BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials PPEs e Laboratory coats gloves face protection as needed Primary barners Class lor BSCs or other physical contamment devices used for all open manipulation of agents PPEs SUMMARY OF RECOMMENDED BIOSAFETY LEVELS FOR INFECTIOUS AGENTS FACILITIES SECONDARY BARRIERS Laboratory bench and sink required BSL 1 plus e Autoclave available BSL 2 plus Physical separation from access corridors Self closing double door access Exhaust air not recirculated Protective laboratory clothing gloves respiratory protection as needed Primary barriers All procedures conducted in Class IM BSCs or Class I or I BSCs in combination with full body air supplied positive pressure personnel suit Negative airflow into laboratory BSL 3 practices plus Clothing change before entering BSL 3 plus Separate building or isolated zone Dedicated supply and exhaust vacuum and decontamination systems Other requirements outlined in the text Dangerous exotic agents which pose high risk of life threatening discase Aerosol transmitted laboratory e infections have occurred or related agents with unknown risk of transmission Shower on exit All material decont
82. require the use of Biosafety Level 3 practices containment equipment and facilities 3 When the instrument indicates that a particular cell contains a positive bottle remove the bottle according to procedures provided in the User s Manual 4 All bottles designated positive should be smeared and subcultured for acid fast bacilli In a biological safety cabinet mix bottle contents disinfect the bottle stopper with an alcohol swab or equivalent and allow to air dry Remove specimen for acid fast staining and subculture using a syringe and needle Disinfect bottle stopper with mycobacteriocidal agent after subculture If the acid fast smear is positive proceed with the Mycobacteria specific identification procedures employed by your institution If the smear is negative for acid fast bacilli but reveals the presence of other microorganisms a Gram stain should be performed If both the acid fast smear and the Gram stain are negative indicating a possible false positive the bottle should be reloaded into the instrument until growth of subculture or redesignation as positive or negative Cultures which were initially determined false positive and were redesignated positive should be smeared and subcultured 5 If non mycobacterial organisms are seen on the Gram stain obtain another specimen for culture if desired The possibility of bacterial septicemia should also be considered 6 Negative cultures may be checked by smear and or subcultured at
83. rifugation speeds create heat that will kill mycobacteria especially in the presence of chemicals Therefore optimal results are obtained with a refrigerated centrifuge The centrifuge must be fast enough to attain a relative centrifugal force RCF of 3000 x g If the RCF is not high enough many tubercle bacilli remain in suspension following centrifugation and are poured off with the discarded supernatant fluid Recent studies have shown that 3000 x g for 15 minutes would sediment 95 of mycobacteria in a digested sputum specimen The specific gravity of tubercle bacilli ranges from 1 07 to 0 79 making centrifugal concentration of specimens ineffective if the RCF is not 3000 x g The relative centrifugal force RCF or g at a given radius is a function of the revolutions per minute rom in a centrifuge Use the nomogram on the following page to determine the relative centrifugal force the revolutions per minute or the radius when only two of these three variables are known To find the unknown value draw a straight line using a ruler through the two known values Read the unknown value from the point of intersection on the column corresponding to that variable For example if the radius of the centrifuge to be used is 160 mm one would need to spin at 5000 rpm to achieve the minimum of 3000 x g TECHNICAL TIPS 1 Check the radius and rpms of the centrifuge to be sure 3000 X g is achieved during concentration of the sample Pour of
84. ry Standards Institute CLSI Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A ISBN 1 56238 669 7 Clinical and Laboratory Standards Institute 940 West Valley Road Suite 1400 Wayne Pennsylvania 19087 1898 USA 2008 Della Latta P ed Mycobacteriology and Antimycobacterial Susceptibility Testing in Isenberg HD ed Clinical Microbiology Procedures Handbook vol 2 Washington DC ASM Press 2004 Garcia L ed in chief Clinical Microbiology Procedures Handbook 2 edition 2007 update Washington DC ASM Press 2007 Global tuberculosis control surveillance planning financing WHO report 2007 Geneva World Health Organization WHO HTM TB 2007 376 Forbes BA Sahm DF Weissfeld AS Diagnostic Microbiology St Louis Missouri Mosby 1998 Cumitech 16A Laboratory Diagnosis of the Mycobacterioses October 1994 ASM press Published article list John A Crump David C Tanner Stanley Mirrett Celeste M McKnight L Barth Reller Duke University Medical Center Durham NC Carolinas Medical Center Charlotte NC C 46 Controlled Comparison of Bactec 13A Bactec Myco F Lytic BacT ALERT MB and Isolator 10 Systems For Detection Of Mycobactermia J Clin Microbiol 2003 41 5 1987 1990 Global Customer Support K5 31JANO09 44 of 76 APPENDIX B Mycobacterial Support Manual 2 T Hong WR Butler F Hollis MM Floyd SR Toney Yi Wei Tang D Steele and RJ Leggia
85. s S Xenopi TOTAL 168 0 183 0 8 0 7 0 28 0 8 0 1 0 1 0 1 0 MB 88 87 4 100 0 85 100 0 87 5 100 0 0 0 100 0 MB LJ 95 2 93 4 100 0 100 0 100 0 100 0 100 0 100 0 100 0 MB 7H11 LJ amp H11 97 6 16 2 96 7 89 6 100 0 50 0 85 57 1 100 0 17 9 100 0 100 0 100 0 100 0 0 0 100 0 100 0 100 0 LJ 55 4 12 1 50 0 42 9 17 9 100 0 100 0 100 0 100 0 The sensitivity of BacT ALERT MP culture bottles alone is 45 higher than LJ slants and 54 higher than 7H11 plates BacT ALERT MP culture bottles show greater recovery of M tuberculosis and the MAC group BacT ALERT MP culture bottles also recover atypical mycobacteria such as M chelonae and M fortuitum in significantly greater numbers than solid media See below and on next page that smear positive samples greater numbers of organisms present in sample have shorter time to detection and identification 510 k data on file at bioM rieux Inc Global Customer Support K5 31JAN09 BacT ALERT Mean Time to Identification Days MAI M TB Other mycobacteria All mycobacteria Direct smear 9 5 18 0 13 6 15 4 14 8 23 2 21 4 18 6 Direct smear 25 0 28 6 0 0 75 0 100 0 0 0 100 0 Combo 13 0 19 3 18 7 16 7 55 of 76 APPENDIX G Mycobacterial Support Manual SEEDED STUDY AVERAGE TIME TO DETECTION
86. s at room temperature with use of a timer Inoculation of tests in correct order MP culture bottle solid media slide 0AO00 w 3 The following techniques help prevent crossover contamination between samples a Use of individual pour tubes for reagents being added to test b Use of syringes or pipettes long enough to reach bottom of tube without touching sides of tube when sampling pellet c Careful recapping of centrifuge tube to include 1 Never having more than one tube top open at any given time 2 Never allowing a tube cap to be placed sample side down on the working surfaces RESOURCES 1 BioM rieux Inc BacT ALERT MP Instructions for Use July 2008 2 Della Latta P ed Mycobacteriology and Antimycobacterial Susceptibility Testing in Isenberg HD ed Clinical Microbiology Procedures Handbook vol 2 Washington DC ASM Press 2004 sect 7 3 Pfyffer GE Mycobacterium General Characteristics Laboratory Detection and Staining Procedures in Murray PR Baron EJ Pfaller MA et al eds Manual of Clinical Microbiology ed 9 Washington DC American Society for Microbiology 2007 Global Customer Support K5 31JAN09 36 of 76 BacT ALERT MP Culture Bottle Procedure Mycobacterial Support Manual Global Customer Support K5 31JANO09 37 of 76 Mycobacterial Support Manual BacT ALERT MB Culture Bottle Procedure BacT ALERT MB Culture Bottle Procedure OBJECTIVES To know about the BacT ALERT MB Culture Bottle an
87. s automated mycobacterial liquid culture system J Med Micro June 1998 47 6 547 53 Presented data 1 ASM 2005 Poster C 034 YF Wang AC Popp M Shapiro Emory University School of Medicine Grady Memorial Hospital Performance Analysis of BacT ALERT MP Bottles and the 7H11 Plates Method for Detection of Mycobacterium tuberculosis 2 ASM 2002 C T Upchurch S B Florence P H Gilligan UNC Hospitals Chapel Hill NC C 48 Comparison of Decontamination Methodologies When Utilizing MB BacT ALERT 3D Mycobacterium Detection System 3 ICAAC 2000 Poster 1616 Karen Rondomanski N Glover N Anderson and Lee Borenstein Olive View UCLA Medical Center Sylmoar CA and Los Angeles County Public Health Laboratory Los Angeles CA Evaluation of the Bactec MGIT 960 and the BacT ALERT 3D Systems for Recovery of Mycobacteria from Clinical Specimens 4 ASM 1998 M F Davila C T Gomez Y Chin and E A Macias Comparison of the MB BacT Mycobacteria Detection System Mycobacteria Growth Indicator Tubes MGIT and Conventional Culture for the Detection of Mycobacteria Species 5 ASM 1998 S F Tidwell MT ASCP T Clark B S Arkansas Department of Health Little Rock AR Reduction of Bacterial Contamination in MB BacT Bottles from Organon Teknika t American Society of Microbiology Annual Meeting Interscience Conference on Antimicrobial Agents and Chemotherapy Global Customer Support K5 31JANO09
88. tion contain mucus such as sputum and gastric lavage Mycobacteria as well as contaminating flora are often present but trapped within the mucus Liquification is achieved by adding chemicals which when vortexed with the specimen break down the mucus and release the organisms Several agents can be used to liquefy a clinical specimen including NALC dithiothreitol sputolysin and enzymes In most procedures liquification release of the organisms from mucin or cells is enhanced by vigorous mixing with a vortex type of mixer in a closed container Following mixing the container should be allowed to stand for 15 minutes before opening to prevent the dispersion of fine aerosols generated during mixing Decontamination Most specimens received for mycobacterial culture contain various amounts of organic debris and a variety of contaminating normal or transient bacterial flora A chemical decontamination process usually effectively kills the contaminants while allowing 1 Forbes BA Sahm DF Weissfeld AS Diagnostic Microbiology St Louis Missouri Mosby 1998 p 726 Global Customer Support K5 31JANO09 13 of 76 Mycobacterial Support Manual Best Practices in Sample Processing recovery of the mycobacteria The high lipid content of the Acid Fast Bacilli cell wall makes the mycobacteria more resistant to both acid and alkaline decontaminating agents Strict adherence to the timed killing period is necessary to maximize recovery
89. tle solid media slide DO NOT attempt to concentrate MAS by using lt 10 mL of reconstitution fluid NAA Pwo The following techniques help prevent crossover contamination between samples 1 Use of individual pour tubes for reagents being added to test Use of syringes or pipettes long enough to reach bottom of tube without touching sides of tube when sampling pellet 3 Careful recapping of centrifuge tube to include a Never having more than one tube top open at any given time b Never allowing a tube cap to be placed sample side down on working surfaces TIPS to REMEMBER All currently available digesting decontaminating agents are to some extent toxic to tubercle bacilli therefore to ensure the survival of the maximum number of bacilli in the specimen the digestion decontamination procedure must be precisely followed In order for enough tubercle bacilli to survive to give a confirmatory diagnosis it is inevitable that a proportion of cultures will be contaminated by other organisms It is also important to note that a laboratory which experiences no contamination is probably using a method that kills too many of the tubercle bacilli The generally accepted breakthrough contamination rate is 3 5 of specimens cultured on nonselective solid media If the rate is less that 3 the procedure is too harsh and might damage the mycobacteria 1 The goal is to inhibit the normal flora but not the hardier mycobacteria It is
90. tter control of the NaOH concentration and sample neutralization step and allow a uniform neutral final pH to be obtained for all samples This will minimize the BacT ALERT MP culture bottle false positive rate while helping to maximize Mycobacterium recovery rates EXERCISE SESSION See Appendix I for discussion The following techniques enhance Time to Detection by increasing the numbers of viable mycobacteria recovered from a sample Proper centrifugation at 3000 xg Immediate decantation after centrifugation Resuspension of centrifuged pellet with lt 2 ml of phosphate buffer Final pH of pellet between 6 8 and 7 5 Use of ONLY 0 5 mL of MAS DO NOT attempt to concentrate MAS by using lt 10 mL of reconstitution fluid oS Se a CLSI Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A Wayne PA Clinical and Laboratory Standards Institute 2008 Appendix b Global Customer Support K5 31JANO9 19 of 76 Mycobacterial Support Manual Best Practices in Sample Processing The following techniques help to control breakthrough contamination Transport specimens to lab promptly and refrigerate immediately Use of proper sample volume Use of NACL NaOH working concentration 2 processing reagent Complete vortexing 20 30 seconds with inversion and use of a timer Incubating 15 minutes at room temperature with use of a timer Inoculation of tests in correct order MP culture bot
91. user may choose to perform additional QC testing prior to use Table 2 Manufacturers Minimum Quality Control Requirements for Commercially Prepared Media Medium Incubation Control Organisms ATCC Expected Results Atmosphere Length No and Temperature Mycobacteria media COz lt 21 days 35 C M tuberculosis H37Ra 25177 Growth Lowenstein Jenson agar M kansasii Group 12478 Growth and Middlebrook broth M scrofulaceum Group II 19981 Growth May be inhibited medium used for recovery of on selective media AFB are exempt from user M intracellulare Group Ill 13950 Growth May be inhibited quality control provided that on selective media manufacturers certify acceptable performance M fortuitum Group IV 6841 Growth using the QC isolates listed E coli 25922 Inhibition partial to complete on selective media ATCC is a registered trademank of the American Type Cuture Collection C15 Laboratory Detection and Identification of Mycobacteria Approved Guideline CLSI document M48 A Wayne PA Clinical and Laboratory Standards Institute 2008 pg 61 gt NCCLS Quality Control for Commercially Prepared Microbiological Culture Media Approved Standard Third Edition NCCLS document M22 A3 ISBN 1 56238 536 4 NCCLS 940 West Valley Road Suite 1400 Wayne Pennsylvania 19087 1898 USA 2004 pg 4 i NCCLS Quality Control for Commercially Prepared Microbiological Culture Media Approved Standard Thi
92. ustomer Support K5 31JAN09 10 of 76 MEDIA OVERVIEW Mycobacterial Support Manual TRAINING SESSION BacT ALERT MB culture bottles MB BacT Enrichment Fluid BacT ALERT Blood Collection Adaptor Caps and Inserts BacT ALERT MB culture bottles GLASS with plastic sleeve MB BacT Enrichment Fluid A i k i A _ e Inoculate 3 5 ml blood into bottle eDirect with needle and syringe eDirect using adapter cap cap PN 210361 insert PN 210362 Sterile transport tube heparin sodium or lithium or SPS sodium polyanetholesulfonate tube Global Customer Support K5 31JANO09 11 of 76 Mycobacterial Support Manual MEDIA OVERVIEW EXERCISE SESSION see Appendix for answers 1 How many BacT ALERT MB culture bottles are in one case 2 What are the storage conditions for the BacT ALERT MB culture bottles and the MB BacT Enrichment Fluid 3 How many bottles can be prepared with one box of MB BacT Enrichment Fluid TIPS to REMEMBER BacT ALERT MB culture bottles are GLASS with a plastic sleeve DO NOT attempt to send these bottles through a pneumatic tube system Because the tops of these bottles are larger than the clinical plastic BacT ALERT culture bottles the adaptor cap for this bottle is also larger and specific for this bottle BacT ALERT MB culture bottles may be loaded into any cabinets BacT ALERT Classic or drawers BacT ALERT 3D The BacT ALERT

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