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MGIT TM Procedure Manual

1. Do not increase PANTA and NaOH concentration at the same time Try one at a time Once the contamination is under control bring the concentration back to the normal recommended concentration Specimen inoculation e Was specimen inoculation carried out inside the safety hood Never inoculate outside a safety cabinet Do not keep specimen tubes close to each other as this may result in cross contamination MGIT Procedure Manual C7 Appendix C e Were the tubes inoculated with the correct amount of specimen 0 5 ml for all systems Adding more of the specimen can affect PANTA concentrations or overwhelm the antibiotics resulting in higher contamination Do not open all the tubes Open one tube at a time for inoculation e Were the inoculated MGIT tubes mixed after the PANTA and specimen have been added Failure to mix may result in contaminating bacteria not coming in contact with the antibiotics Other factors e Was the elevated contamination rate occurring in general or can isolated incidences of increased contamination be identified Particular days A special situation may be responsible such as construction pollution a dust storm or dusty environment Particular season High contamination rate in summer is common Improve transport time and conditions High contamination associated with one person Some technicians are more experienced or careful than others Need retraining e Was contamination c
2. which is non radiometric and offers the same rapid sensitive and reliable methods of testing as the BACTEC 460 TB System BBL MGIT System is the manual system while BACTEC MGIT 960 MGIT 960 is the fully automatic system for detection of mycobacterial growth and drug susceptibility testing of M tuberculosis Numerous studies have been carried out using MGIT System for primary isolation of mycobacteria as compared with LJ and Ogawa media 17 18 19 20 21 22 23 24 25 26 and with BACTEC 460 TB 27 28 29 30 31 32 33 34 35 24 36 25 37 Go Similarly testing for drug susceptibility by MGIT has been thoroughly evaluated 2 MG susceptibility testing for PZA produces results similar to the BACTEC 460 TB system Some investigators have also evaluated MGIT 960 for second line and M avium complex drug susceptibility testing Better performance of MGIT as compared with other commercially available TB liquid culture and molecular amplification systems has also been reported 282 80 B Principle of the BACTEC MGIT 960 System 1 MGIT medium The MGIT Mycobacteria Growth Indicator Tube consists of liquid broth medium that is known to yield better recovery and faster growth of mycobacteria The MGIT contains 7 0 ml of modified Middlebrook 7H9 broth base This medium is terminally sterilized by autoclaving An enrichment MGIT OADC Oleic acid Albumin Dextrose and Catalase or MGIT 960 Growth Supplement is added to make
3. Appendix B Procedure for Ordering Cultures You may order these cultures from the American Type Culture Collection ATCC at the following address Specify the name of the organism and its ATCC number Refer to the ATCC catalog for details American Type Culture Collection 10801 University Boulevard Manassas VA 20110 2209 USA Phone 1 703 365 2700 Fax 1 703 365 2750 E mail news atcc org Users outside the United States should investigate the import regulations of their respective governments and obtain custom and or other government clearances as necessary prior to ordering materials 2 McFarland turbidity standard Already prepared McFarland Standards are available from BD The following procedure is given in case the commercial standard is not available a b Prepare 1 solution of chemically pure sulfuric acid in water Prepare 1 solution of chemically pure barium chloride in water Mix 0 1 ml of barium chloride solution with 9 9 ml of sulfuric acid solution in a clean properly washed test tube Seal the tube with tape or wax This is equal to McFarland No 1 Standard Add 0 05 ml of barium chloride to 0 95 ml of sulfuric acid or add 5 ml water to 5 ml of well mixed suspension of McFarland No 1 Standard will yield McFarland No 0 5 Accurate measurements are critical to achieve a workable McFarland Standard When comparing any turbidity with a McFarland Standard shake the tubes well Use same type an
4. Similarly inoculate 0 5 ml from Tube 4 for M fortuitum and Tube 5 for M kansasii into each of the two labeled MGIT tubes Mix e Enter the inoculated tubes in the MGIT 960 instrument Take the tubes out when indicated positive by the instrument Retrieve data for time to positive e Expected results lt M tuberculosis Tube fluorescence positive in 6 to 10 days gt M kansasii Tube fluorescence positive in 7 to 11 days gt M fortuitum Tube fluorescence positive in 1 to 3 days If the above criteria are not met repeat the test If QC test still does not give satisfactory results check the viability of the inoculum age of the culture if stored frozen and other procedures If everything meets the established specifications contact Technical Services at BD Diagnostic Systems f Precautions e Use freshly prepared suspension adjusted to McFarland 0 5 standard If frozen 70 C 5 C thaw prior to use for each quality control testing Do not store or refreeze once thawed e All work should be carried out in a proper biological safety cabinet e All materials should be sterilized by autoclaving prior to disposal e Follow all the recommended safety precautions 3 Quality control of laboratory procedures There should be periodic quality control checks of all the reagents used such as NaOH NALC and buffer as well as for procedures followed in the laboratory For better contamination monitoring it is important to include a negativ
5. The BACTEC MGIT 960 susceptibility test provides results similar to those obtained by BACTEC 460 Radiometric TB System within approximately the same timeframe The BACTEC MGIT 960 SIRE susceptibility test concentrations are slightly less than those used in the method of proportion on solid media in order to avoid false susceptibility results The BACTEC MGIT 960 susceptibility testing was evaluated thoroughly by comparing with Middlebrook 7H10 medium as well as with BACTEC 460TB System 5 41 4245 44 45 46 47 49 50 51 3 Reagents BACTEC MGIT 960 SIRE Kit for critical concentrations contains the following drugs in lyophilized form Each kit contains one each of S I R and E drug vial and 8 vials of MGIT 960 SIRE Supplement a Drugs gt Streptomycin approximate lyophilized drug per vial 332ug gt INH approximate drug per vial 33 2 ug gt Rifampin approximate drug per vial 332 ug gt Ethambutol approximate drug per vial 1660 ug b SIRE supplement The SIRE supplement vial differs from the MGIT Growth Supplement and contains per liter of purified water the following gt Bovine albumin 50 0 g gt Dextrose 20 0 g gt Catalase 0 03 g gt Oleic acid 0 6 g c Storage Upon receipt re
6. the specimens should be expectorated sputum and not saliva with a volume of about 2 10 ml each 2 Transportation Specimens should be transported to the laboratory as quickly as possible Delays in transportation especially in hot weather result in an increase in contaminating bacteria that result in higher contamination rate of the medium Specimens should be transported in a container such as an ice box in which temperature is maintained as low as possible This is especially important in countries with high ambient temperatures 3 Storage Upon receipt the specimens should be refrigerated and processed as soon as possible D Digestion Decontamination and Concentration Numerous procedures for digestion and decontamination have been in use throughout the world Some procedures are known to be compatible with egg based media only and may not be used with any other medium not containing egg yolk These procedures include Zephiran Trisodium Phosphate Z TSP Sodium Lauryl Sulphate Cetylpyridinium chloride CPC or other quaternary ammonium compounds It is extremely important to follow the standard procedure for decontamination recommended for MGIT in order to obtain optimal results Detection of growth in MGIT is based on an oxygen sensor system and high concentration of N Acetyl L Cysteine NALC or sodium hydroxide NaOH may result in false fluorescence Processing of specimens may vary according to their type The following is
7. 3 hours or overnight Do not overheat or expose smear to UV light e Perform all the above procedures in a biological safety cabinet Handle the smear carefully since mycobacteria may still be viable 2 Staining methods Commercially prepared staining kits are available for example BD Ziehl Neelsen Staining Kit BD Kinyoun s Staining Kit BBL Two Step Ouick Stain BBL Fluorescent Staining Kit see Appendix A for details These stains would give optimal results because they have been guality controlled However if the stains are to be made in the laboratory follow procedures given in Appendix B If commercially prepared stains are used follow manufacturer s recommendations Information about different stains is given in Appendix B a _ Ziehl Neelsen staining This method is used for staining smears made from specimen if fluorochrome staining is not available It is also used to stain fluorochrome positive smears for confirmation and for staining smears made from positive cultures Flood the slide with carbol fuchsin stain You may stain by covering the smear with filter paper and flooding the smear with the stain 8 Heat gently until steam rises electric stainer may be used for heating Do not allow the stain to boil or dry Keep adding stain as it dries during heating Stain for 5 10 minutes allow cooling Wash gently with water Decolorize with acid alcohol for 2 minutes or until no more color appears with acid alcoh
8. How will the final results be analyzed How will the reproducibility of the test system be evaluated It helps to test reproducibility of the test method If reproducibility is to be evaluated select a small number of test strains 5 10 with a few 2 5 resistant strains included in this testing Set up each isolate 2 5 times each on a separate day Set up the test and the reference method with freshly prepared inoculum each time and use the same inoculum for both methods which should be carried out at the same time c Preparation of inoculum e Work under a biological safety cabinet e From a positive 7 ml MGIT tube use the following guidelines for details refer to Section III A 4 C MGIT Procedure Manual D 2 Appendix D Day 0 the day a MGIT tube is positive by the instrument Re incubate at least one more day Day 1 or 2 one or two days after the instrument is considered positive Use undiluted for the susceptibility testing inoculation Day 3 4 or 5 dilute 1 5 and use for the susceptibility testing inoculation More than 5 days subculture in MGIT and follow the above criteria Procedures e Once a MGIT tube is instrument positive make an AFB smear and confirm the presence of typical cords of AFB without any contamination e Observe growth in the tube There should be detectable particulate growth in the tube e Make sure it is not contaminated turbid e Make sure it is a pure culture of M tuberc
9. Indicator Tube manual 4 ml carton of 25 tubes 245113 BBL MGIT Mycobacteria Growth Indicator Tube manual 4 ml carton of 100 tubes 245116 BBL amp MGIT OADC 15 ml carton of 6 vials Each vial sufficient for 25 MGIT Tubes manual 245122 BBL MGIT Mycobacteria Growth Indicator Tube 7 ml for BACTEC MGIT 960 carton of 100 tubes 245114 BBL amp MGIT PANTA Antibiotic Mixture lyophilized carton of 6 vials Each vial sufficient for 25 MGIT tubes 245124 BACTEC MGIT Growth Supplement Kit 6 vials 15 ml each BACTEC MGIT lyophilized PANTA Antibiotic Mixture 6 vials Each Growth Supplement PANTA vial sufficient for 15 18 MGIT tubes for BACTEC MGIT 960 220908 BBL Lowenstein Jensen Medium Slants package of 10 20 x 148 mm tubes with cap 220909 BBL Lowenstein Jensen Medium Slants carton of 100 20 x 148 mm tubes with cap 221174 BBL Middlebrook and Cohn 7H10 Agar package of 20 295939 BBL Middlebrook 7119 Broth 8 ml package of 10 tubes 221818 BBL Normal Saline 5 ml package of 10 231106 BBL Taxo X Factor Strips 1 vial 30 discs 240862 BBL MycoPrep Specimen Digestion Decontamination Kit ten 75 ml bottles of NALC NaOH solution and 5 packages of phosphate buffer 240863 BBL6 MycoPrep Specimen Digestion Kit ten 150 ml bottles 01 NALC NaOH solution and 10 packages of phosphate buffer 297297 McFarland Standard 0 5297298 McFarland Standard 1 0 needed
10. MGIT medium as compared to solid media If the amount of growth on solid media is very low e g 1 2 colonies there may be only a 50 chance of recovering the isolate in the MGIT or any other medium Corrective measures should be taken if the number of colonies is higher on solid medium and the MGIT culture is negative If there is an overall decrease in the culture positivity rate investigate the following parameters a Incubation If an incubator is used confirm that the temperature of the incubator is 37 C 1 C by placing a calibrated thermometer in various locations throughout the incubator or instrument drawers Monitor the readings several times each day until heating stability is determined Check temperature of the MGIT 960 by retrieving the information from the instrument Too frequent opening of the incubator door or the instrument drawers may have some adverse effect on the incubation temperature In extremely warm climates the incubation temperature may be difficult to maintain if the laboratory temperature is not well controlled Mycobacteria which require optimum temperature other than 37 C may fail to grow at 37 C Majority of mycobacterial species grow well at 37 C 1 C They may grow slowly or may not grow if the temperature drops below 35 C Mycobacteria that require temperature other than 37 C may not grow in the MGIT 960 instrument Specimens suspected of having these mycobacteria must be inoculated into two tubes
11. Tuberc Lung Dis 2000 4 866 870 1 Goloubeva V Lecocq M Lassowsky P et al Evaluation of mycobacteria growth indicator tube for direct and indirect drug susceptibility testing of Mycobacterium tuberculosis from respiratory specimens in a Siberian prison hospital J Clin Microbiol 2001 39 1501 1505 0 Idigoras P Beristain X Iturzaeta A et al Comparison of the automated nonradiometric BACTEC MGIT 960 system with Lowenstein Jensen Coletsos and Middlebrook 7H11 solid media for recovery of mycobacteria Eur J Clin Microbiol Infect Dis 2000 19 350 354 2 Jayakumar KV Forster T Kyi MS Improved detection of Mycobacterium spp using the BACTEC MGIT 960 system Br J Biomed Sci 2001 58 54 158 Lee JJ Lin CB Wang JD et al Comparative evaluation of the BACTEC MGIT 960 system with solid medium for isolation of mycobacteria Int J Tuberc Lung Dis 2003 7 569 574 23 Macondo EA Ba F Toure Kane NC et al Improvement of tuberculosis diagnosis by the Mycobacteria Growth Indicator Tube MGIT in a developing country laboratory Bull Soc Pathol Exot French 2000 93 97 100 24 Samra Z Kaufman L Bechor J et al Comparative study of three culture systems for optimal recovery of mycobacteria from different clinical specimens Eur J Clin Microbiol Infect Dis 2000 19 750 754 MGIT Procedure Manual Section IV References 25 Somoskovi A Kodmon C Lantos A et al Comparison of recoveries of Mycobacterium tuberculosis using the aut
12. a general outline of procedures for different types of clinical specimens MGIT Procedure Manual 14 1 Sputum Proper sputum collection is extremely critical for best results and early morning specimens are preferred The specimen should be expectorated sputum and not saliva that often would not yield correct results A specimen should be between 2 10 ml in volume Ideally from a new patient three specimens should be collected on consecutive days and should be processed separately WHO recommends two morning specimens and a third spot specimen when a patient visits the clinic Pooled specimens are not recommended a NaOH NALC procedure This is the standard recommended procedure to be used with MGIT which is also recommended by CDC In this procedure the initial concentration of NaOH is 4 This 4 NaOH solution is mixed with an equal quantity of sodium citrate solution 2 9 to make a working solution NaOH concentration in this solution is 2 When an equal quantity of NaOH NALC citrate and sputum are mixed the final concentration of NaOH in the specimen is 1 For the procedure for the preparation of NaOH NALC see Appendix B Commercially prepared NaOH NALC sodium citrate solution and phosphate buffer are also available BD MycoPrep Use of MycoPrep would minimize the use of non standard digestion solutions Cat No 240862 240863 Materials and Methods Materials Required Disposable 50 ml plastic tubes Falcon tu
13. et al Bacteriological investigation of unusual mycobacteria isolated from immuno compromised patients Diagn Microbiol Infect Dis 1993 6 321 323 13 Tenover FC Crawford JT Huebner RE et al The resurgence of tuberculosis is your laboratory ready J Clin Microbiol 1993 31 767 770 MGIT Procedure Manual Section IV References Bird BR Denniston MM Huebner RE et al Changing practices in mycobacteriology a follow up survey of State amp Territorial Public Health Laboratories J Clin Microbiol 1996 34 554 559 I5 Siddiqi SH Hwangbo CC A new antimicrobial mixture polymixin B amphotericin B naildixic acid trimethoprim and azlocillin for effective suppression of non mycobacterial contamination during primary isolation of mycobacteria 1986 ASM Annual Meeting Washington DC Abstract U35 Abe CK Hirano K Wada M et al Comparison of the newly developed MB redox system with Mycobacteria Growth Indicator Tube MGIT and 2 Ogawa egg media for recovery of mycobacteria in clinical specimens Kekkaku Japanese 1999 74 707 713 17 Apers L Mutsvangwa J Magwenzi J et al A comparison of direct microscopy the concentration method and the Mycobacteria Growth Indicator Tube for the examination of sputum for acid fast bacilli Int J Tuberc Lung Dis 2003 7 376 381 8 Chien HP Yu MC Wu MH et al Comparison of the BACTEC MGIT 960 with Lowenstein Jensen medium for recovery of mycobacteria from clinical specimens Int J
14. from wooden applicator sticks Try not to take any medium when removing growth Transfer the growth into the tube with broth and glass beads Tighten the cap and Vortex the tube for 1 2 minutes to break the clumps The turbidity of the suspension should be greater than the McFarland 1 0 standard Let the suspension stand undisturbed for 20 minutes Carefully transfer the supernatant suspension with a pipette into another sterile tube Avoid taking any growth which has settled on the bottom Let this tube stand for another 15 minutes undisturbed Carefully take the supernatant out with a pipette without disturbing the sediment and transfer into another sterile tube The turbidity of this suspension should be greater than McFarland 0 5 standard Adjust the turbidity of this suspension to McFarland 0 5 standard by adding sterile saline and adjusting by visual comparison Do not make it below this standard Dilute the above suspension 1 5 by adding 1 0 ml of the suspension to 4 0 ml of sterile saline Mix well and use it as the inoculum for drug susceptibility testing Inoculation and incubation Label 5 MGIT tubes for each test culture Label one for GC growth control without drug one for STR one for INH one for RIF and one for EMB Aseptically add 0 8 ml of BACTEC 960 SIRE Supplement to each of the MGIT tubes Use only MGIT SIRE Supplement and not MGIT Growth Supplement Aseptically add 0 1 ml 100 microliter or properly recon
15. it should be neutralized with NaOH before transportation or storage c Bronchial washings All other pulmonary specimens such as bronchial washings BAL may be treated as sputum If the specimen is up to 10 ml in volume process the whole specimen For larger volumes concentrate the specimen by centrifugation 3000x g 15 20 minutes If the specimen is thick or mucoid liquefy by adding a small quantity of NALC powder 50 100 mg After centrifugation resuspend the sediment in 5 ml sterile water and decontaminate like sputum d Laryngeal swabs Transfer the swab into a sterile centrifuge tube and add 2 ml sterile water If necessary break off the swab stick so the cap of the centrifuge tube can be placed on it and tightened Add 2 ml of NaOH NALC solution replace the cap and mix well in a vortex mixer Let stand for 15 minutes Remove the swab by with forceps squeezing the liquid out of the swab and discarding it Fill the tube with phosphate buffer Mix and centrifuge at about 3000x to 3500 g for 15 20 minutes Discard the supernatant fluid and resuspend the sediment in 1 2 ml sterile buffer Use this suspension for smear and culture e Tissue Tissue biopsies are generally collected aseptically and therefore decontamination procedures are not required Homogenize the tissue in a tissue grinder with a small quantity of sterile saline or water 2 4 ml All steps must be done in a biological safety cabinet BSC and all equipment must
16. one incubated at 37 C and the other incubated in another incubator at the optimum temperature required by the expected mycobacteria species Decontamination procedure e Confirm that purity and concentration of all the reagents used in the digestion decontamination procedure are satisfactory Do not use tap water Use distilled deionized water only for preparation of reagents e tis better to start with freshly prepared reagents MGIT Procedure Manual Cl Appendix C Do not use reagents that are not compatible with the MGIT System such as benzalkonium chloride Zephiran or sodium lauryl sulfate High pH of the specimen inoculated into MGIT medium may influence the performance of MGIT adversely Do not expose the specimen to the decontamination reagent for longer than the recommended time Check if MGIT tubes are positive by visual growth but negative by fluorescence b Centrifugation Relative Centrifugal Force RCF should be 3 000 5 000 x g A lower force decreases the recovery of mycobacteria Make sure the centrifuge is giving required RCF Generation of heat during centrifugation also lowers the recovery as at higher temperature NaOH becomes more lethal for bacteria Avoid generation of excessive heat A refrigerated centrifuge is recommended If refrigerated centrifuge is not available use chilled phosphate buffer which is added before centrifugation c _ Use of PANTA Check that PANTA is reconstituted with the
17. or high contamination rate follow the instructions for troubleshooting Appendix C Ouality Control for the reagents and products used in the isolation as well as for the test procedure is critically important for mycobacteriology laboratories see Section II K Ouality Control G Work up of Positive Cultures 1 AFB smear from a positive MGIT tube Once a MGIT tube is positive by fluorescence or by visual observation prepare a smear and stain with carbol fuchsin stain Procedure Use 8 clean 6 Mix the broth by vortexing and then by using a sterile pipette remove and aliquot Place 1 2 drops on the slide and spread over a small area approx 1 x 1 cm Let the smear air dry Heat fix the smear by passing it over a flame a few times or by using an electric warmer at 65 C 70 C for 2 hours to overnight Do not leave the smear openly exposed to the UV light of the safety cabinet e Stain the smear with Ziehl Neelsen Kinyoun s or BBL Quick Stain Fluorochrome stain is NOT recommended Air dry but do not blot dry Place a drop of oil on the stained and completely dried smear and screen under a low power objective to locate stained bacteria Switch to an oil immersion objective lens for detailed observation If the broth appears turbid or contaminated irrespective of AFB smear results subculture on a blood or chocolate agar or TSI to rule out the presence of contaminating bacteria If the smear is nega
18. or supplemented as reguired to meet the performance criteria There is a fluorescent sensor embedded in silicone on the bottom of the tube The tube is flushed with 10 CO at the time of filling and then capped with polypropylene screw caps Keep the caps closed until you are ready to make any addition to the medium Open the cap for as little time as possible b MGIT growth supplement enrichment MGIT growth supplement is provided for the BACTEC MGIT 960 7 ml tube For manual MGIT a different enrichment BBL MGIT OADC 15 ml is used The enrichment must be added to the MGIT medium prior to inoculation of a specimen MGIT growth supplement contains 15 ml of the following approximate formula gt Bovine Albumin 50 0 gm gt Dextrose 20 0 gm Catalase 0 03 gm Oleic Acid 0 1 gm Polyoxyethyline state POES 1 1 gm MGIT growth supplement or OADC enrichment is a sterile product Handle aseptically Do not use if turbid or if it appears to be contaminated Do not leave MGIT tube caps open after adding OADC It is important to add the growth supplement in the biological safety cabinet to avoid contaminating the medium c MGIT PANTA Contamination can be reduced by supplementing the medium with a mixture of antimicrobial PANTA prior to the inoculation of specimen Each vial of MGIT PANTA for MGIT 960 contains a lyo
19. proper volume The high concentration of PANTA may inhibit growth of some NTM bacteria especially if the number is low in the specimen 2 Delay in the detection time Detection time can range from 24 hours to six weeks for MGIT and weeks for LJ medium If the average detection time established in published studies or in a particular setting of a laboratory is significantly longer follow these instructions Digestion decontamination procedure Decrease the NaOH concentration and or time of exposure to NaOH Higher concentrations of NaOH or longer exposure time will prolong the detection time of mycobacteria High pH of the final inoculum will prolong the detection time MGIT Procedure Manual C 2 Appendix C e Check if the incubation temperature is within specifications Lower temperature would delay detection e In a few instances too high concentration of PANTA may delay the detection of certain strains of NTM especially if the starting number is low Procedures Check e Water used for preparation of reagents should be pure distilled deionized e All the reagents used should be sterile e All pipettes and tubes should be sterile e All inoculations should be made in the biological safety hood e Growth Supplement PANTA mixture should be added to MGIT tubes just prior to inoculation e It is important that the addition of MGIT OADC or MGIT Growth Supplemen PANTA should be done inside a biological cabinet Leave the
20. room temperature Lower temperatures reduce the digestion decontamination process of NAOH NALC 2 Specimens other than sputum extra pulmonary a Pus and other mucopurulent specimens If the specimen is thick or mucoid and less than 10 ml in volume digest and decontaminate with NaOH NALC method similar to the procedure used for sputum specimens If the specimen is not thick it may be treated with 2 4 NaOH The concentration of NaOH depends upon the contaminating bacteria expected to be present in the specimen If the volume is over 10 12 ml process only 10 ml or first concentrate by centrifugation at 3000x g for 15 20 minutes In such a situation if the specimen is thick liquefy the specimen by adding a small quantity of NALC only 50 100 mg powder and mix well After the concentration step resuspend the sediment in 5 ml sterile water decontaminate with NaOH and concentrate again by configuration Always resuspend the sediment pellet in buffer to reduce the pH MGIT Procedure Manual 18 b Gastric aspirates Concentrate by centrifugation before decontaminating Resuspend the sediment in about 5 ml of sterile water and decontaminate with NaOH NALC or 2 4 NaOH as recommended for sputum After decontamination concentrate again prior to inoculation of the sediment into culture media Due to the low pH gastric aspirates should be processed as soon as possible within 4 hours of collection If the specimen cannot be processed quickly
21. species are expected to be present incubate further for 2 3 weeks MGIT Procedure Manual 27 f Detection of positive growth The instrument signals a tube positive for growth and an indicator green light shows the exact location of the positive tube in the drawer of the instrument At this point the tube should be removed and scanned outside the instrument The tube should be observed visually Mycobacterial growth appears granular and not very turbid while contaminating bacterial growth appears very turbid Growth especially of the M tuberculosis complex settles at the bottom of the tube Information about the time to detection of positive growth can be retrieved from the unloaded positives report Time to detection of positive growth depends on several factors such as the number of viable AFB inoculated into MGIT tube the type of species of mycobacteria such as M tuberculosis and M bovis that grow more slowly than NTM such as M avium complex certain types of specimens such as specimens from extra pulmonary sites usually take a longer time to turn positive due to lower numbers of AFB present in the specimens the treatment status of patients also plays an important role Specimens from chronically treated patients with drug resistant TB take a longer time to grow processing of specimen influences the positivity as well A high pH or very low pH may cause injury or death to mycobacteria during processing o
22. specimen number by a marker Incubate these subcultures at 35 C 1 C and observe after 24 48 hours If contaminating growth appears confirm again by gram and ZN stain If contamination is confirmed with negative AFB smear from the broth discard the specimen and report as contaminated If contamination is confirmed with positive AFB smear from the broth repeat the isolation procedure see below How to control high contamination rate The following are steps to help reduce a high contamination rate during isolation of mycobacteria from clinical specimens Review all the procedures and make sure all recommended steps are followed closely If high contamination persists take the following measures Increase the NaOH concentration not more than 1 590 final concentration in the specimen The increase in NaOH is known to decrease contamination rate Do not increase the exposure time more than 25 minutes to NAOH NALC solution Increase the concentration of PANTA PANTA concentration may be increased by reconstituting with a smaller volume of Growth Supplement However the increase of PANTA concentration should be carefully evaluated since higher concentration of some antimicrobials present in PANTA may adversely affect growth of some species of mycobacteria other than M tuberculosis Imstead of 15 0 ml use 10 0 ml to reconstitute PANTA Add the regular 0 8 ml volume in the MGIT tube Do not change the NaOH concentration and PA
23. susceptibility test is a gualitative procedure to test susceptibility of M tuberculosis complex against PZA Results are obtained within 4 to 21 days The MGIT 960 medium is a modified 7H9 broth with a reduced pH of 5 9 The detection of growth is achieved by the oxygen sensor at the bottom of the tube in the same way as the one in the regular MGIT tube and the principle of the detection of resistance is the same as for SIRE drug susceptibility testing please refer to Section III A 2 3 Reagents The BACTEC MGIT 960 PZA medium tube contains 7 ml of broth It consists of 5 9 g of modified Middlebrook 7H9 broth and 1 25 Casein peptone per liter of purified water adjusted to pH 5 9 The BACTEC MGIT PZA Drug Kit contains two vials of 20 000 ug of lyophilized PZA and 6 vials of PZA supplement Each vial of the supplement contains 15 ml of enrichment with the following formula per liter of water gt Bovine albumin 50 0 g gt Dextrose 20 0 g gt Polyoxyethyline state POES l lg gt Catalase 0 03 g Olecic acid 0 1 g Storage instructions PZA medium may be stored at 2 25 C Do not freeze avoid exposure to light and do not use if found turbid Upon receipt PZA drug vials should be stored at 2 8 C Once reconstituted the antibiotic solution may be kept frozen at 20 C or colder for up to
24. test AST is a rapid broth based procedure it compares best with BACTEC 460 radiometric method from a workflow standpoint However there are some technical differences in the two broth based methods and results of the two methods would compare the best if these differences are understood and when the recommended procedures for BACTEC MGIT 960 and the reference method are strictly followed Some of the procedures are already included in the main drug susceptibility section of this manual and are repeated briefly for convenience These guidelines are meant to re emphasize the key points of the BACTEC MGIT 960 procedures b Planning the evaluation e_ First review the published data and look for accuracy and reproducibility of the new system Look for the comparative antimicrobial susceptibility testing AST data with BACTEC MGIT 960 and the reference method for both resistant and susceptible strains e Evaluate the system in your laboratory by comparing the BACTEC MGIT 960 method with a reference method Since BACTEC MGIT 960 is a broth based system it is often compared with the BACTEC 460TB system e BACTEC MGIT 960 susceptibility test can be set up from growth on solid medium or in liguid medium However it is recommended that initially it should be done from growth in the BACTEC MGIT 960 medium A standardized inoculum is critical for susceptibility testing and inoculum from a fresh positive MGIT tube is more easily standardized as compare
25. the medium complete This Growth Supplement is essential for growth of many mycobacteria especially those belonging to M tuberculosis complex Addition of the MGIT PANTA is necessary to suppress contamination MGIT Procedure Manual 10 2 Principle of detection and drug susceptibility testing In addition to Middlebrook 7H9 liquid media the MGIT tube contains an oxygen quenched fluorochrome tris 4 7 diphenyl 1 10 phenonthroline ruthenium chloride pentahydrate embedded in silicone at the bottom of the tube During bacterial growth within the tube the free oxygen is utilized and is replaced with carbon dioxide With depletion of free oxygen the fluorochrome is no longer inhibited resulting in fluorescence within the MGIT tube when visualized under UV light The intensity of fluorescence is directly proportional to the extent of oxygen depletion MGIT tubes may be incubated at 37 C and read manually under a UV light or entered into a MGIT 960 instrument where they are incubated and monitored for increasing fluorescence every 60 minutes Growth of bacteria as well as mycobacteria increases the fluorescence In case of M tuberculosis at the time of positivity there are approximately 10 10 colony forming units CFU per ml of medium The instrument declares a tube negative if it remains negative for six weeks 42 days The detection of growth can also be visually observed by the presence of a non homogeneous light turbidity or sm
26. tubes are evaluated If the GU of the drug containing tube is gt 100 then the result is interpreted as Resistant and if it is less than 100 it is Susceptible e Susceptibility results and GU values are printed in the Unloaded AST Set Report e GU values around 100 in the drug containing tube at the time when the growth control tube GU is around 400 may be due to borderline resistance typically caused by an isolate with a MIC near the test concentration but the instrument will interpret strictly according to the above rule e For AST sets entered in the instrument as undefined drugs the instrument marks the AST set complete when the GU of the growth control tube reaches gt 400 The Unloaded AST Set Report will print GU values but no interpretations will be provided A manual interpretation using the above rules should be made e GU values can be retrieved from the instrument Vial Inventory Report for all ongoing AST sets e Reports are given qualitative results such as Susceptible or Resistant according to the CLSI NCCLS recommendations e According to the CDC and CLSI recommendations always test at the critical concentration e Testing of higher concentrations is optional Clinical relevance of higher concentration testing is non defined MGIT Procedure Manual D 6 Appendix D e Usually higher concentrations are tested when a patient isolate is resistant to the critical concentrations
27. with the stain covering the whole smear all the time no filter paper no heating 8 After 15 minutes rinse the slide gently with water Drain excess water Flood the slide with 0 5 acid alcohol decolorizer and leave for 2 minutes Gently rinse the slide with water again Drain Cover the smear with potassium permanganate solution counter stain and leave for 2 minutes Do not over expose the smear to counter stain Rinse with water again Air dry and examine the smear under 10x then 40x objective using a UV light microscope for acid fast bacilli Caution Use chlorine free water as chlorine may interfere in the fluorescence Preferably use distilled or deionized water The time for counter stain is critical Do not exceed 2 minutes with potassium permanganate Do not blot dry 3 Microscopy It is important to examine smears very carefully Use a good binocular light microscope with oil immersion 100x objective and a 10x eye piece total magnification 1000x for ZN smears Examine a minimum of 100 fields for each smear before reporting as negative For smears found 3 to 4 only a few fields may need to be examined Report results as soon as possible Below is the recommended procedure of smear examination Lines with arrows indicate movement of the field observation by moving the slide under the lens of the microscope MGIT Procedure Manual 23 4 Reporting Report smear results as soon as the
28. 2 40 607 610 55 Walters SB Hanna BA Testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by Mycobacterium Growth Indicator Tube method J Clin Microbiol 1996 34 1565 1567 Aono A Hirano K Hamasaki S et al Evaluation of BACTEC MGIT 960 medium for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide PZA compared with the results of pyrazinamidase assay and Kyokuto PZA test Diagn Microbiol Infect Dis 2002 44 347 352 Pfyffer GE Palicova F Rusch Gerdes S Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide with the non radiometric BACTEC MGIT 960 system J Clin Microbiol 2002 40 1670 1674 Bastian I Rigouts L Palomino JC et al Kanamycin susceptibility testing of Mycobacterium tuberculosis using Mycobacterium Growth Indicator Tube and a colorimetric method Antimicrob Agents Chemother 2001 45 1934 1936 59 Piersimoni C Nista D Bornigia S et al Evaluation of new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the Mycobacteria Growth Indicator Tube J Clin Microbiol 1998 36 64 67 57 Alcaide F Benitez MA Escriba JM et al Evaluation of the BACTEC MGIT 960 and the MB BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA AccuProbe J Clin Microbiol 2000 38 398 401 8 Katila ML Katila P Erkinjuntti Pekkanen R Accelerated detect
29. Appendix C e Was the Growth Supplement added to the lyophilized PANTA with a sterile transfer device e Did PANTA completely dissolve in the solution IfPANTA is not completely dissolved this will cause contamination problem e Was the Growth Supplement added to PANTA inside a safety hood Addition on the bench top outside a safety hood may introduce environmental contamination e Was Growth Supplement PANTA added to the tubes by following the recommended procedures MGIT 960 0 8 ml tube Do not open all the MGIT tubes at one time Add into the tubes with a sterile transfer device Use of a repeat pipettor with sterile cotton plugged tip helps in reducing contamination To ensure maximum potency of the antimicrobials PANTA should be freshly reconstituted and added to the MGIT tube on the day the medium will be inoculated Be sure to mix MGIT tubes after all additions Mix by inverting the tube e What if a high contamination rate persists Ifa high contamination rate persists the PANTA concentration may be increased up to a maximum of 2 times the recommended concentration by reconstituting the PANTA with smaller quantities of reconstituting fluid Growth Supplement First try increasing the PANTA concentrations 25 35 by reconstituting the PANTA with a smaller quantity of water manual MGIT or Growth Supplement MGIT 960 Do not change the quantity of reconstituted PANTA supplement that is added to the MGIT tube
30. Drug Susceptibility Testing SIRE 1 Introduction Antimicrobial susceptibility testing is critical in prescribing an effective drug regime for a tuberculosis patient especially in areas where drug resistance incidence is high It is also important in the follow up of patients who are on antimicrobial therapy but are not responding to therapy Drug susceptibility testing is an integral part of the WHO DOTS Plus program The BACTEC MGIT 960 susceptibility testing for Streptomycin S Isoniazid I Rifampin R and Ethambutol E called SIRE is a rapid and qualitative procedure for establishing susceptibility of M tuberculosis for the four drugs using critical test concentrations In addition higher test concentrations for S I and E high E not available in US are also available in case of testing against higher concentrations is indicated 2 Principles of the test Isolated cultures from TB patients are subjected to growth in the presence of a known concentration of a test drug A control is also included with no addition of the drug If the patient s isolate grows in the control but does not grow in presence of the drug it is considered susceptible On the other hand if it grows in both the tubes then it is considered to be resistant to that drug There are several methods for susceptibility testing but the most common one is the proportion method In the proportion method resistance is established at the 1 level for most of the dru
31. Inoculate 0 5 ml of the culture suspension to the PZA tube using a sterile pipette For growth control inoculation first dilute the inoculum 1 10 by adding 0 5 ml of the culture suspension the one used for the drug tube to 4 5 ml of sterile saline Mix well by tightening the cap and inverting at least 5 6 times Add 0 5 ml of this diluted suspension into the tube labeled GC Note For PZA susceptibility test the inoculum for the control is diluted 1 10 and not 1 100 as in SIRE AST Tighten the caps and gently invert both the MGIT tubes several times to mix Place them in a two AST Set Carriers refer to the BACTEC MGIT 960 User s Manual for further information with the seguence of first GC and the PZA Enter the PZA set into the instrument using AST set entry feature refer to the BACTEC MGIT 960 User s Manual for further information Make sure the GC is placed first and PZA second in the AST Set Carrier Select PZA as the drug in 2nd tube AST set carrier definition when performing the AST set entry Check the purity of the inoculum by streaking a loopful of the culture suspension onto blood agar plate If a blood agar plate is not available use Chocolate agar BHI agar Incubate and check for growth after 48 hours If growth appears on the streaked plate discontinue the susceptibility test and do not use results of this susceptibility test Repeat testing after obtaining a pure culture Precautions All the additions and ha
32. NTA at the same time Try one procedure at a time and document improvement of results If there seems to be a common source of contaminating bacteria same kind of bacteria contaminating repeatedly check sterility of all reagents It is a good practice to dispense small guantities of reagents and use only one at a time Leftovers should be discarded or re sterilized Try to reduce time between collection of specimens and processing If a specimen needs to be stored use refrigeration Transport specimen with ice and in an insulated chest especially in hot weather Inverting the tube during the decontamination process helps decontaminate the inside surface of the top of the tube MGIT Procedure Manual 31 b If there is a persistent Pseudomonas contamination problem oxalic acid procedure is known to be more efficient for killing these bacteria However it has not been validated for MGIT Azlocillin in PANTA is very effective in the inhibition of pseudomonas growth increasing the PANTA concentration may help Isolation of mycobacteria from contaminated or mixed cultures Decontamination of contaminated culture Usually more than one specimen is collected from a patient and it is not necessary to salvage a contaminated specimen if other specimens from the same patient are positive and not contaminated However if it is critical to have results on a particular specimen that was contaminated the contaminated broth may be r
33. Prepared forthe Foundation for Innovative New Diagnostics FIND MGIT DIAGNOSTICS Procedure Manual For BACTEC MGIT 960 TB System Also applicable for Manual MGIT M ycobacteria Growth Indicator Tube MGIT Culture and Drug Susceptibility Demonstration Projects by Salman H Siddiqi Ph D BD Fellow Sparks Maryland USA and Sabine Rusch Gerdes Ph D Director National Reference Center for Mycobacteria Borstel Germany July 2006 TABLE OF CONTENTS Section I Principle of Procedures A B Introduction Principle of the BACTECTM MGITTM 960 TB System 1 MGIT medium 2 Principle of detection and susceptibility testing Section II Procedure for Primary Isolation A B C Introduction Important Safety Precautions Specimen Handling 1 Collection 2 Transportation 3 Storage Digestion Decontamination and Concentration 1 Sputum a NaOH NALC procedure b Other procedures c Important points 2 Specimens other than sputum extra pulmonary Pus and other mucopurulent specimens Gastric aspirates Bronchial washings Laryngeal swabs Tissue Urine Other body fluids 20 6 Smears for Acid Fast Bacteria AFB 1 Smear preparation 2 Staining methods a Ziehl Neelsen staining b Kinyoun s staining c Two step staining d Fluorochrome acid fast staining 3 Microscopy 4 Reporting MGIT Procedure Manual TABLE OF CONTENTS F Preparation and Inoculation for Cult
34. T tubes Addition of drugs to the medium Addition of inoculum Loading MGIT tubes in the instrument Interpretation of results i How to evaluate BACTEC MGIT 960 susceptibility results 2 Susceptibility testing and second line drugs hoe ho Roop MGIT Procedure Manual PREFACE The purpose of this procedure manual is to provide additional procedures and comprehensive instructions which may not be included in the package inserts of the BACTECTM MGITTM 960 System These measures and instructions should help in starting a new liguid culture system in a laboratory especially in developing countries This procedure manual provides guidelines for the BBL amp MGIT System which is a manual system and for BACTEC MGIT 960 TB System which is an automatic instrument system Because contamination of liguid media is a concern for new users special emphasis is given and guidelines for contamination control have been provided in different sections of the manual Every laboratory using the Mycobacteria Growth Indicator Tube MGITTM System should keep this manual readily available and should use this document as a reference for mycobacteriology procedures particularly for the MGIT System Optimal performance is achievable only if these procedures are strictly followed Further changes in the procedure or products if needed will be communicated with replacement pages which should be inserted into your copy of this manual For further procedural detai
35. al Avg Time to Detection Smear Negative Culture Positive Total Avg Time to Detection Smear Positive Culture Negative Total Smear Negative Culture Negative Total Positive Control Avg Time To Detection Contamination Total Avg Time To Detection VVVVVV Smear Positive smear of the clinical specimen Culture Positive calculate for both MGIT and any other medium used culture positive confirmed for AFB If there is an abrupt shift in any category of specimen data it would indicate some change in the laboratory practices or reagents Increase in contamination rate indicates that all the procedures should be reviewed and corrective measures should be taken to achieve satisfactory results If culture positivity or time to detection in MGIT is similar or lower as compared to the solid medium procedures need to be re evaluated since overall MGIT performance is expected to be better with earlier time to detection MGIT Procedure Manual 39 4 Record keeping Record the lot numbers for MGIT tubes MGIT OADC or MGIT Growth Supplement MGIT PANTA and other reagents Keep a record of the batch of specimens processed at one time date of inoculation person who did the work time to positivity by fluorescence smear results from positive tube contamination etc Compare results of MGIT with those of solid medium MGIT Procedure Manual 40 Section III Drug Susceptibility Testing A Primary
36. all granular flaky appearance in the medium Growth of some NTM most commonly rapid growers results in light turbidity while contaminating bacteria generally produce heavy turbidity Drug susceptibility testing can be performed based on the same principle Two MGIT tubes are inoculated with the test culture A known concentration of a test drug is added to one of the MGIT tubes and growth is compared with the MGIT tube without the drug growth control If the test drug is active against the isolated mycobacteria it will inhibit the growth and thus there will be suppression of fluorescence while the growth control will grow uninhibited and will have increasing fluorescence Growth is monitored by the BACTEC 960 instrument which automatically interprets results as susceptible or resistant MGIT Procedure Manual 11 MGIT Procedure Manual 12 Section II Procedure for primary isolation A Introduction Mycobacteria Growth Indicator Tube BBL MGIT contains modified Middlebrook 7H9 broth base When supplemented with MGIT Growth Supplement and PANTA it provides an optimum medium for growth of a majority of mycobacterial species All types of specimens pulmonary as well as extra pulmonary except blood can be inoculated into MGIT for primary isolation of mycobacteria Urine specimens have not been evaluated by BD but other investigators have reported successful isolation of mycobacteria from urine specimens Mucoid specimens are expec
37. aused by a common contaminant or various contaminants Establish if the contamination is coming from the environment from a constant source or from specimens Usually contamination with a specific type of bacteria or mycobacteria comes from a common source such as the water supply or contaminated reagent s If one particular contaminant persists check the sterility of reagents Also check the laboratory environment for a possible source Run negative control to check contamination during the processing Quality control e Are you carrying out regular Quality Control testing with negative and positive controls Recommendation Process 5 ml sterile buffer negative control along with regular batch of specimens processed in a day Process the negative control in the same way as clinical specimens and inoculate into MGIT tubes This would indicate if there is a source of contamination during the processing MGIT Procedure Manual C 8 4 Appendix C Periodic sterility testing of the reagents especially a freshly made batch is required to keep a check on contamination source from the reagents Use blood agar plate or any other suitable bacteria medium for checking contamination and Middlebrook agar or LJ medium for mycobacterial contamination check Environmental contamination may be reduced by thoroughly disinfecting the lab working inside a biosafety hood for all the additions and other processes and fixing the source
38. be sterile Decontaminate the homogenized specimen following the same NaOH NALC procedure as in sputum After resuspension of the sediment with phosphate buffer inoculate 0 5 ml MGIT tube If the tissue grinder is not available use a mortar and pestle Tissue may also be placed in a Petri dish with sterile water 2 4 ml and be torn apart with the help of two sterile needles Work under the hood and use sterilized materials MGIT Procedure Manual 19 1 Urine Isolation of mycobacteria from urine specimens has not been validated due to a very small number of urine specimens in BD clinical trials Some investigators have successfully used BACTEC 460 TB and MGIT medium for isolation of mycobacteria from urine Asa routine isolation method a totally voided early morning urine specimen is used for mycobacterial culture Pooled or mid stream urine specimens are not recommended The specimen is concentrated by centrifugation using several 50 ml centrifuge tubes with screw caps for at least 20 25 minutes Resuspend the sediment in each tube with 1 2 ml sterile water and then pool together total volume 5 10 ml Decontaminate the concentrated specimens with 4 NaOH for 15 20 minutes After decontamination proceed in a manner similar to sputum g Other body fluids Body fluids such as CSF synovial fluid and pleural fluid are collected aseptically and thus can be inoculated into MGIT medium without decontamination with the addition of PANTA Ho
39. bes Sterile NAOH NALC sodium citrate solution preferably MycoPrep Phosphate buffer pH 6 8 0 067M Commercially prepared MycoPrep or lab prepared and sterilized Centrifuge with a minimum 3000 3500 force and safety shield refrigerated centrifuge is preferred o Vortex mixer shaker Timer Pipettes transfer pipettes or a pipettor with cotton plugged pipette tips MGIT Procedure Manual 15 b If specimen is not collected in a 50 ml centrifuge tube transfer it to a 50 ml centrifuge tube with a screw cap Add NaOH NALC sodium citrate solution in a volume equal to the quantity of specimen Tighten the cap Vortex lightly or hand mix for about 15 30 seconds Invert the tube so the whole tube is exposed to the NAOH NALC solution Wait 15 20 minutes up to 25 minutes maximum after adding the NaOH NALC solution Vortex lightly or hand mix invert every 5 10 minutes or put the tubes on a shaker and shake lightly during the whole time Make sure the specimen is completely liguefied If still mucoid add a small guantity of NALC powder 30 35 grams directly to the specimen tube Mix well At the end of 15 20 minutes add phosphate buffer pH 6 8 up to the top ring on the centrifuge tube plastic tube has a ring for 50 ml mark Mix well lightly vortex or invert several times Addition of sterile water is not a suitable alternative for the phosphate buffer Centrifuge the specimen at a speed of 3000 g or m
40. blood in a specimen would inactivate NALC Vigorous shaking may also inactivate NALC Make sure that NALC powder is not expired e How are the reagents used Bulk reagents if used repeatedly become contaminated Cross contamination may also occur Reagents should be kept in aliquots Once used the leftover should be discarded or re sterilized if permitted Be careful in adding the reagent to the specimen tube Avoid touching the tube or creating any aerosol e Is the time for digestion decontamination 15 20 minutes 15 20 minutes of exposure to the reagent is ideal but up to 25 minutes may not hurt mycobacteria Less than 15 minutes is not long enough and will result in high contamination MGIT Procedure Manual C5 Appendix C More than 25 minutes would kill more contaminating bacteria but may injure mycobacteria which will result in the increase in time to detection In case of persistent high contamination time of exposure may be extended to a total of 25 minutes but it is preferred to increase the NaOH concentration 4 6 than increasing the time Do not increase NaOH concentration and time to exposure at the same time In summary longer exposure time of NaOH concentration means lower contamination and slower detection time Shorter exposure time or lower concentration of NaOH means a higher contamination rate and a shorter detection time You need to consider these factors e How often is the specime
41. control with 1 100 dilution of the inoculum MGIT Procedure Manual D 7 BD has not validated drugs other than SIRE and PZA However two studies including one multicenter study have been carried out in Europe for testing several secondline anti tuberculosis drugs with good reproducible results see Section III C If the Set Carrier is entered as undefined the growth control GC tube is monitored until a GU of 400 is reached At that point in time the AST set is marked as complete by the instrument Take out the susceptibility test carrier after scanning Print the Unloaded AST Set Report which will have GU values for the drug tubes Growth Control GU gt 400 gt Ifthe GU of the drug containing tube is 100 or more the result is reported as Resistant Ifthe GU of the drug containing tube is less than 100 the result is reported as Susceptible Remember the Instrument Inventory Report prints GU values for all ongoing AST sets MGIT Procedure Manual D 8
42. ction of bacterial growth with carbon 14 labeled glucose Radiology 1969 92 154 155 gt Kirihara JAL Hillier SL Coyle MB Improved detection times of Mycobacterium avium complex and M tuberculosis with the BACTEC radiometric system J Clin Microbiol 1985 22 841 845 Morgan MA Horstmeier CD DeYoung DR Roberts GD Comparison of a radiometric method BACTEC and conventional culture media for recovery of mycobacteria from smear negative specimens J Clin Microbiol 1983 18 384 388 7 Roberts GD Goodman NL Heifets L et al Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid fast smear positive specimens J Clin Microbiol 1983 18 689 696 s Siddiqi SH Libonati JP Middlebrook G Evaluation of a rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis J Clin Microbiol 1981 13 908 912 Siddiqi SH Hawkins JE Laszlo A Inter laboratory drug susceptibility testing of Mycobacterium tuberculosis by radiometric procedure and two conventional methods J Clin Microbiol 1985 22 919 923 10 Takahashi H Foster V Detection and recovery of mycobacteria by a radiometric procedure J Clin Microbiol 1983 17 380 381 Siddiqi SH Libonati JP Carter ME et al Enhancement of mycobacterial growth in middlebrook 7H12 medium by polyoxyethylene stearate Current Microbiol 1988 17 105 110 6 5100101 SH Laszlo A Butler WR
43. d size of tube for both the test and the McFarland Standard MGIT Procedure Manual B 2 3 Appendix B AFB stains Note Already prepared stains are available from BD If it is not possible to obtain commercially prepared stains follow these procedures to prepare the stains a Ziehl Neelsen stain Solution A Basic fuchsin 1 0 gm in 10 ml of 9590 ethyl alcohol Dissolve completely Solution B 5 0 gm phenol in 95 ml of 959e ethyl alcohol Mix gently Note The concentration of Basic fuchsin may differ in different procedures recommended in the literature Higher Basic fuchsin concentration helps in staining AFB better Filter the final staining solution if particles or precipitate is observed at any time Use distilled deionized water or filter the tap water through a bacterial filter In some instances high numbers of mycobacteria are present in tap water and may result in false positivity of smear Counter Stain Solution A 0 3 g Methylene blue chloride in 30 ml of 95 ethyl alcohol Dissolve completely Solution B 0 01g potassium hydroxide in 100 ml distilled deionized water Mix solutions A and B Filter if necessary to remove particles Kinyoun s stain Solution A Dissolve 4g of Basic fuchsin in 20 uL of 95 alcohol completely Solution B Dissolve 8g phenol crystals in 100 uL of distilled deionized water gentle heating may be required Mix solutions A amp B Filter with filter paper to remove particles Acid A
44. d to inoculum prepared from growth on solid media e Develop a protocol for this evaluation The protocol should include the following details Start and finish dates of study MGIT Procedure Manual Di Appendix D The personnel who would carry out actual testing Preferably one person who is well experienced in susceptibility testing should be responsible for the whole evaluation study Selection of a reference method which has been used successfully and routinely in your laboratory The reference method could be BACTEC 460TB system Middlebrook agar 7H10 or 7H11 or Lowenstein Jensen LJ medium Which drug concentrations will be used in the test and what will be the reference method The concentrations should have established eguivalency for both critical and higher concentrations How many total cultures are to be tested during the planned evaluation and how many cultures are to be tested in each set up Select a reasonable number of cultures preferably 20 50 with some known resistant strains 8 10 Preferably select some freshly isolated cultures and some from frozen stock cultures How will the discordant results be handled Cultures with discordant results may be retested with both methods or with a different method Another option is that an arbiter could be arranged who would test these in a different laboratory with the same or a different method How will the borderline resistant cultures be handled
45. dium follow the procedure for suspension preparation as described above The suspension may be stored in aliquots frozen for up to six months at 70 C 10 C for details of QC strain preparation see Section II K 2 If the QC batch fails that is the pan susceptible H3Rv shows some resistance then all the results obtained within that batch become invalid and the testing should be repeated MGIT Procedure Manual 51 C Secondline Drug Susceptibility Testing Procedures for secondline susceptibility testing in MGT are not provided by BD However recently two major studies have been carried out to evaluate susceptibility testing against secondline drugs with MGIT 960 These studies have established that the MGIT 960 system can be efficiently used for testing secondline drugs and have also provided procedures and the test concentrations 6 MGIT Procedure Manual 52 Section IV References Isenberg HD Editor Clinical Microbiology Procedure Handbook Vol 1 American Society for Microbiology Publisher Washington D C Kent PT Kubica GP Public Health Microbiology a Guide for the Level III Laboratory Centers for Disease Control Division of Laboratory Training and Consultation Atlanta GA US Department of Health and Human Services US Government Printing Office 1985 3 Middlebrook O Cohn ALL Bacteriology of tuberculosis Laboratory Methods Am J Pub Health 1958 48 844 853 DeLand FH Wagner RN Jr Early dete
46. e by the following observations a Rate of growth Generally M tuberculosis M bovis and to some extent M kansasii are slow growers and take a longer time to turn positive in a MGIT tube as compared to other mycobacteria NTM b In liquid medium growth M tuberculosis appears granular while growth of most NTM does not show flakes or granules rather it forms uniform slight turbidity except M kansasii c Smear made from a positive MGIT broth also helps in tentative differentiation of M tuberculosis complex from other mycobacteria Cultures belonging to the TB complex form typical clumps and serpentine cords while other mycobacteria appear as loose smaller clumps and cording or single cells M kansasii may be difficult to differentiate because its morphology is closer to M tuberculosis The identification of isolates grown in MGIT tube by molecular methods such as AccuProbe GenProbe Corp has been reported with good results amp 6 70 According to published reports AccuProbe testing of positive specimens can be performed within 24 hours of confirmation of MGIT Procedure Manual 33 positivity The lateral flow immunochromatography test CapiliaTB test has also been reported for differentiation of the M tuberculosis complex from NTM For complete speciation other methods such as biochemical testing can be used Although HPLC analysis has been successfully carried out on positive 12B vials HPLC analysis of grow
47. e control with a batch of specimens to be processed This could be done on a daily or weekly basis Periodically a positive control may also be included to monitor growth performance of a QC organism MGIT Procedure Manual 38 a Positive and negative controls For the negative control use 5 ml of phosphate buffer and for the positive control use 5 ml of M tuberculosis suspension McFarland 0 5 turbidity diluted 1 500 see quality control procedure Process negative and positive controls along with clinical specimens using the same digestion decontamination and concentration methods Inoculate into fresh MGIT tubes and incubate similarly to other specimens The positive control should show positive growth and time to detection should be within a specified time with each testing this is established by data collected from the positive control after several tests The negative control should show no growth within the incubation protocol period If negative control shows positive fluorescence check for the presence of bacteria mycobacteria If positive for growth investigate procedures and all the reagents for possible source of contamination b Quality control with laboratory data For overall performance of laboratory procedure periodic analysis of results helps in monitoring the lab efficiency and establishing good laboratory practices Every 3 to 6 months calculate the following statistics Smear Positive Culture Positive Tot
48. e negative control All the controls should be examined before the slides from clinical specimens The positive control should show good staining of AFB while the negative control should not show any AFB Certain mycobacteria other than M tuberculosis especially the rapid growers are easily decolorized and may give faint staining reactions To monitor the quality of MGIT Procedure Manual 35 microscopy technigues have a second person look at a selected number of positive and negative smears as well as those that have very few AFB on the smear Compare results of the two technicians For external guality control EOC follow the standard procedures established in the laboratory 2 Quality control QC testing of MGIT medium Every new lot of MGIT medium and every new lot of the enrichment should be quality control tested by the user upon receipt and before it is used routinely a OC strains Cultures The following three mycobacterial cultures are recommended for guality control testing gt M tuberculosis H37Rv ATCC 27294 gt M kansasii ATCC 12478 gt M fortuitum ATCC 6841 If the ATCC reference strains of M kansasii or M fortuitum cannot be obtained then a laboratory isolate which is well characterized may be used as a quality control strain Well characterized strains will be available from the mycobacterial strain bank of TDR WHO in late 2006 b Preparation of culture suspension e Subculture the above mycobac
49. ed 2000 124 82 86 z Bergmann JS Woods GL Reliability of mycobacteria growth indicator tube for testing susceptibility of Mycobacterium tuberculosis to ethambutol and streptomycin J Clin Microbiol 1997 35 3325 3327 7 Bergmann JS Woods GL Mycobacterial growth indicator tube for susceptibility testing of Mycobacterium tuberculosis to isoniazid and rifampin Diagn Microbiol Infect Dis 1997 28 153 156 48 Cambau E Truffot Pernot C Boulahbal F et al Mycobacterial Growth Indicator Tube versus the proportion method on Lowenstein Jensen medium for antibiotic susceptibility testing of Mycobacterium tuberculosis Eur J Clin Microbiol Infect Dis 2000 19 938 942 MGIT Procedure Manual Section IV References a Huang TS Tu HZ Lee SS et al Antimicrobial susceptibility testing of Mycobacterium tuberculosis to first line drugs comparisons of the MGIT 960 and BACTEC 460 Systems Ann Clin Lab Sci 2002 32 142 147 Reisner BS Gatson AM Woods GL Evaluation of Mycobacteria Growth Indicator Tubes for susceptibility testing of Mycobacterium tuberculosis to isoniazid and rifampin Diagn Microbiol Infect Dis 1995 22 325 329 Tortoli E Benedetti M Fontanelli A et al Evaluation of automated BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to four major anti tuberculous drugs comparison with the radiometric BACTEC 460TB method and the agar plate method of proportion J Clin Microbiol 200
50. eprocessed to recover mycobacteria 6 Transfer the entire MGIT broth into a 50 ml centrifuge tube Add an equal quantity of 4 sterile NaOH solution Mix well and let stand for 15 20 minutes mixing and inverting the tube periodically Add phosphate buffer pH 6 8 after 15 20 minutes up to 40 ml mark Mix well Centrifuge at least at 3000x g for 15 20 minutes Pour off the supernatant fluid Re suspend the sediment in 0 5 ml of buffer and mix well Inoculate 0 5 ml into a fresh MGIT tube supplemented with MGIT growth supplement PANTA Leave inoculated tubes at room temperature for 30 minutes and the place in the instrument and follow for observation of growth Isolation of mixed mycobacterial culture on Middlebrook Agar Plate AFB positive cultures with more than one mycobacterial species may be separated by streaking a small loopful of positive broth on a Middlebrook 7H10 or 7H11 agar plate Spread the loopful on the full surface of the plate to achieve isolated colonies Incubate the inoculated plate at 37 C 1C in a plastic bag Periodically observe for growth up to 6 weeks MGIT Procedure Manual 32 d Cross contamination Cross contamination of mycobacteria from specimen to specimen is also known in mycobacteriology laboratories Usually it happens during the processing of specimens especially at the time when a NaOH NALC solution is added to the specimen or when a buffer is added to the tubes Aerosol generation or s
51. es 7 Hasegawa N Miura T Yamaguchi K et al New simple and rapid test for culture confirmation of Mycobacterium tuberculosis complex a multi center study J Clin Microbiol 2002 40 908 912 72 Levidiotou S Papamichael D Gessouli E et al Detection of mycobacteria in clinical specimens using the mycobacteria growth indicator tube MGIT and the Lowenstein Jensen medium Microbiol Res 1999 154 151 155 75 TDR TB Website http www who int tdr diseases tb default htm 74 CLSI Website http www nccls org 75 Kruuner A Yates MD Drobniewski FA Evaluation of MGIT 960 based antimicrobial testing and determination of critical concentrations of first and second line antimicrobial drugs with drug resistant clinical strains of Mycobacterium tuberculosis J Clin Microbiol 2006 44 811 818 76 Rusch Gerdes S Pfyffer GE Casal M et al Multi center laboratory validation of the BACTEC MGIT 960 technigue for testing susceptibilities of Mycobacterium tuberculosis to classical second line drugs and newer antimicrobials J Clin Microbiol 2006 44 688 692 MGIT Procedure Manual MGIT Procedure Manual Appendix A Section IV Appendices APPENDIX A Supplies from Becton Dickinson amp Company BD The following is the list of supplies from BD related to mycobacteriology work 1 Supplies for Culture Work Catalog number Description 290020 Falcon Sputum Collection System 245111 BBL MGITTM Mycobacteria Growth
52. f the specimen Thus it takes longer for revival and growth of viable mycobacteria In some instances as many as 60 70 of the mycobacteria are killed during processing loss of mycobacteria during centrifugation is also significant If the centrifuge generates heat it will accelerate killing of mycobacteria during centrifugation Insufficient centrifugation speed may not bring down all mycobacteria into the sediment since mycobacteria being hydrophobic are difficult to concentrate It has been reported that in a broth system most of the mycobacterial species grow better and faster compared to the solid media 5 This is especially true for slow growing mycobacteria such as the M tuberculosis complex particularly from treated patients M avium which is a slow growing NTM on solid medium grows much faster in liguid medium MGIT Procedure Manual 28 Nofe In some instances especially if mycobacterial growth is extremely slow or there is less oxygen consumption during mycobacterial growth there may be growth in the MGIT broth without the presence of fluorescence It is recommended that at the termination of incubation protocol all negative tubes should be observed visually for turbidity and growth before discarding If there is any suspicion of growth an AFB smear and subculture should be done This eliminates chances of reporting false negatives If MGIT results are not satisfactory due to poor recovery delay in detection
53. for growth of M haemophilum MGIT Procedure Manual A 1 Appendix A 2 Supplies for AFB smears 212522 TB Stain Kit K For staining mycobacteria by 1 TB Carbolfuchsin KF the Kinyoun cold acid fast 1x250 ml TB Decolorizer procedure 1x250 ml TB Brilliant Green 1 x 250 ml 212520 TB Stain Kit ZN For staining mycobacteria by 1 TB Carbolfuchsin ZN the Ziehl Neelsen hot acid 1 x 250 ml TB Decolorizer fast procedure 1 x 250 ml TB Methylene Blue 1 x 250 ml 212315 TB Quick Stain Kit For a more rapid version of 1 TB Quick Stain Carbolfuchsin staining mycobacteria by the 1x250ml TB Ouick Stain Methylene Blue cold acid fast procedure 1x250 ml 212519 TB Fluorescent Stain Kit M For staining mycobacteria by 1 TB Auramine M the Morse Blair Weiser and 1 x 250 ml TB Decolorizer TM Sproat fluorescent procedure 1 x 250 ml TB Potassium Permanganate 1 x 250 ml 212521 TB Fluorescent Stain Kit T For staining mycobacteria by 1 TB Auramine Rhodamine T the Truant Brett and Thomas 1 x250 ml TB Decolorizer TM fluorescent procedure 1 x 250 ml TB Potassium Permanganate 1 x 250 ml 212514 TB Auramine M For staining mycobacteria by 4 x 250 ml the Morse Blair Weiser and Sproat fluorescent procedure 212515 TB Auramine Rhodamine T For staining mycobacteria by 4 x 250 ml the Truant Brett and Thomas fluorescent procedure 212523 TB Brilliant Green K For staining mycobacteria by 4 x 250 ml the Kinyoun cold and Ziehl Neelsen h
54. frigerate the lyophilized drugs at 2 8 C Reconstitute prior to use Once opened and reconstituted the leftover drug solutions may be frozen in aliquots at 20 C or lower and stored for up to 6 months or up to the date of original expiry whichever comes sooner Once thawed discard the leftover and do not store or refreeze MGIT Procedure Manual 42 4 Procedures a Reconstitution of lyophilized drugs Reconstitute each critical concentration drug vial with 4 ml of sterile distilled deionized water Mix thoroughly and make sure the drug is completely dissolved b Addition of a drug to the medium Add 0 1 ml 100 uL of reconstituted drug solution into each of the labeled BACTEC MGIT 960 tubes This will result in the following critical concentration of drugs in the medium gt Streptomycin 1 0 pg ml of medium gt Isoniazid 0 1 ug ml of medium gt Rifampin 1 0 ug ml of medium gt Ethambutol 5 0 ug ml of medium c Preparation of the inoculum Inoculum from the MGIT tube It is important that the growth is within the following recommended timeframe The day a MGIT tube is positive by the instrument is considered Day 0 The tube should be kept incubated for at least one more day Day 1 before being used for the susceptibility testing may be incubated in a separate incubator at 37 C 19C A positive tube may be used for drug susceptibility testin
55. g up to and including the fifth day Day 5 after it becomes instrument positive A tube that has been positive for more than 5 days should be subcultured in a fresh MGIT tube supplemented with MGIT 960 Growth Supplement and should be tested in a MGIT 960 instrument until it is positive Use this tube from one to five days of instrument positivity as described above If growth in a tube is on Day 1 or Day 2 mix well vortex to break up clumps Leave the tube undisturbed for about 5 10 minutes to let big clumps settle on the bottom Use the supernatant undiluted for inoculation of the drug set If growth is on Day 3 4 or 5 mix well to break up the clumps Let the large clumps settle for 5 10 minutes and then dilute 1 0 ml of the positive broth with 4 0 ml of sterile saline This will be a 1 5 dilution Use this well mixed diluted culture for inoculation MGIT Procedure Manual 43 Inoculum from growth on solid medium It is important to have fresh growth on a solid medium such as an LJ slant within 15 days of appearance of growth on the medium Older cultures may result in unreliable susceptibility test results Add 4 ml of BBL Middlebrook 7H9 broth to a clean sterile tube approximately 16 x 128 mm in size with 8 10 glass beads Use growth on solid medium which is no more than 15 days old within 15 days of appearance of positive growth Scrape as many colonies as possible with the help of a Sterile loop or a sterile spatula made
56. gs This means that if 1 or more of the total test bacterial population is resistant to a drug it is considered as resistant for clinical purposes Historically proportion method has used Middlebrook agar solid medium After 3 to 4 weeks of incubation the percentage of colonies on the drug medium as compared with the drug free medium is calculated to establish resistance In 1980 a broth based proportion method known as BACTEC 460TB radiometric susceptibility testing was introduced In this method a radiometric medium BACTEC 12B with a C labeled substrate is used The bacterial inoculum in the control is a hundredfold less than the inoculum in the drug containing medium The CO produced during growth and metabolism of mycobacteria in this medium is measured and designated as the Growth Index GI Once the GI in the control reaches 30 usually after 4 6 days incubation maximum 12 days comparison of GI values in the drug containing and drug free medium establishes the proportion of resistance MGIT Procedure Manual 41 The BACTEC MGIT 960 susceptibility test was also established with similar principles with the increase in the fluorescence in the sensor measured automatically and designated as growth value GV If a drug is added to the medium which is bacteriostatic or bactericidal to the test mycobacteria it inhibits growth and thus there is little or no oxygen consumption therefore little or no fluorescence of the sensor
57. hnique was widely used for blood culture using the BACTEC 460 instrument In 1980 this technique was introduced commercially for mycobacterial recovery from clinical specimens and drug susceptibility testing A large number of clinical trials were carried out to compare the radiometric BACTEC 460 TB System with solid media for primary isolation and drug susceptibility testing Several evaluations of the BACTEC 460 TB System published between 1980 and 1985 demonstrated excellent results with significant time savings 210 especially from smear negative specimens The BACTEC 460 TB System has been reported to yield 15 20 increased culture positivity of clinical specimens as compared to conventional solid media such as LJ medium with an average time to detection of positive growth from 8 to 14 days as compared to 3 to 5 weeks on solid media The introduction of the BACTEC 460 TB System revolutionized laboratory testing for mycobacteria and has established itself as the gold standard for culture and susceptibility testing The high efficiency of the BACTEC TB System is due to the use of liquid medium Moreover a growth enhancing substance is added to the medium to further reduce the detection time Since the introduction of the BACTEC 460 TB System it has been established that liquid medium is far superior to solid media for recovery time to detection and drug susceptibility testing Certain species of mycobacteria are reported to grow in liquid med
58. icient for 40 tests 245119 BD BACTEC MGIT 960 Manual SIRE Kit sufficient for 80 tests 245157 BD BACTECTM MGITTM 960 IR Kit sufficient for 40 tests 245128 BD BACTECTM MGITTM 960 PZA Drug Kit sufficient for 50 tests 245115 DB BACTEC MGITTM 960 PZA Tubes carton of 25 tubes 245125 BD BAACTECTM MGIT 960 Streptomycin 4 0 Kit sufficient for 20 tests 245126 BD BACTECTM MGIT 960 Isoniazid 0 4 Kit sufficient for 20 tests 245127 BACTECTM MGITTM 960 Ethambutol 7 5 Kit sufficient for 20 tests MGIT Procedure Manual A 4 APPENDIX B Miscellaneous Procedures amp Information 1 Strains of mycobacteria commonly used ATCC No Strain 21294 M tuberculosis H37Rv Susceptible to all TB Drugs 35820 M tuberculosis H37Rv Streptomycin resistant 35821 M tuberculosis H37Rv PAS resistant 35822 M tuberculosis H37Rv INH resistant 35826 A f tuberculosis H37Rv Cycloserine resistant 35827 M tuberculosis H37Rv Kanamycin resistant 35828 M tuberculosis H37Rv Pyrazinamide resistant 35829 M tuberculosis 113 7Rv Thiacetazone Amithiozone resistant 35830 M tuberculosis H37Rv Ethionamide resistant 35837 M tuberculosis H37Rv Ethambutol resistant 35838 M tuberculosis 113 7Rv Rifampin resistant 35721 M bovis 35775 M kansasii 35785 M scrofulaceum 13950 M intracellulare 35717 M avium American Type Culture Collection Refer to these ATCC strain numbers when ordering MGIT Procedure Manual B 1
59. ide PZA Susceptibility Testing 1 Introduction Susceptibility testing against PZA is always carried out at a lower pH of the medium since pyrazinamide PZA is active only at the low pH in vitro There are two methods widely used for PZA susceptibility testing In the proportion method Middlebrook 7H10 agar medium at pH 5 5 is used with 25 50 ug ml PZA Colony counts on the drug free and drug MGIT Procedure Manual 47 containing medium determine susceptibility However the pH of 5 5 is detrimental to mycobacterial growth and a significant number of test results cannot be determined because of poor growth or lack of growth The other method is the radiometric BACTEC 460TB method Here the BACTEC 12B medium is modified by reducing the pH to 6 0 At this pH mycobacteria grow better than at pH 5 5 To compensate for the increase in the pH the concentration of PZA is increased to 100 ug ml PZA susceptibility testing with BACTEC 460TB system has proven to be satisfactory and has been recommended by the Clinical and Laboratory Standards Institute CLSI previously known as NCLS The BACTEC MGIT 960 PZA test method is developed on the same principle as the BACTEC 460 method except that it is a non radiometric method and results are automatically interpreted by the instrument Results obtained by the MGIT method correlate well with those obtained by the BACTEC 460 method gt gt 2 Principles of the test The BACTEC MGIT 960 PZA
60. ies of NaOH and sodium citrate solution Prepare only as much volume as can be used in a day Add NALC powder to achieve a final concentration of 0 5 for 100 ml NaOH Na citrate solution add 0 5 g NALC powder Mix well and use the same day NALC activity is lost if left standing for more than 24 hours MGIT Procedure Manual B 4 Appendix B b Sodium hydroxide solution Prepare 4 NaOH solution by dissolving 4g of NaOH in 100 ml distilled deionized water Sterilize by autoclaving This solution can be stored and used for decontamination of nonmucoid contaminated cultures and specimens c Phosphate buffer pH 6 8 0 067 M e Dissolve 9 47g of anhydrous disodium phosphate NazHPO in 1000 ml 1 liter distilled deionized water using a volumetric flask e Dissolve 9 07 g monopotassium phosphate KH2PO in 1000 ml 1 liter distilled deionized water using a volumetric flask e Mix equal quantities of the two solutions Check the pH Adding more solution A will raise the pH more solution B will lower the pH The final pH should be 6 8 e Sterilize by autoclaving MGIT Procedure Manual B 5 MGIT Procedure Manual B 6 APPENDIX C Procedures for Troubleshooting 1 Decrease or no recovery of mycobacteria First determine the number and types of Mycobacterium sp that were missed in the MGIT medium and recovered in conventional media Generally there should be a higher number of mycobacterial species isolated in
61. inoculum d Inoculation and incubation Testing at higher drug concentrations Results Reporting Ouality control OC oo O ON Pyrazinamide PZA Susceptibility Testing 1 Introduction 2 Principles of the test 3 Reagents 4 Procedures a Reconstitution of lyophilized PZA drug b Preparation of the inoculum c Inoculation and incubation Results Reporting 7 Quality control nM Secondline Drug Susceptibility Testing Section IV References MGIT Procedure Manual TABLE OF CONTENTS Section V Appendices Appendix A Supplies from Becton Dickinson amp Company BD 1 Supplies for culture work 2 Staining kits 3 Supplies for drug susceptibility testing Appendix B Miscellaneous Procedures and Information 1 Stains of mycobacteria commonly used 2 McFarland turbidity standard 3 AFB stains a Ziehl Neelsen stain b Kinyoun s stain c Auramine O fluorescent fluorochrome stain 4 Reagents for digestion decontamination a NaOH NALC reagents b Sodium hydroxide solution c Phosphate buffer pH 6 8 0 067 M Appendix C Procedures for Troubleshooting 1 Decrease or no recovery of mycobacteria 2 Delay in the detection time 3 Guidelines to control high contamination 4 Cross contamination Appendix D Guidelines for Susceptibility Testing 1 Initial start and evaluation of BACTEC MGIT 960 susceptibility testing Introduction Planning the evaluation Preparation of inoculum Addition of supplement into MGI
62. ion and identification of mycobacteria with MGIT 960 and COBAS AMPLICOR systems J Clin Microbiol 2000 38 960 964 0 Williams Bouyer N Yorke R Lee HI et al Comparison of the BACTEC MGIT 960 and ESP Culture System II for growth and detection of Mycobacteria J Clin Microbiol 2000 38 4167 4170 MGIT Procedure Manual Section IV References Yan JJ Huang AH Tsai SH et al Comparison of the MB BacT and BACTEC MGIT 960 system for recovery of mycobacteria from clinical specimens Diagn Microbiol Infect Dis 2000 37 25 30 l Centers for Disease Control and Prevention National Institutes of Health Biosafety in microbiological and biomedical laboratories HHS Publication No CDC 99 8395 US Government Printing Office Washington 1999 Also available at www cdc gov od ohs biosfty bmbl4 bmbl4toc htm 4 Kent PT Kubica GP The sputum digestion process in mycobacteriology centrifugation efficacy and digestant toxicity Annual Meeting of American Society for Microbiology St Louis MO 1984 Abstract U2 Fadda G Roe SL Recovery and susceptibility testing of Mycobacterium tuberculosis from extra pulmonary specimens by the BACTEC radiometric method J Clin Microbiol 1984 19 720 721 4 Harris G Rayner A Blair J et al Comparison of three isolation systems for the culture of mycobacteria from respiratory and non respiratory samples J Clin Pathol 2000 53 615 618 65 Hanna BA Walters SB Bonk SJ et al Recovery of myc
63. ison of BACTEC MGIT 960 and BACTEC 460 for culture of Mycobacteria Diagn Microbiol Infect Dis 2000 38 123 126 A0 Witebsky FG Keiser JF Conville PS et al Comparison of BACTEC 13A medium and Du Pont Isolator for detection of mycobacteria J Clin Microbiol 1988 26 1501 1505 4l Abe C Aono A Hirano K Evaluation of the BACTEC MGIT 960 system for drug susceptibility testing of Mycobacterium tuberculosis isolates compared with the proportion method on solid media Kekkaku Japanese 2001 76 657 662 42 Ardito F Posteraro B Sanguinetti M et al Evaluation of BACTEC Mycobacteria Growth Indicator Tube MGIT 960 automated system for drug susceptibility testing of Mycobacterium tuberculosis J Clin Microbiol 2001 39 4440 4444 0 Adjers Koskela K Katila ML Susceptibility testing with the manual Mycobacteria Growth Indicator Tube MGIT and the MGIT 960 System provides rapid and reliable verification of multidrug resistant tuberculosis J Clin Microbiol 2003 41 1235 1239 4 Bemer P Palicova F Rusch Gerdes S et al Multi center evaluation of fully automated BACTEC Mycobacteria Growth Indicator Tube 960 system for susceptibility testing of Mycobacterium tuberculosis J Clin Microbiol 2002 40 150 154 a Bergmann JS Fish G Woods GL Evaluation of BBL MGIT Mycobacteria Growth Indicator Tube AST SIRE System for antimycobacterial susceptibility testing of Mycobacterium tuberculosis to 4 primary anti tuberculosis drugs Arch Pathol Lab M
64. ith confirmation by AFB smear Preferably after completion of identification M tuberculosis complex or MOTT bacilli CDC recommendation within an average of 14 days Speciate mycobacteria later and report c Upon completion of the drug susceptibility test susceptible or resistant to each test drug CDC recommendation within an average of 28 days d Culture Negative upon completion of the incubation protocol 42 days I _ Performance Characteristics Generally liguid media are reported to yield more positive cultures than the solid media with significant savings in time to detection of positive growth Numerous studies have been presented in scientific meetings or have been published in journals comparing performance of BACTEC MGIT 960 with BACTEC 460TB System and with conventional solid media 17 18 19 20 21 22 24 25 26 32 66 72 MGIT Procedure Manual 34 J Limitations of the Procedure a Colony morphology and pigmentation cannot be observed in a liguid medium b Even if a single viable contaminating bacterium survives the decontamination and PANTA inhibition it may contaminate the entire medium Contamination may mask mycobacterial growth c A positive culture from a clinical specimen cannot be correlated with colony forming units CFU present in the specimen which sometimes is used to establish important NTM infection d MGIT tube that appears positive may contain a mixed growth of more than one type of m
65. ium only thus failing to be detected on solid media In 1993 the Centers for Disease Control and Prevention CDC recommended that every clinical laboratory must use a liquid medium to isolate mycobacteria in conjunction with solid media A follow up survey indicated an increasing trend of using liquid medium for achieving rapid and maximum recovery of mycobacteria from clinical specimens MGIT Procedure Manual 9 Liguid media is more prone to contamination with bacteria that are commonly present as normal flora in certain types of clinical specimens and sometimes survive the decontamination process Thus addition of antimicrobials is needed to suppress contamination in liquid media With the BACTEC 460 TB System an antimicrobial mixture called PANTA Polymyxin B Amphotericin B Nalidixic Acid Trimethoprim Azlocillin is used for this purpose and reduces the contamination rate close to that generally experienced with solid media Some PANTA formulation is also used in newer liquid media that have been developed in recent years One of the disadvantages of the BACTEC 460 TB System is the use of C Labeled radioactive substrate Because of the strict regulations of handling and waste disposal of radioactive material it became necessary to develop a non radiometric technique for mycobacterial culture and susceptibility testing Becton Dickinson and Company BD developed a new system called Mycobacteria Growth Indicator Tube MGIT
66. lcohol Add 3 ml of concentrated hydrochloric acid to 95 ml of 95 ethyl alcohol Mix gently Counter Stains Methylene Blue Dissolve 0 3 gm Methylene blue chloride in 100 ml of distilled deionized water MGIT Procedure Manual B 3 Appendix B c Auramine O fluorescent fluorochrome stain Solution A Auramine O Dissolve 0 1g of Auramine in 10 ml 95 ethanol Solution B Phenol Dissolve 3 0 g of phenol crystals in 87 ml of distilled deionized water Mix solution A and Acid Alcohol Add 0 5 ml of concentrated hydrochloric acid in 100 ml of 70 ethanol Potassium permanganate Dissolve 0 5g of potassium permanganate KMn04 in 100 ml distilled deionized water 4 Reagents for digestion decontamination a _ NaOH NALC reagents Note This is the recommended procedure for MGIT System Commercially prepared NaOH NALC MycoPrep is available from BD These reagents are thoroughly guality control tested and yield the best results The following procedures are described in case these reagents are to be prepared in the laboratory Preparation e Prepare 4 NaOH solution by dissolving 4g NaOH pellets into 100 ml distilled deionized water Sterilize by autoclaving Concentration of NaOH may be varied 3 6 NaOH solution at the beginning e Prepare 2 9 sodium citrate solution by dissolving 2 9 g sodium citrate 21120 in 100 ml distilled deionized water Sterilize by autoclaving Mixing Prior to use mix equal quantit
67. ls used conventionally in mycobacteriology laboratories please refer to Clinical Microbiology Procedure Handbook Section 7 and Public Health Mycobacteriology Level 111 Guide CDC Handbook MGIT Procedure Manual 7 MGIT Procedure Manual Section I Principle of Procedure A Introduction Demonstration of acid fast bacilli AFB in a smear made from a clinical specimen provides a preliminary diagnosis of mycobacterial disease while the isolation of mycobacteria on culture provides a definite diagnosis of tuberculosis or disease due to mycobacteria other than M tuberculosis MOTT bacilli or non tuberculous mycobacteria NTM As much as 50 60 of AFB culture positive clinical specimens may fail to reveal AFB on smear made from the specimen As a consequence culture techniques play a key role in the diagnosis of mycobacterial disease Egg based media such as Lowenstein Jensen LJ or Ogawa have been used for cultivation of mycobacteria for several decades In 1958 Middlebrook and Cohn described an agar based medium to permit more rapid detection of mycobacterial growth However it still reguired an average of 3 4 weeks to recover mycobacteria from clinical specimens In 1969 Deland and Wagner developed a technigue for semi automated detection of the metabolism of bacteria by measuring the CO liberated during the growth and decarboxylation of C labeled substrate incorporated in the growth medium This radiometric tec
68. m clinical specimens of a university hospital with low incidence of tuberculosis J Clin Microbiol 2001 39 3764 3767 33 Palaci M Ueki SY Sato DN et al Evaluation of Mycobacteria Growth Indicator Tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens J Clin Microbiol 1996 34 762 764 Pinheiro MD Ribeiro MM Comparison of the BACTEC 460TB system and the BACTEC MGIT 960 system in recovery of mycobacteria from clinical specimens Clin Microbiol Infect 2000 6 171 173 355 Rohner P Ninet B Benri AM et al Evaluation of the BACTEC 960 automated non radiometric system for isolation of mycobacteria from clinical specimens Eur J Clin Microbiol Infect Dis 2000 19 715 717 A Scarparo C Piccoli P Rigon A et al Evaluation of the BACTEC MGIT 960 in Comparison with BACTEC 460TB for detection and recovery of Mycobacteria from clinical specimens Diagn Microbiol Infect Dis 2002 44 157 161 MGIT Procedure Manual Section IV References 37 Tortoli E Cichero P Piersimoni C et al Use of BACTEC MGIT 960 for recovery of mycobacteria from clinical specimens Multi Center study J Clin Microbiol 1999 37 3578 3582 38 Tsuyuguchi K Ikeda T Nakatani K et al Evaluation of the Mycobacteria Growth Indicator Tube system for detection and quantification of mycobacteria from clinical specimens Kekkaku Japanese 2003 78 389 393 0 Whyte T Cormican M Hanahoe B et al Compar
69. n mixed after addition of the digestion decontamination reagent Mixing of specimen should be done two to four times after the addition of digestion decontamination reagent Too much vortexing oxidizes the reagent and makes it less efficient Hand mixing is better Invert the tube a couple of times so the lip of the tube is well decontaminated Insufficient mixing does not allow the reagent to mix with the specimen and may cause higher contamination Is the phosphate buffer used to QC the sample to 50 ml obtained commercially MycoPrep sterile or autoclaved by the facility Addition of buffer is better than water Water is not recommended for MGIT If autoclaved in house check sterility and validate autoclaving procedure Addition of PANTA e Check storage conditions and expiry date of lyophilized PANTA refrigerated at 2 8 C Improper preparation or storage of PANTA can affect the performance or optimal concentrations e Is reconstituted PANTA stored properly and used in a timely manner MGIT 960 2 8 C within 5 days May not be frozen Manual MGIT 2 8 C within 72 hours May be stored at 20 C for up to 6 months must not exceed expiration date Must be used once thawed and may not be refrozen e Was PANTA reconstituted properly MGIT 960 15 ml of Growth Supplement per vial Measure out 15 ml of Growth Supplement as the vial has little more than a 15 ml capacity MGIT Procedure Manual C 6
70. ndicate that the results are ready Scan the susceptibility Set Carrier and print the report The instrument printout indicates susceptibility results for each drug Results are gualitative Susceptible S Resistant R or indeterminate X The instrument interprets results at the time when the growth unit GU in growth control reaches 400 within 4 13 days At this point the GU values of the drug vial are evaluated 5 Susceptible the GU of the drug tube is less than 100 R Resistant the GU of the drug tube is 100 or more MGIT Procedure Manual 46 X Error indeterminate results when certain conditions occur which may affect the test such as GU of the control reaches gt 400 in less than 4 days In such situations the test should be repeated with pure actively growing culture which is confirmed to be M tuberculosis complex Certain drug resistant strains grow very slowly in the medium and the results may not be achieved within 13 days with the standard inoculum In such a case the inoculum should be increased by decreasing the dilution of the culture suspension in order to get reportable results 7 Reporting Results must be reported as soon as they are available When reporting results it is important to include the name of the method used the type of drug and its level of concentration In case of resistance check the medium visually and make sure that the test culture is not contaminated look for
71. ndling of cultures should be done only inside a biosafety cabinet To avoid contamination use properly sterilized tubes reagents and other items Proper reconstitution of PZA drug and accurate addition of the drug to the medium is essential for getting correct results Preparation of inoculum is critical It should be as homogeneous as possible with the least amount of mycobacterial clumps Dilution 1 10 of the culture suspension for the growth control and mixing is critical Use only PZA Supplement and PZA medium for the PZA susceptibility test Make sure the tubes in the AST set carrier are placed in the proper seguence i e GC PZA MGIT Procedure Manual 50 5 Results The BACTEC 960 instrument will monitor the inoculated media and will give results within 4 21 days Growth Control reaches GU 400 or more once the test is complete At this point the susceptibility set can be removed after scanning and a report can be printed The susceptibility report will be S susceptible or R resistant If the GC tubes become positive in less than 4 days or remain negative up to 21 days or if some other conditions occur which may affect the test results the instrument report will show an Error X In such situations the test needs to be repeated The instrument interpretation of results is based on GU values as described for SIRE drugs Section III A 5 6 Reporting Report results as susceptible or resistant indicati
72. ng the method and concentration of the drug used Mono resistance to PZA is uncommon In case mono resistance is observed with a clinical isolate repeat the test and report results only when it is confirmed Cultures which are contaminated or belong to an NTM species or are a mixed culture of M tuberculosis and other mycobacteria will give erroneous results Strains of M bovis including M bovis BCG are also naturally resistant to PZA 7 Quality control It is extremely important to periodically perform a quality control of drug susceptibility testing for PZA The minimum requirement is to test each new batch of reagents such as PZA drug or MGIT PZA medium If the QC batch fails all the results obtained within that batch as well as the new batch of a reagent should be thoroughly reviewed and the testing should be repeated Use M tuberculosis H37Rv ATCC American Type Culture Collection number 27294 as a QC strain which is susceptible to all anti tuberculosis drugs It is not necessary to include a resistant strain as most of the resistant strains against a drug which are available from ATCC and other culture collections are highly resistant and do not give any added benefit in the quality control The test procedure for QC organisms is the same as described above for clinical isolates The inoculum could be from a freshly grown culture in the MGIT medium or on a LJ slant If the suspension of QC bacteria is made from growth on solid me
73. obacteria from blood in Mycobacteria Indicator Tube and Lowenstein Jensen slant after lysis centrifugation J Clin Microbiol 1995 33 3315 3316 OP Lu D Heeren B Dunne WM Comparison of the Automated Mycobacteria Growth Indicator Tube System BACTEC 960 MGIT with Lowenstein Jensen medium for recovery of mycobacteria from clinical specimens Am J Clin Pathol 2002 118 542 545 5 Tu HZ Chang SH Huaug TS et al Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification Mycobacterium tuberculosis complex Mycobacterium avium complex and Mycobacterium kansasii Ann Clin Lab Sci 2003 33 179 183 5 Banaiee N Bobadilla del Valle M Riska PF et al Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages J Med Microbiol 2003 52 557 561 Kontos F Petinaki E Nicolaou S et al Multicenter evaluation of the fully automated BACTEC MGIT 960 system and three molecular methods for the isolation and the identification of mycobacteria from clinical specimens Diagn Microbiol Infect Dis 2003 46 299 301 7 Kontos F Petinaki E Gitti Z et al Combined use of the fully automated BACTEC MGIT 960 System and a PCR restriction fragment length polymorphism analysis for routine detection and identification of mycobacteria from clinical samples J Microbiol Methods 2003 52 137 140 MGIT Procedure Manual Section IV Referenc
74. of contamination if established Cross contamination Cross contamination of mycobacteria from one tube to another is common in mycobacteriology laboratories Take the following precautions to prevent such cross contamination Use daily aliquots of NaOH NALC Citrate solution and PBS Any leftover should be discarded The buffer may be sterilized again by autoclaving Keep the specimen tubes tightly closed and clean them from outside prior to vortexing or shaking Pour decontamination reagents or buffer slowly on the side of the tube without causing any splashing Do not touch the container of reagents to the lip of the tube at the time of addition After vortexing wait awhile before opening the cap so that aerosol generated during the mixing settles down Open cap of specimen tube very gently to avoid aerosol generation When adding reagents to the tube open one tube at a time Do not keep all the tubes open at the same time Do not place tubes too close to each other in the rack MGIT Procedure Manual C 9 MGIT Procedure Manual APPENDIX D Guidelines for Susceptibility Testing 1 Initial start and evaluation of BACTEC MGIT 960 susceptibility testing a Introduction The following are the suggested guidelines for those BACTEC MGIT 960 users who would like to evaluate this system for susceptibility testing and compare it with a reference method such as the BACTEC 460TB method As the BACTEC MGIT 960 susceptibility
75. oid outside contamination Keep the specimen in cool conditions during transport preferably in an insulated ice box Transport to lab as guickly as possible gt Upon receipt keep in a cool place preferably in a refrigerator gt Process the specimen as soon as possible Specimen guality and guantity e What is the quality of the specimen being digested and decontaminated Too watery is not satisfactory It is mostly saliva and not the coughed up sputum This may yield poor AFB recovery but would not contribute to contamination If it is too mucoid it may need additional mucolytic reagent If not completely liquefied during the processing it may contribute to higher contamination During the digestion procedure if specimen is found not completely liquefied add a small quantity of NAC powder e What is the volume of the specimen being digested and decontaminated Should be 2 0 10 0 ml Lower volumes may yield fewer positive results Higher volumes may contribute to higher contamination Should not be pooled Pooled specimens may result in high contamination Specimen processing e What is the method of specimen digestion decontamination NALC NaOH is the method of choice Recommended NaOH concentration of 4 is ideal final concentration in the specimen 1 Increase in NaOH usually results in lowering the contamination rate Higher NaOH concentration up to 1 5 in the specimen is acceptable in situations whe
76. ol Wash gently with water Drain excess water Pour Methylene Blue counter stain on the smear and leave for 2 minutes Wash gently with water Drain excess water Air dry and observe under microscope Do not blot dry as it may remove smear accidentally MGIT Procedure Manual 21 b Kinyoun s staining The procedure for staining is the same as with the Ziehl Neelsen method except heating carbol fuchsin is not necessary since it is a cold staining method see Appendix B Kinyoun s stain is used in place of the Ziehl Neelsen ZN method c Two step staining The BD Quick Staining Kit is available and does not require heating or decolorizing see Appendix A This is a superior method because the decolorizing step has been eliminated by adding the decolorizing agent into the counter stain This is a replacement for Ziehl Neelsen or Kinyoun s staining method The procedure for staining is the same as the Kinyoun s method except there is no decolonization step After washing the carbol fuchsin stain apply the counter stain Follow the procedure recommended by the manufacturer d Fluorochrome acid fast staining This method is recommended for quick screening of large numbers of specimens for the presence of acid fast bacteria AFB Commercially prepared stains are available for fluorescent staining BD Brand Fluorescent Stain see Appendix A Among their advantages fluorochrome stained smears are much quicker to
77. omated BACTEC 960 system the BACTEC 460 TB system and Lowenstein Jensen medium J Clin Microbiol 2000 38 2395 2397 Zaruba R Kralova M Evaluation of the effectiveness of the BACTEC MGIT automatic system for culture of mycobacteria in comparison with classical methods of culture Epidemiol Microbiol Immunol 2002 51 66 70 27 Ardito F Sanguinetti M Posteraro B et al Comparison of the mycobacteria growth indicator tube with radiometric and solid culture for isolation of mycobacteria from clinical specimens and susceptibility testing of Mycobacterium tuberculosis New Microbiol 2000 23 151 158 28 Badak FZ Kiska DL Setterquist S et al Comparison of Mycobacteria Growth Indicator Tube with BACTEC 460 for detection and recovery of Mycobacteria from clinical specimens J Clin Microbiol 1996 34 2236 2239 2 Flanagan PG Williams R Paull A Comparison of two automated systems for the isolation of mycobacteria from clinical specimen Eur J Clin Microbiol Infect Dis 1999 18 912 914 30 Hanna BA Ebrahimzadeh A Elliott LB et al Multicenter evaluation of the BACTEC MGIT 960 system for recovery of mycobacteria J Clin Microbiol 1999 37 748 752 31 Kanchana MV Cheke D Natyshak I et al Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria Diagn Microbiol Infect Dis 2000 37 31 36 32 Leitritz L Schubert S Bucherl B et al Evaluation of BACTEC MGIT 960 and BACTEC 460TB systems for recovery of mycobacteria fro
78. ommonly used and has not been validated for MGIT It is recommended only for urine specimens or those specimens which cannot be processed by NaOH or NaOH NALC method due to persistent problems with bacteria contamination Zephirain Trisodium Phosphate Method Z TSP This method is compatible with egg based media only and does not work with any other solid or liquid media DO NOT USE THIS METHOD FOR MGIT Cetylpyridinium Chloride CPC Method A mixture of 1 0 CPC and 2 0 NaCl is used as a transport medium as well as decontamination reagent This is a slow acting decontamination reagent This method is not compatible with a non egg based medium DO NOT USE THIS METHOD FOR MGIT Benzalkonium Chloride and Lauryl Sulfate Methods These methods are not compatible with non egg based media and should not be used with the MGIT system Important points NaOH is bactericidal for contaminating bacteria It is also harmful for mycobacteria but to a much lesser extent NaOH also helps in liquefying the specimen NALC only liquefies the specimen and has no decontamination properties The final pH of the specimen concentrate greatly affects the recovery and time to detection of mycobacteria High pH will lower the positivity rate and increase the time to detection of positive culture High pH may also cause transient false fluorescence Keep the pH as close to neutral as possible It is not necessary to neutralize the processed specimen es
79. ore for 15 20 minutes Use of refrigerated centrifugation at a higher speed is known to increase recovery of mycobacteria After centrifugation allow tubes to sit for 5 minutes to allow aerosols to settle Then carefully decant the supernatant into a suitable container containing a mycobactericidal disinfectant Make sure the sediment is not lost during decanting of the supernatant fluid Add a small quantity 1 2 ml phosphate buffer pH 6 8 and resuspend the sediment with the help of a pipette or vortex mixer Use the resuspended pellet for making smears and for inoculation of MGIT tubes and other media Other procedures These methods are routinely used in laboratories for other culture systems NaOH Method Sodium hydroxide NaOH alone Petroff s Method is used with the starting concentration of 3 4 NaOH A higher concentration of NaOH could be toxic to mycobacteria and could affect the oxygen sensor adversely The procedure is the same as the one for NaOH NALC Add buffer after 20 minutes of decontamination and after centrifugation to reduce the pH MGIT Procedure Manual 16 Oxalic Acid Method Conventionally oxalic acid 5 aqueous solution is used only for those specimens which have a persistent Pseudomonas contamination problem Neutralization of the specimen with an alkali after digestion and decontamination is preferred This method has not been validated for MGIT Sulfuric Acid Method This method is not c
80. ortexing e Using a suspension that is too heavy or too light or with low viability may alter the AST results d Addition of supplement into MGIT tubes e Work under a biological safety cabinet to avoid outside contamination e Use BACTEC MGIT 960 Growth Supplement for growth and BACTEC MGIT 960 SIRE Supplement for susceptibility testing Using the wrong supplement will alter the AST results e Use of automatic pipettor with sterile tips for adding the supplement helps the workflow and would reduce the chance of contamination e Open one tube at a time and do not leave the cap off too long e Close the cap and mix well e Addition of drugs to the medium e Work under a biological safety cabinet e When reconstituting a drug vial make sure the drug is completely mixed in the solution Partially dissolved drug solution may give false resistance e Add exactly 100 uL 0 1 ml of a drug to the properly labeled tube Use a properly calibrated sterile pipette or a pipettor with sterile tip with a cotton plug MGIT Procedure Manual D 4 Appendix D e Do not add drug to the growth control e Always follow the seguence of G S I R E and make sure each drug is added to its respective labeled tube e Use a separate pipette or pipette tip for each drug e Do not leave the MGIT tube cap open for too long f Addition of inoculum e Work under a biological safety cabinet e Add 0 5 ml of the appropriate inoculum into each drug con
81. ory is discharged to the outdoors the ventilation is balanced to provide directional airflow into the room access to the room is restricted when work is in progress and the practices and eguipment recommended for BSL 3 are followed This includes use of proper protective gowns gloves and respirator masks approved by OSHA while handling specimens and mycobacterial cultures International Safety Standards along with the local specifications may also be followed Use an appropriate mycobacterial disinfectant such as Amphyl6 for cleaning the work area The CDC states With so many disinfectants available it is important to consult the product brochures to make certain the disinfectant is bactericidal for mycobacteria MGIT Procedure Manual 13 Prior to use examine all MGIT tubes for evidence of damage Do not use any tube that is cracked or has other defects Do not use a tube if the medium is discolored cloudy or appears to be contaminated Comprehensive reviews of laboratory safety procedures may be found in recognized publications of the Centers for Disease Control the American Society for Microbiology or other International and National guidelines C Specimen Handling 1 Collection Specimens should be collected in clean preferably sterile containers with a tight fitted lid or cap At least two morning specimens collected on separate days should be processed for each new case For patients with respiratory symptoms
82. ot acid fast procedures MGIT Procedure Manual A 2 2 212518 Supplies for AFB smears continued TB Carbolfuchsin KF For staining mycobacteria by the Kinyoun cold acid fast procedure Appendix A 4 x 250 ml 212511 TB Carbolfuchsin ZN For staining mycobacteria by the Ziehl Neelsen hot acid fast procedure 4 ml 212517 212512 TB Decolorizer TB Decolorizer TM For staining mycobacteria by the Kinyoun cold and Ziehl Neelsen hot acid fast procedures For staining mycobacteria by the Truant Brett and Thomas and the Morse Blair Weiser and Sproat fluorescent procedures 4 x 250 ml 4 x 250 ml 212516 TB Methylene Blue For staining mycobacteria by the Kinyoun cold and Ziehl Neelsen hot acid fast procedures 4 x 250 ml 212513 TB Potassium Permanganate For staining mycobacteria by the Truant Brett and Thomas and the Morse Blair Weiser and Sproat fluorescent procedures 4 x 250 ml 212316 TB Ouick Stain Carbolfuchsin For a more rapid version of staining mycobacteria by the cold acid fast procedure 3 x 250 ml 212317 TB Quick Stain Methylene Blue MGIT Procedure Manual For a more rapid version of staining mycobacteria by the cold acid fast procedure 3 x 250 ml A 3 Appendix A 3 Supplies for drug susceptibility testing Catalog number Description 245123 BD BACTEC MGITTM 960 SIRE Kit suff
83. particularly in the case of INH gt e If an isolate is resistant at the low level of INH and susceptible at the high concentration it is considered low level resistance CLSI e Isolates with low level resistance may give inconsistent results when repeatedly tested with the same method or even another method e If a new patient isolate is found resistant the resistance should be reconfirmed by the same or different method e Mono resistance especially in cases of Rifampin Ethambutol and PZA is rare and should be confirmed before reporting i Howto evaluate BACTEC MGIT 960 susceptibility results e Carry out parallel testing with BACTEC 460TB or another reference method and BACTEC MGIT 960 using the same culture inoculum Carefully follow the recommended procedure for both methods Use the critical concentrations of drugs e With discrepant results first check the purity of the culture e Ifthe culture is pure repeat testing with both methods e If possible retest with the critical as well as with the higher concentrations S I E with both methods This would help in determining if the culture is borderline resistant e Include a QC strain such as H37Rv with each susceptibility test set up H37Rv strain should yield reportable results with complete susceptibility to all the test drugs at the critical concentrations If any discrepancy is observed with this strain discard all the results obtained in that set up e Analy
84. pecially with the NaOH NALC method Some laboratories routinely neutralize the processed specimen The neutralization step needs to be controlled very carefully MGIT Procedure Manual 17 With NaOH NALC digestion do not agitate the tube vigorously Extensive aeration causes oxidation of NALC and makes it ineffective e If the specimen has some blood mixed with it do not use NaOH NALC method because NALC does not work in the presence of blood Use the NaOH method instead Mycobacteria being hydrophobic are hard to centrifuge down Lower centrifugation speed g force would not sediment mycobacteria very well and some bacteria would be lost during decanting the supernatant which will affect the positivity rate Higher centrifugation speeds and longer time maximum 25 minutes result in a better concentration of mycobacteria which positively affects smear and culture positivity Temperature increase during centrifugation increases the killing effect on mycobacteria which will decrease the positivity rate and increase time to detection A refrigerated centrifuge with at least 3000x g force is ideal If a refrigerated centrifuge is not available avoid temperature build up especially if the room temperature is high Add refrigerated chilled phosphate buffer before centrifugation which should help in keeping the temperature low Other reagents during the digestion decontamination step should not be refrigerated but kept at
85. philized mixture of the antimicrobials with the concentrations at the time of production as follows gt Polymyxin B 6 000 units lt Amphotericin B 600 ug gt Nalidixic Acid 2 400 ug gt Trimethoprim 600 ug gt Adzlocillin 600 ug For manual MGIT the procedure for adding PANTA differs but the final concentrations of PANTA antimicrobials in the medium are the same in both the systems MGIT Procedure Manual 25 2 Procedures a _ Reconstituting PANTA Reconstitute MGIT PANTA with 15 0 ml MGIT growth supplement Mix until completely dissolved Add 0 8 ml of this enrichment to each MGIT tube The enrichment with reconstituted PANTA should be added to the MGIT medium prior to inoculation of specimen in MGIT tube Do not add PANTA enrichment after the inoculation of specimen Do not store MGIT tube after the addition of enrichment PANTA b _ Inoculation of MGIT medium e Label MGIT tubes with specimen number e Unscrew the cap and aseptically add 0 8 ml of MGIT growth supplement PANTA to each MGIT tube Use of an adjustable pipettor is recommended e Using a sterile pipette or a transfer pipette add up to 0 5 ml of a well mixed processed concentrated specimen to the appropriately labeled MGIT tube Use separate pipette or pipette tip for each specimen e Immediately recap the tube tightly and mix by inverting the t
86. plashing during the addition causes cross contamination by contaminating the next tube or by contaminating the reagent stock solution Touching the lip of the specimen tube with the reagent container during pouring or adding of the reagent may also lead to high contamination Sometimes stock solution of a reagent gets contaminated with mycobacteria commonly found in water M gordonae M xenopi Aliquoting small quantities reduce the chances of cross contamination In the event of a cross contamination episode all reagents equipment and biosafety cabinets must be thoroughly checked 3 Sub culturing a positive MGIT tube It is always helpful to subculture a MGIT tube positive for mycobacteria on an LJ slant At the time positive mycobacterial growth is detected there is sufficient biomass to use this growth for making a smear performing drug susceptibility testing or performing other tests such as species identification However growth on solid medium is important for observation of colony morphology and chromogenicity for biochemical testing and speciation or for a future reference For subculture inoculate approximately 0 1 0 2 ml of a well mixed positive MGIT broth on an LJ medium This is especially important if MGIT is used as a stand alone system All subcultures should be incubated at 37 C 19C and be examined periodically until good growth is detected 4 Identification of isolated mycobacteria Tentative differentiation may be mad
87. r some of the suspension to another sterile tube and adjust the turbidity to McFarland 0 5 standard This is the working suspension for QC testing This suspension may be frozen in small aliquots 1 2 ml in appropriate tubes vials at 70 C 10 C The frozen suspension may be used up to 6 months Once thawed do not refreeze c Preparation of dilutions Dilute the working suspension McFarland 0 5 freshly prepared or frozen 1 5 by taking 1 0 ml of suspension and adding into 4 0 ml of sterile water or saline Mix well Tube 1 Dilute two more times 1 10 by adding 0 5 ml of suspension Tube 1 into 4 5 ml of sterile water or saline Tube 2 Mix well and then again add 0 5 ml from Tube 2 to 4 5 ml of sterile saline or distilled deionized water Tube 3 Mix well Final dilution 1 500 Tube 3 Stop here for M tuberculosis and use Tube 3 for QC testing For M fortuitum further dilute Tube 3 1 10 Take 0 5 ml of suspension from Tube 3 and add to 4 5 ml of sterile water or saline and mix well Final dilution 1 5000 Tube 4 Use Tube 4 for QC testing For M kansasii dilute Tube 4 once again 1 10 by adding 0 5 ml from Tube 4 to 4 5 ml of sterile saline water mix well Final dilution 1 50 000 Tube 5 Use Tube 5 for QC testing MGIT Procedure Manual 37 d JInoculation incubation e Supplement MGIT medium with Growth Supplement and PANTA as recommended e Inoculate 0 5 ml from Tube 3 to each of two MGIT tubes for M tuberculosis
88. re contamination is a serious problem Once the contamination problem is under control try to lower the NaOH concentration gradually and bring it to the recommended concentration MGIT Procedure Manual C 4 Appendix C Certain digestion decontamination procedures are not compatible for liquid media such as Cetyl pyridinium chloride transport and processing method e Are reagents made in house or bought Commercially prepared reagents such as MycoPrep are quality controlled However the NaOH concentration in these is the standard 4 If an increase in NaOH is needed additional NaOH becomes necessary If prepared in the laboratory check the concentration of the reagents carefully Jf sterilized in the lab check autoclaving procedures and sterility of reagents e If method is NAOH NALC is the solution made daily NaOH and sodium citrate solutions may be prepared sterilized and kept for a long time Once NALC is added the solution is only effective for 24 hours and should be made fresh daily e How much NALC is added to the NaOH and Na citrate Check NALC concentration It should be at least 0 590 An insufficient amount of NALC will not digest the specimens enough to allow NaOH to come in contact with contaminating bacteria NALC helps in digestion but does not decontaminate NaOH does liguefy as well as decontaminate a specimen Add additional NALC in case of thick and highly mucoid specimens Presence of
89. rsely affect the performance of the MGIT medium e Incubation Incubation Temperature All inoculated MGIT 7mL tubes should be entered in the BACTEC MGIT 960 instrument after scanning each tube please refer to the BACTEC MGIT 960 Instrument Manual for details It is important to keep the cap tightly closed and not to shake the tube during the incubation This helps in maintaining the oxygen gradient in the medium The instrument maintains 37 C 1 C temperature Since the optimum temperature for growth of M tuberculosis is 37 C make sure the temperature is close to 37 C Note If a specimen is suspected of containing mycobacteria which require an optimum temperature other than 37 C for example M haemophilum M marinum M chelonae and M ulcerans require 30 C then two sets of media should be inoculated one in the instrument at 37 C and the other in an outside incubator at 30 C These tubes can be monitored by using a UV light source Wood s lamp and can also be checked visually refer to the BACTEC MGIT 960 Manual Specimens from skin and open wounds should always be inoculated into duplicate MGIT tubes one for 37 C and the other for 30 C Length of incubation MGIT tubes should be incubated until the instrument flags them positive After a maximum of six 6 weeks the instrument flags the tubes negative if there is no growth Some species such as M ulcerans and M genavense may require extended incubation time If such
90. screen which offers time and labor savings over Ziehl Neelsen or other carbol fuchsin methods Screening can be done faster using lower magnification 250x to 450x magnification compared to the ZN staining 800x 1000x magnification and thus a larger area can be covered within the same timeframe Fluorochrome staining is considered more sensitive than the ZN staining method for detecting AFB on a smear However it requires an expensive UV microscope and since the stained smears are not stable they should be read preferably the same day overnight refrigerated storage is acceptable It is recommended that fluorochrome smears that are positive for AFB should be re stained with any of the carbol fuchsin staining methods to confirm the results looking for AFB morphology which cannot be easily detected in fluorochrome stains This practice should be followed at least during the initial phase of starting fluorochrome staining and also for periodic quality control checking of this method Fluorochrome staining is not recommended for smears made from positive cultures Two fluorescent stains commonly used are Auramine O and Auramine Rhodamine With Auramine O staining mycobacteria appear bright yellow fluorescent color while with Auramine Rhodamine staining mycobacteria develop yellow orange fluorescent color The staining procedure is the same for both stains MGIT Procedure Manual 22 Procedures Shake the bottle of stain and flood the slide
91. six months but should not to exceed the original expiration date Once thawed do not store or refreeze MGIT Procedure Manual 48 The PZA supplement should be stored at 2 8 C upon receipt Avoid freezing or overheating and use prior to the expiration date Minimize exposure to light 4 Procedures a Reconstitution of lyophilized PZA drug Reconstitute each of the PZA drug vials with 2 5 ml of sterile distilled deionized water Mix well The reconstituted drug solution will contain 8000 ug ml of PZA b Preparation of the inoculum The PZA susceptibility test is recommended for a pure culture of M tuberculosis complex The test culture should be thoroughly checked for its purity and a confirmed identification of M tuberculosis Preparation from a positive MGIT tube Use a freshly positive MGIT tube as described in the section for SIRE testing Please refer to Section III A 4 Day 0 the day a MGIT tube is positive by the instrument Re incubate Day 1 or 2 one or two days after instrument positive Use undiluted for the susceptibility testing inoculation Day 3 4 or 5 mix well and dilute 1 5 by adding 1 0 ml of positive broth in 4 0 ml of sterile saline Mix well Use this for the susceptibility testing inoculation Day 6 and onward subculture in a fresh MGIT tube and follow the above guidelines Caution Avoid mycobacterial clumps by mixing the growth well vortex and let it stand for 5 10 minutes Take the s
92. stituted STR drug in the STR labeled tube Similarly add other drugs in the other labeled tubes It is important to add the correct amount of drug to each tube If possible use a well calibrated micropipette for each addition Use a separate pipette or micropipette tip for each drug Do not add any drug to the GC tube MGIT Procedure Manual 44 Concentration of drug Volume added to Final concentration in Drug after reconstitution MGIT tube MGIT tube STR 83 ul ml 100 ml 1 0 pl ml INH 8 3 ul ml 100 ml 0 1 pl ml RIF 83 ul ml 100 ml 1 0 ul ml EMB 415 plml 100 ml 5 0 ul ml The drugs should be reconstituted using 4 ml sterile deionized or distilled water to achieve the indicated concentrations Calculations of the dilution factor for MGIT medium 7 0 ml of medium 0 8 ml of SIRE Supplement 0 5 ml of inoculum 8 3 ml Addition of 0 1 ml of the drug solution in 8 3 ml of the medium 1 83 dilution Aseptically add 0 5 ml of the well mixed culture suspension inoculum into each of the drug containing tubes using a pipette Do not add to the control For the control first dilute the test culture suspension 1 100 by adding 0 1 ml of the test culture suspension to 10 0 ml of sterile saline Mix well by inverting the tube 5 6 times Use this diluted suspension to add 0 5 ml into the growth control tube Tighten the caps and mix the inoculated broth well by gently inverting the tube several times Susceptibility tes
93. t Set Carriers are provided in different numbers of drug combinations For a routine SIRE test with critical concentration a Set Carrier of five tubes is used refer to BACTEC MGIT 960 User s Manual for details Place labeled tubes in the correct sequence in the set carrier GC STR INH RIF EMB Enter the susceptibility set carrier into the BACTEC MGIT 960 instrument using the susceptibility test set entry feature Refer to the BACTEC MGIT 960 User s Manual AST Instructions Ensure that the order of the tubes in the AST Set Carrier conforms to Set Carrier definitions For example GC STR INH RIF EMB for the SIRE standard testing If you need to check purity of the inoculum streak the test culture suspension onto a blood agar plate If blood agar is not available use chocolate agar or BHI agar Incubate at 35 C 19C for 48 hours and check if there is any growth If growth appears do not set up the susceptibility test It may be important to establish the purity of culture before setting up susceptibility test particularly if contamination is suspected MGIT Procedure Manual 45 5 Testing at higher drug concentrations It is recommended that SIRE drugs should always be tested at the critical concentration However in certain situations testing at a higher concentration is indicated This is important in those isolates which have a low level of resistance that is an isolate is resistant at the critical concentra
94. taining MGIT tube Do not add to the Growth control e Dilute the inoculum 1 100 for SIRE or 1 10 for PZA and inoculate 0 5 ml to the growth control tube e Make sure the inoculum is well homogenized and well mixed e Use a separate sterile pipette or sterile tip with cotton plug for each test culture e Tighten the caps and mix the medium by inverting three to four times g Loading MGIT tubes in the instrument e Make sure the tubes are in the proper AST Set Carrier and in the proper order for example Growth Control Streptomycin INH Rifampin and Ethambutol C S I R E or in case of PZA Control PZA e Select the proper Set Carrier definition when entering the AST set in the instrument as the instrument can read the tubes only according to the defined Set Carrier order e There are 2 3 4 5 and 8 AST Set Configurations available MGIT Procedure Manual D 5 Appendix D h Interpretation of results e BACTEC MGIT 960 SIRE AST is a 4 13 day and MGIT 960 PZA is a 4 21 day qualitative test Results are interpreted and reported by the instrument as S susceptible or R resistant or X uninterpretable e The BACTEC MGIT 960 instrument continuously monitors the fluorescence of tubes in terms of Growth Units GU Predefined algorithms compare the GU of a drug containing tube with the growth control tube e When the growth control reaches a GU of gt 400 between 4 13 days for SIRE and 4 21 days for PZA the drug containing
95. ted to contain contaminating bacteria as normal flora and must be digested liquefaction and decontaminated before inoculation On the other hand aseptically collected body fluids or tissue biopsies do not need to be decontaminated However since it is difficult to maintain sterile conditions throughout the collection of specimens it is recommended that all specimens be decontaminated Aseptically collected specimens need only light decontamination Clinical specimens collected in large volumes of more than 10 ml require centrifugation before decontamination to reduce the overall volume and to concentrate mycobacteria present in the specimens into a smaller volume After decontamination the specimen should be centrifuged again and the sediment used for preparation of smear and inoculation for culture B Important Safety Precautions Perform all procedures such as processing of specimens smear preparation inoculum preparation making dilutions inoculation of media and subculturing in a suitable biological safety cabinet in a room dedicated for mycobacterial work The CDC has recommended a Biosafety Level BSL 2 laboratory with negative air pressure and with an appropriate ventilation system for mycobacterial work More recently the CDC has recommended that work involving manipulation of TB cultures such as DST be done in a BSL 3 laboratory However this work may be done in a BSL 2 laboratory providing the exhaust air from the laborat
96. teria on several LJ slants e Incubate at 37 C 1 C e Observe growth visually e As soon as there is good confluent and pure growth use this growth for making suspension e Growth should appear within 10 15 days of subculturing and should be used within this period Aged cultures would not give reliable results e Remove growth from the slant by carefully scraping the colonies off the slant with a sterile loop or sterile spatula made from wooden applicator sticks Take extreme precaution not to scrape off any culture medium which gives false turbidity measurement MGIT Procedure Manual 36 Transfer growth into a Screw cap tube containing 4 ml of sterile 7H9 broth and glass beads 6 10 beads 1 2 mm diameter which helps to break up clumps Tube A Vortex the tube for at least 1 2 minutes Make sure the suspension is well dispensed and very turbid greater than McFarland 1 turbidity Let the suspension stand undisturbed for 20 minutes Using a transfer pipette carefully transfer the supernatant to another sterile screw cap glass tube Tube B Avoid picking up any sediment Let this stand undisturbed for 15 minutes Carefully transfer the supernatant into another screw cap glass tube Tube C without taking any sediment Adjust the turbidity of suspension in Tube C to McFarland 0 5 turbidity standard by adding more 7H9 broth or sterile saline deionized water and mix well If the suspension is too turbid transfe
97. th in the MGIT tube has not been published yet H Results Reporting If viable mycobacteria are present in an inoculated specimen they will grow in the MGIT medium and will be detected visually as well as by fluorescence Report results only when a MGIT tube is positive by the instrument and smear made from the positive broth is also positive for AFB Do not report a positive culture unless smear made from the positive tube is definitely positive for AFB In rare cases a MGIT tube may be negative in the instrument but will be positive by AFB smear and or subculture In such a case report positive results Reports should be sent as soon as results are ready In case the identification requires additional time results may be reported as culture positive for AFB identification pending If possible it is better to identify the M tuberculosis complex by molecular probe or other rapid tests and report results once identification is known Negative cultures should be reported after completing the incubation protocol of the instrument and visual observation of the negative tubes Contaminated cultures must be reported as contaminated after confirmation by smear or subculture on bacterial medium Conventionally reports are sent at the following points a Smear from specimen fluorochrome or ZN Report positive or negative and the staining method used CDC recommendation within 24 hours of receipt of specimen b Culture Positive w
98. tion but susceptible at the higher concentration of the drug Thus many laboratories first test at the critical concentration of a drug and if an isolate is found resistant then the higher concentration is tested Only Streptomycin INH and Ethambutol are tested at the higher concentration Among these three drugs INH is the most important drug as clinicians may choose to continue INH in the therapeutic regime if the isolated culture of a patient is resistant at the critical concentration and susceptible at the higher concentration called low level resistance The following higher concentrations of S I and E are available for BACTEC MGIT 960 System BACTEC MGIT STR 4 0 Kit contains one vial of lyophilized streptomycin 664 hg and two vials of SIRE Supplement BACTEC MGIT INH 0 4 Kit contains one vial of lyophilized isoniazid 66 4 ug and two vials of SIRE Supplement BACTEC MGIT EMB 7 5 Kit contains one vial of lyophilized ethambutol 1245 ug and two vials of SIRE Supplement For the higher concentration of drugs reconstitute the lyophilized drug with 2 0 ml of sterile deionized water and then add 0 1 ml to the MGIT medium The final concentration of the drug in the medium should be 4 0 ug ml of medium gt INH e 0 4 ug ml of medium lt EMB 7 5 ug ml of medium 6 Results The instrument monitors the entered susceptibility test set Once the test is complete within 4 to 21 days the instrument will i
99. tive for AFB and the tube does not appear to be contaminated i e broth is clear re enter the tube into the instrument for further monitoring Repeat AFB smears after 1 3 days MGIT Procedure Manual 29 2 Dealing with contamination Liguid media are more prone to contamination than solid media It is extremely important to process specimens with extreme care adhering very closely to procedures and recommendations Following are guidelines for controlling excessive media contamination for further details please refer to Appendix C 3 Troubleshooting a _ Bacterial contamination The incidence of contamination with bacteria other than mycobacteria varies from laboratory to laboratory depending upon several factors According to the CDC guidelines up to 5 contamination rate is acceptable in cultures of clinical specimens on solid media A general recommendation is that 5 290 is acceptable for all media types However for liquid media slightly higher contamination may be accepted up to 7 8 Very low contamination rate less than 3 may indicate too harsh a decontamination process which would also affect growth of mycobacteria and may reduce the positivity rate and increase time to detection of positive mycobacterial culture On the other hand a higher contamination rate above 8 may be due to the following reasons Improper or under decontamination of specimen Very mucoid specimens that are hard to liquefy may res
100. tube open for as little time as possible Leaving the tube open especially on an open bench top would increase the contamination rate 3 Guidelines to control high contamination The normal acceptable contamination rate is 590 2 for solid media For liguid media a slightly higher contamination rate is expected Bacterial contamination is also an indicator of level of effective decontamination procedure High contamination rate indicates improper decontamination procedure while too low contamination indicates over treatment of the specimen that could also lower the culture positivity rate or increase the detection time If in the MGIT it is more than 7 8 then the decontamination procedure is not satisfactory and corrective measures should be taken If there is a problem of increased contamination flowing guidelines would be helpful Some of the procedures are already included in the main section of this manual and are repeated here briefly for convenience Specimen collection and transport e Are specimens collected in sterile containers e What are the conditions for specimen transport and storage prior to processing MGIT Procedure Manual C 3 Appendix C Is transport time too long The longer the transport time the more likely the probability of contamination Are weather conditions too hot Higher contamination is more likely in hot weather if specimen is transported Collect specimens in clean and sterile containers to av
101. turbidity and put 1 drop of the medium on an agar plate or that the test culture belongs to NTM In case of unexpected results or mono resistance against Rifampin PZA or Ethambutol repeat the test to verify resistance 8 Quality control QC It is extremely important to perform a guality control of drug susceptibility testing periodically The minimum reguirement is to test each new batch of reagents such as SIRE drugs or MGIT medium If the batch OC fails all the results obtained within that batch as well as the new batch of a reagent should be thoroughly reviewed and the testing should be repeated Use M tuberculosis H37Rv ATCC American Type Culture Collection number 27294 as a OC strain which is susceptible to all anti tuberculosis drugs It is not necessary to include a resistant strain as most of the resistant strains against a drug which are available from ATCC and other culture collections are highly resistant and do not give any added benefit in the guality control The test procedure for OC organisms is the same as described above for clinical isolates The inoculum should be from a freshly grown culture in the MGIT medium or on LJ slant In case the suspension of OC bacteria is made from growth on solid medium follow the procedure for suspension preparation as described above The suspension may be stored in aliquots frozen for up to 6 months at 70 C 10 C for details of OC strain preparation see Section II K 2 B Pyrazinam
102. ube compared to a drug free tube Growth Control The BACTEC MGIT 960 instrument continually monitors tubes for increasing fluorescence Analysis of fluorescence in the drug containing tube compared to the fluorescence of the Growth Control tube is made by the instrument to determine susceptibility results for SIRE and PZA at the concentrations provided by BD Predefined algorithms compare the growth unit GU in the drug containing tube to the growth unit in the Growth Control tube A result of susceptible or resistant is reported by the instrument Susceptibility tests Set Carriers with different configurations are provided The scanning of these Set Carriers carrying the inoculated MGIT tubes tells the 960 instrument what drugs are being tested Since the instrument software is designed for SIRE and PZA drug susceptibility testing only other drugs if inoculated would not be recognized by the instrument However there is a feature built into the system that can accept undefined drugs If the Set Carrier is entered as undefined drugs the instrument will monitor the susceptibility indicate when the test is ready but will not interpret the results An undefined drug susceptibility test is to be interpreted manually With the undefined entry feature you can test any secondary or new anti tuberculosis drug Make sure the tubes are properly labeled with the name of the test drug and concentration The first tube in the set carrier should always be the growth
103. ube several times e Wipe tubes and caps with a mycobactericidal disinfectant and leave inoculated tubes at room temperature for 30 minutes e Work under the biologic safety cabinet for the specimen inoculation c Inoculation of additional media It is customary to use two different types of media for maximum recovery of mycobacteria With the MGIT system maximum recovery of mycobacteria may be achieved by using an additional solid medium most commonly an egg based medium such as LJ is used The decision to use MGIT medium alone or in combination with an additional conventional solid medium should be made after reviewing each institution s own experience and reguirements Usually 0 1 to 0 25 ml of processed concentrated specimen is inoculated onto solid medium d Precautions e One of the major sources of contamination in MGIT medium is environmental contaminants introduced during addition of growth supplement MGIT Procedure Manual 26 e Make all additions inside a biosafety hood e Do not open several tubes at a time e Open MGIT tube for as short a period of time as possible e A repeat pipettor is very helpful when adding the growth supplement e Always recap the tube tightly If the cap is left loose it may affect the detection of fluorescence e Volumes greater than 0 5 ml of decontaminated specimen may disturb the pH of the medium and may cause false fluorescence This may also increase contamination or otherwise adve
104. ulosis If mycobacteria other than M tuberculosis NTM are present alone or with M tuberculosis AST results will not be reliable e Mix the positive MGIT broth well by vortexing Leave for 5 10 minutes to allow large clumps to settle Take inoculum from the supernatant broth e Ifa tube is positive longer than 5 days or received with unknown biomass subculture into a fresh 7ml MGIT tube Follow procedure described below Mix tube by inversion gt Make a 1 100 dilution of the positive tube using a saline or 7H9 broth gt Inoculate a MGIT tube supplemented with Growth Supplement without PANTA with 0 5 ml of 1 100 diluted specimen Cap tube tightly mix by inversion Enter tube into instrument Vv e A too heavy or too light inoculum may give unsatisfactory results From growth on solid medium e Use freshly grown cultures within 14 days after colonies appear on the medium e Follow the procedure carefully Make a very homogeneous suspension with turbidity comparable to McFarland 0 5 standard MGIT Procedure Manual D 3 Appendix D Precautions e Use a pure culture of M tuberculosis with no contamination and no mixed cultures of different mycobacteria e Make sure the suspension is well dispersed Presence of large clumps may influence susceptibility test results e Do not scrape off medium along with the growth It will give false turbidity e When making dilutions mix well by inverting at least 4 5 times or by v
105. ult in high contamination Long storage and transportation time of the specimen after collection In such situations especially in hot weather bacteria tend to overgrow and then are hard to kill by routine decontamination procedure e Use of non sterile materials such as pipettes tubes etc Sometimes if reagents are prepared stored in bulk and used for long periods of time they may become contaminated For details of troubleshooting refer to Appendix C 3 Detection of contamination Growth of contaminated bacteria will result in positive fluorescence It is important to observe all fluorescent positive MGIT tubes visually for turbidity and to make an AFB smear If a MGIT tube broth is heavily turbid contamination is suspected even if the AFB smear is positive Usually contaminating bacteria cause heavy turbidity although M tuberculosis growth appears as particles without any significant turbidity while some of the NTM may produce light turbidity Contamination may be confirmed by the following method e Make a smear and stain with Ziehl Neelsen stain Presence of non acid fast contaminated bacteria on smear confirms contamination MGIT Procedure Manual 30 Sub culture a loopful of blood agar If blood agar is not available use chocolate agar or brain heart infusion BHI agar plate Several specimens 4 may be carefully inoculated on a plate small streak for each specimen properly labeled Divide the plate and identify
106. upernatant broth for inoculation preparation Preparation from growth on a solid medium Follow the same procedure as described for SIRE susceptibility testing Scrape off as many colonies as possible from the surface of the solid medium using a sterile loop or wooden applicator stick Transfer into a sterilized tube containing 4 5 ml of sterile 7H9 broth with 8 10 glass beads Tighten the screw cap and vortex the broth for 1 2 minutes Leave the culture suspension undisturbed for 20 minutes Carefully remove the supernatant fluid and transfer to a fresh sterile tube Vortex again and leave undisturbed for 15 minutes Transfer the supernatant fluid into a third sterile tube Adjust the turbidity of the suspension to McFarland 0 5 standard by gradually adding sterile saline For susceptibility test inoculation dilute this suspension 1 5 by adding 1 0 ml of the suspension to 4 0 ml of sterile saline Use this diluted suspension for setting up the susceptibility MGIT Procedure Manual 49 c Inoculation and incubation Label two MGIT PZA tubes one as GC growth control and one as PZA drug containing Using a pipette aseptically add 0 8 ml of PZA supplement to each of the two tubes Aseptically add 0 1 ml 100 uL of the reconstituted drug into the PZA tube If possible use a micropipette Try to be as accurate as possible in adding the drug This will give you 100 pg PZA per ml of the medium Do not add drug to the GC tube
107. ure 1 Reagents a MGIT medium b MGIT growth supplement enrichment c MGIT PANTATM 2 Procedures a Reconstituting PANTA b Inoculation of MGIT medium c Inoculation of additional media d Precautions e Incubation f Detection of positive growth G Work up of Positive Cultures 1 AFB smear from a positive MGIT tube 2 Dealing with contamination a Bacterial contamination b Isolation of mycobacteria from contaminated or mixed cultures c Isolation of mixed mycobacterial culture on Middlebrook Agar Plate d Cross contamination 3 Sub culturing a positive MGIT tube 4 Identification of isolated mycobacteria H Results Reporting I Performance Characteristics J Limitations of the Procedure K Ouality Control 1 Ouality control of AFB smear staining 2 Ouality control OC testing of MGIT medium OC strains Preparation of culture suspension Preparation of dilutions Inoculation incubation Expected results Precautions 3 Quality control of laboratory procedures a Positive and negative controls b Quality control with laboratory data 4 Record keeping oeocp MGIT Procedure Manual TABLE OF CONTENTS Section III Drug Susceptibility Testing A Primary Drug Susceptibility Testing SIRE 1 Introduction 2 Principles of the test 3 Reagents a Drugs b SIRE supplement c Storage 4 Procedures a Reconstitution of lyophilized drugs b Addition of a drug to the medium c Preparation of the
108. wever since sterility is not guaranteed it is recommended these specimens should be lightly decontaminated If the specimen volume is more than 10 ml concentrate by centrifugation at about 3000 3500x g for 15 20 minutes Liquefy thick or mucoid specimens prior to centrifugation by adding NALC powder 50 100 mg After centrifugation resuspend the sediment in about 5 ml of saline and then decontaminate following the procedure similar to that for sputum Isolation of mycobacteria from blood specimens has not been evaluated thoroughly A few studies have been published or presented where blood was used with MGIT System after lysis centrifugation BACTEC Myco F Lytic medium is recommended for isolation of mycobacteria and fungi from blood samples E Smears for Acid Fast Bacteria AFB 1 Smear preparation Prepare smears from all processed specimens before inoculation into medium Details of the procedure are given in the CDC Procedure Handbook or any other reference mycobacteriology book The procedure is outlined as follows a After digestion decontamination concentration and resuspension of the pellet mix the specimen well with a pipette and place about one drop or 2 3 loopfulls on a clean microscope slide b Spread the smear about 112 cm x 1 cm c Allow the smear to air dry completely MGIT Procedure Manual 20 d Heat fix the smear either by passing over the flame three to four times or by heating on a slide warmer at 65 75 C for 2
109. y are available Negative as well as positive results should be reported The CDC recommends the smear result should be reported within 24 hours of receiving the specimen 2 There are different criteria for degree of positivity which may be followed to guantitatively report the number of AFB seen on a smear One of the guantitative reporting procedures recommended by the CDC is as follows Number of AFB Seen Report 0 Negative 1 2 AFB Whole Smear Doubtful positive Confirm by observing another smear from the same specimen or from another specimen from the same patient 1 9 AFB 100 Field 1 1 9 AFB 10 Field 2 1 9 AFB Field 3 gt 9 Field 4 If smear positivity is doubtful with only 1 2 AFB seen on the whole smear stain and examine another smear made from the same patient Doubtful fluorochrome stained positive smears should be confirmed by Ziehl Neelsen or any other carbol fuschin method It is important to run a positive and a negative guality control slide with each batch of stains See Section I K for Quality Control F Preparation and Inoculation for Culture 1 Reagents a MGIT medium The MGIT 960 tube contains 7 0 ml of modified 7H9 broth base Note The manual MGIT tube is different in that it contains 4 0 ml of the medium The approximate formula per 1000 ml of purified water contains Modified Middlebrook 7H9 broth base 5 9 gm Casein peptone 1 25g MGIT Procedure Manual 24 Adjusted and
110. ycobacteria Faster growing mycobacteria may develop positive fluorescence prior to slower growing mycobacteria Therefore it is important to subculture positive MGIT tube on a Middlebrook agar plate if there is any indication of the presence of more than one species of mycobacteria on the AFB smear made from the culture e Sometimes excessive carryover of reducing agent or alkali may cause false fluorescence of the sensor for a short time f The use of PANTA antibiotic mixture although necessary for suppression of contaminating bacteria may have some inhibitory effect on some mycobacteria other than M tuberculosis complex This inhibition varies from species to species and isolates within a species However overall isolation of NTM is higher in liquid as compared to solid media K Quality Control 1 Quality control of AFB smear staining Some laboratories only perform quality control with a fresh batch of stain However it is recommended to include a positive control and a negative control with each batch of slides for staining Prepare smears from positive cultures of M tuberculosis H37Rv ATCC 27294 or H37Ra ATCC 21577 mycobacteria other than M tuberculosis complex NTM may also be used for positive control A suspension equivalent to McFarland number 0 5 1 0 turbidity standards must be used for making a positive smear Smears can also be made from growth on solid medium Bacterial suspension such as E coli can be used for th
111. ze the data for susceptible resistant and those which had shown borderline results For BACTEC 460TB the GI values can indicate the borderline situation while 960 does not use borderline designation The BACTEC MGIT 960 GU values may be helpful in establishing borderline resistance MGIT Procedure Manual D 6 Appendix D e Borderline resistant cultures are isolates with an MIC near the test concentration Usually borderline resistant isolates yield inconsistent results and may cause discrepancies when two methods are compared These cultures are found susceptible when tested at the higher drug concentration of a test drug e Calculate sensitivity and specificity of the new system as compared to the reference method e Look at the repeat testing and evaluate the reproducibility of both methods e False resistance by the test method as compared to the reference method is considered a major error while false susceptibility by the test method as compared to the reference method is considered to be a much graver error since it would be detrimental for patient therapeutic management e Calculation of positive and negative predictive values may not represent the real situation as these values depend upon the prevalence of drug resistance in a population 2 Susceptibility testing for second line drugs The BACTEC MGIT 960 susceptibility test is based on growth of the Mycobacterium tuberculosis isolate in a drug containing t

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