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Mycobacteriology Laboratory Manual

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4. D If the subsequent ZN is positive and BAP negative proceed as in step 4 above taking note of the additional days hours of incubation to report a final TTD on the lab worksheet and Appendix B If the subsequent ZN is positive and is contaminated proceed as in step 3 above taking note of the additional days hours of incubation to report a final TTD on the lab worksheet and Appendix B If the subsequent ZN smear is still negative and the BAP is contaminated proceed as in step 2 above regardless of the original TTD for this culture If the subsequent ZN smear is still negative and the BAP still has no growth re incubate the MGIT tube either in the MGIT machine if within 5 hours of removal or in a 37 1 incubator if more than 5 hours have passed since removal for the full 42 day protocol If the machine signals positive again prior to the end of the protocol repeat the ZN and BAP tests and proceed as specified in step 1 3 or 4 above If still negative re incubate offline for the remainder of the 42 day protocol Repeat the ZN and BAP tests after the 42 day incubation period and proceed as in steps 1 3 or 4 above i If after 42 days the ZN is negative and the BAP has no growth visually inspect the tube for signs of growth e g turbidity and test the MGIT broth with an MPT MTB 64 antigen test Document whether or not the tube is turbid and the result of the identification
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6. ASIA qi pelqng Buiusei9g 33 9NDDVAIL 1 CIV NIWVINO S3ALISOd ATU 201 XIIN3ddV K10j amp 1oqe APPENDIX N LABORATORY VISITOR LOG Page of Lab Name isssssse Renee nenne nennen Lab Director Name ott e t ss acted oett s Address locations Date of Visit Record each day of visit on a separate line Printed Name of Visitor Title of Visitor Purpose of Visit Signature of Visitor Printed Name of Laboratory Personnel Initials Date format dd mmm yyyy 147 Mycobacteriology Laboratory Manual
7. O e C O 31 Date Reviewed by Signature Comments Date format dd mmm yyyy Equip Number Location room or equivalent Temperature Range 37 1 Thermometer If emperature is out of range inform the supervisor and record any action taken below Month Yea odores Day Temp 1 Time Taken Initials Temp 2 Time Taken Initials Action Maintenance Adjustments Notes 1 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 8 9 20 21 22 23 24 25 26 27 28 29 30 31 Date Reviewed by Signature Comments Date format dd mmm yyyy Mycobacteriology Laboratory Manual 112 Mycobacteriology Laboratory Manual Equip Number Location room or equivalent Temperature Range 37 1 Thermometer 5 10 Note If temperature is out of range inform the supervisor and record any ac io
8. ovp 6 uper pasa 4 pasa Mou 41045 4 5 4 5 yumm demo eM WD 25509 DO 8 159 8 134 Drug Name Drug Name Drug Name Purity Potency Purity Potency Purity Potency Drug Lot Number Drug Lot Number Drug Lot Number Amount Amount Used Remaining Amount Amount Amount Amount Used Remaining Used Remaining pee Date Date format dd mmm yyyy Mycobacteriology Laboratory Manual APPENDIX G Results Results gt Interna Positive Negative 2 Lot Number bal era Control Control Control QC Pass Date rd Positive Positive Positive Yes No inii Negative Negative Negative IMTB strain 2Strain tested Supervisor tested positive negative Review control control Date Initials Brand Lot Received Expiry Quantity Date Manufacturer Number Date Date Received Opened Date format dd mmm yyyy 135 Mycobacteriology Laboratory Manual sjou 240q JosiAJednc 48 6HZ unjnds Bau jojuo
9. Inoculate 0 5 ml from Tube 3 to a MGIT tube Mix well Enter the inoculated tube in the MGIT 960 instrument Remove the tube when indicated positive by the instrument Retrieve data for time to detection from the MGIT printout 16 3 Reagent Quality Control Periodic quality control of all reagents is critical for ensuring confidence in laboratory results For the clinical trial the Sponsor requires the following reagent quality control procedures for staining reagents sputum digestion decontamination reagents immunochromatographic identification tests and extraction buffer and drug susceptibility reagents In addition record reagent name batch number date prepared and expiry date on all reagent containers 16 3 1 Acid fast Stains Fluorescent and Ziehl Neelsen Methods Frequency Each batch of patient tests and each new batch of in house prepared reagents or each new lot new shipment of commercial reagents Controls Smears with known positive M tuberculosis H37Rv or H37Ra and negative non acid fast bacteria control organisms Acceptable results Correct results as expected for positive and negative controls a Positive controls must demonstrate the presence of acid fast bacilli b Negative controls must be clearly negative with no acid fast bacilli present Corrective Actions If either control result is unacceptable do not report patient results a Repeat controls if acceptable repeat patient
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11. Mycobacteriology Laboratory Manual 15 3 Study Data Reporting Record on Appendix B whether or not an isolate was shipped 1 If neither MGIT nor LJ culture was positive for MTB complex mark No 2 If culture was positive for complex and isolate shipped mark Yes and record aliquot number isolate number and date of shipment for each as applicable 3 If culture was positive for MTB complex but isolate unavailable for shipping e g culture contaminated and could not be purified mark No and add explanation in the Shipping Comments section For all other visit intervals which do not require an isolate to be shipped mark N A on Appendix B 16 QUALITY ASSURANCE Purpose Quality assurance is a critical component of laboratory testing as it ensures accuracy and consistency of laboratory test processes throughout the examination of a sputum specimen from the point of collection to result reporting and database entry In addition good record keeping and periodic monitoring of the data generated will help guarantee that all lab procedures are performing properly Principle The examination and monitoring of multiple parameters involved in testing are guided by standards mandated by accrediting agencies and the quality assurance guidelines in the Good Clinical Laboratory Practice GCLP standards GCLP standards are recommended for international laboratories participating in clinical research and help assure
12. the morning meal except for hospitalized ed by site staff The second specimen will be collected at the site by the study staff e to spontaneously expectorate a samp to produce sputum during the visit two additional attempts e during site collection If a patient is unable o collect the specimen must be made over the next 48 hours be performed from both MGIT and 1 media when growth is detected from both Susceptibility testing for first line anti TB drugs and key second line drug s utilized at each site second line DST only T system ates recovered from a positive MGIT or LJ culture if MGIT not available will be s for storage at room temperature or at 4 C until the end of the study Long term will be stored in duplicate in 7H9 broth w ess otherwise instructed by the sponsor 3 SPECIMEN FLOWCHART Receipt and login of At specified time points mu culture is negative DUE contaminated specified time points Section 4 Laboratory Biosafety and Infection Control and Section 16 Quality Assurance apply to all procedures in the flowchart Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 4 LABORATORY BIOSAFETY AND INFECTION CONTROL Purpose Transmission of tuberculosis including drug resistant TB is a recognized occupational risk for laboratory person
13. Mycobacteriology Laboratory Manual 96 Mycobacteriology Laboratory Manual Procedure 1 Select 10 slides from those read in the previous month and that represent a range from negative to scanty 1 2 and 34 2 Ask technicians to read and record results in a blinded manner 3 Review results for consistency and any discrepancies among technicians 16 4 7 Analysis of Laboratory Data Strongly recommended It is strongly recommended that laboratory data be recorded and analyzed to help assure that specimen processing procedures as well as mycobacterial isolation rates are performing properly After initially analyzing the records for three to six months an overall average or normal trend for an individual laboratory workload can be determined Monthly Data Monitors Forms Appendix is provided for recording these data If there is significant change or deviation from normal results in any of the parameters all procedures must be reviewed and corrective measures instituted Frequency Each parameter should be calculated monthly Culture Positivity Rate 1 Procedure a Count the number of positive MGIT cultures that are smear positive Count the total number of positive MGIT cultures Use these values to calculate the MGIT isolation rate from smear positive specimens smear positive total culture positive reported in the month x 100 Count the number of positive MGIT cultures that are smear negative Cou
14. 2 Controls pH paper or pH meter 3 4 Acceptable results pH for phosphate buffer is 6 8 Corrective Actions If pH for buffer cannot be adjusted to 6 8 discard batch notify supervisor immediately and prepare new batch using new reagent powder if necessary Documentation Record reagent details and pH on Reagent Media QC Form Appendix E If QC results are not acceptable prepare an Appendix K form to document the corrective action 16 3 3 Immunochromatographic Identification Tests Frequency a Each new lot or shipment of kits and each new prepared lot of extraction buffer b Weekly or with each batch of patient tests if testing is performed less frequently Controls a Internal reagent control in device b Positive control Culture of M tuberculosis reference strain H37Rv or H37Ra in MGIT broth c Negative control Culture of a MOTT strain e g a well characterized strain of M avium complex in MGIT broth or broth from an uninoculated MGIT tube Acceptable results Correct results as expected for all controls a Internal control line is visible b M tuberculosis must result in a positive test c MOTT strain or uninoculated broth must result in a negative test Corrective actions If any control result is unacceptable do not report patient tests a Repeat test with new controls if acceptable repeat patient tests b If repeat results still unacceptable notify supervisor imme
15. Ann Acker Women s Health Laboratory San Antonio Texas United States of America Microbiology Consultants Qinghua Zou Beijing China J Abdel Latif Eissa Cairo Egyp inical Laboratory Services Johannesburg South Africa unqi Zhang Shanghai China Cheng Wang Beijing C Erki Sala Kohtla J rve Estonia Karmen Laurson Ta hina Somaia linn Estonia Maris Kauk Tallinn Estonia Toru Mori Tokyo Japan Hyukmin Lee Kyunggi do Korea Kyoung Ho Roh Daejeon Korea Arta Balode Riga Latvia Gintaras Makstutis Siauliai Lithuania Silvija Kiveryte Vilnius Lithuania Jo Moldova Ivan Sabogal Lima Peru Esperanza Cabrera Manila Philippines Del anta Miciuleviciene Vilnius Lithuania Valentina Voroj bit Chisinau Manila Philippines Kavindhran Velen Pretoria South Africa ia Ontengco The Global Laboratory Initiative GLI Secretariat and Core Group members as well as representatives from the WHO GLI TB Supranational Reference Laboratory Network are thanked for reviewing and endorsing the final version of this manual Karin Weyer Christopher Gilpin Jean Diego Zallocco Fuad Mirzayev Wayne Van Gemert Tom Shinnick Rick O Brien Rumina Hasan Heather Alexander Armand Van Deun Amy Piatek Sabine R sch Gerdes Maria Alice Telles Maarten van Cleeff Lucia Barrera Daniela Cirillo Harald Hoffmann Sven Hoffner Michael lademarco and Richard Lumb e Otsuka FIGH IBACK T
16. Mycobacteriology Laboratory Manual 4 The medium is not contaminated or changed in its appearance e g color change with LJ media blood agar hemolyzed etc 5 Agar is not detached from the sides of the tube plate frozen or softened 6 Tubes plates are sufficiently and equally filled 7 Media is not excessively moist or dehydrated If any problems are found notify supervisor contact vendor immediately and withhold media from patient use until issues are resolved QC of Commercial Media Inventory It is important to monitor the performance of commercial media closely during use and if necessary perform complete quality control to ensure the recovery of isolates is satisfactory and as expected QC of Commercial LJ Media Commercial LJ media has been demonstrated to deteriorate over time and its ability to support growth can be compromised Therefore commercially sourced LJ media that has been stored in the lab 26 weeks should be tested according to the sterility performance checks in Section 16 2 1 2 1 QC Protocol for LJ Medium QC of Commercial MGIT Medium Becton Dickinson BD the manufacturer of MGIT reagents recommends QC testing of BACTEC MGIT medium and MGIT 960 Growth Supplement upon receipt before putting into routine use to ensure that the performance characteristics of the medium once supplemented with the OADC PANTA mixture are acceptable At a minimum the Sponsor requires the QC procedure outlined i
17. Study Labels Disposable gloves Refrigerator with detached thermometer Cooler Cold packs 11 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual Forms 3 l T 2 Laboratory Specimen Requisition Form this is a site specific form used for routine laboratory requests Appendix A Specimen Transfer Form Appendix D Equipment Temperature Record Form or use site specific form if available and equivalent Sputum Collection Collect specimens in appropriate sterile disposable containers Screw caps must fit tightly to avoid leakage 50 ml polypropylene centrifuge tubes are preferred Discuss the following collection procedures with the patient Emphasize the nature of the desired specimen Inform the patient that nasal secretions and saliva are not sputum Explain that the desired specimen is produced by a deep cough and is thick mucoid white yellow and sometimes blood tinged It is from the lower airways and lung Instruct the patient not to touch the inside of the collection container or lid with their fingers or other objects Positively identify the patient Prepare two study labels three for patient collected samples with the screening and or subject ID number date and time of collection sputum specimen number 1 or 2 or N A if Visit 2 or Visit 3 and visit number for which the specimen is being collected Place one study label on tube container
18. aee Masked Qum Dae Pa BD MGIT Supplement BD MGIT PANTA BD MGIT Supplement BD MGIT PANTA BD MGIT Supplement BD MGIT PANTA Date format dd mmm yyyy Mycobacteriology Laboratory Manual Lot Batch QC pass Results Date Media Expiry Quantity Date Results Prepared Date of growth Sterility alae dig lec Initials Batch Prepared Initials 102 103 10 4 Control Initials MTB strain Unino Monthly tested Bae Supervisor positive media Review control date initials New Lot Batch Media Component Brand Lot Received Expiry Quantity Date Put P Manufacturer Number Date Date Received in Use L J Media Base Glycerol KH PO Sodium glutamate Malachite green Date format dd mmm yyyy 125 Mycobacteriology Laboratory Manual 126 Appearance Done Tech Brand lot Received Expiration Quantity Not ut QC pass Manufacturer Number Date Date Received Acceptable Acceptable Yes No Date Monthl Comments corrective Review actions D date initials Date format dd mmm yyyy Explain in comments section Mycoba
19. jue Jeqd 94 ny suawioads sod gIW PIOL penodau 0101 9 a01 LOW sueuieds sod g1w 1040 periode seunjno POL f1 IOW A LISW LLOW Jo sisojndiaqny jo uoup os spou AXXA MalAay JosiAsadng sem 109A 142 APPENDIX J STORAGE LOG FORMS Freezer Location room or equivalent Rack Box Position Screening ID Subject ID lob Sputum Aliquot of pis Date shipped Accession Specimen Collection Frozen Eder Number Box Number Number Number if applicable Number Number Tech Initials Supervisor Review Date Initials Sputum specimen number should be 1 or 2 or N A if from visits 2 or 3 Aliquot should be 1 4 or N A if from visits other than 2 or 3 Date format dd mmm yyyy Mycobacteriology Laboratory Manual Rack Number if applicable Box Number Position in Box Screening ID Number Subject ID Number Visit Number lab Accession Number Spulum Spec
20. Date format dd mmm yyyy Equip Number Location room or equivalent Temperature Range 2 8 Thermometer If emperature is out of range inform the supervisor and record any action taken below Month Yea odores Day Temp 1 Time Taken Initials Temp 2 Time Taken Initials Action Maintenance Adjustments Notes 1 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 8 9 20 21 22 23 24 25 26 27 28 29 30 31 Date Reviewed by Signature Comments Date format dd mmm yyyy 109 Mycobacteriology Laboratory Manual 110 Mycobacteriology Laboratory Manual Equip Number Location room or equivalent Refrigerator Temp Range 2 8 Freezer Temp Range 20 C 2 C Thermometer Thermometer Note If temperature is out of range inform the supervisor and record any ac ion taken be Month Year D Refrigerator Freezer Temp 1 Temp 1 Time Taken Initials Refrigerator Temp 2 Freezer Temp 2 Time Taken Initials Action Adjustments Maintenance Notes 5
21. If colony counts are lower than the acceptable range check the preparation of the MTB suspension especially if prepared from a frozen stock Loss of viability is a consideration when freezing low concentrations of MTB Documentation Record results on the Reagent Media QC form Appendix E If QC results are not acceptable prepare an Appendix K form to document the corrective action Preparation of MTB Culture Suspension and Working Dilution Subculture a M tuberculosis QC strain H37Rv or H37Ra onto several LJ slants Incubate the tubes at 37 1 and observe growth visually Use colonies showing good confluent and pure growth within 10 15 days of first appearance Old cultures do not give reliable results Remove growth from the slant by carefully scraping the colonies off the slant with a sterile loop or sterile wooden applicator stick Take extreme precaution not to scrape off any culture medium which gives false turbidity measurement Transfer growth into a screw cap tube containing 4 ml of sterile 7H9 broth and 6 10 glass beads 2 mm diameter and vortex well Let tube sit for 30 minutes for clumped organisms to settle Transfer the supernatant fluid to another sterile tube avoid transferring any of the sediment and let the suspension stand for another 15 minutes Transfer the supernatant to a new tube and adjust turbidity of this suspension to a McFarland No 1 standard using 7H9 broth Prepare
22. Negative amp BAP Growth Action Refer to Flow chart 2 Early Positive ZN Negative Negative amp no visual growth in MGIT tube Action Refer to Flow chart 3 43 Mycobacteriology Laboratory Manual 44 Mycobacteriology Laboratory Manual Flow Chart 2 Contaminated MGIT Cultures Y ZN Positive ZN Negative Action Report ZN negative BAP contaminated culture contaminated original TTD from MGIT printout and final TTD from Appendix M Flow Chart 2A Suspected MOTT Cultures positive signal Original MGIT culture Suspected MOTT from Flow Chart 1 ZN Positive BAP Negative Rapid ID test Negative or invalid Action Reincubate for 48 hours and retest with Rapid ID test If MTB positive from rapid ID test Report ZN positive BAP negative Positive for MTB original TTD from MGIT printout If MTB negative or invalid from rapid ID test Action Perform HAIN GenoType Mycobacterium CM or GenoType MTBDRplus If MTB positive on GenoType Mycobacterium CM or GenoType MTBDhplus test Report ZN positive BAP negative Positive for MTB original TTD from MGIT printout If positive for MOTT on Genolype Mycobacterium CM test or MTB negative on GenoType MTBDRplus Report ZN positive BAP negative No TB growth but posit
23. Record results on the Reagent Media QC form Appendix E If contamination is seen prepare an Appendix K form to document the corrective action Performance QC 1 Frequency Each new batch of prepared medium 2 Controls Number of tubes equivalent to 1 396 of total volume prepared tested with 107 and 10 dilutions of M tuberculosis H37Rv or H37Ra in 7H9 broth e g 296 of a 100 ml batch of medium is 4 tubes in total 2 tubes inoculated with each of two working dilutions See procedure below Mycobacteriology Laboratory Manual 82 Mycobacteriology Laboratory Manual Es Acceptable Results Growth in all tubes is consistent with the appearance of M tuberculosis flocculent granular within 1 2 weeks 4 Corrective Actions a If tubes do not show growth notify supervisor immediately discard entire batch and prepare new media b If tubes show overall turbidity suggesting bacterial growth confirm with ZN stain and or subculture to solid media c If sterility check was acceptable and only inoculated tubes are contaminated repeat performance QC with fresh culture of M tuberculosis Documentation Record results on the Reagent Media QC form Appendix E If QC results are not acceptable prepare an Appendix K form to document the corrective action Preparation of MTB Culture Suspension and Dilutions Prepare dilutions as above in Section 16 2 1 2 1 QC Protocol for 1 Medium Inoculation
24. Store all fluorescent stained sputum smears in a slide box until the end of the study 2 Label these slides with study label and sample details after staining and smear is dry 9 ACID FAST BACILLI MICROSCOPY AFB EXAMINATION Purpose The purpose of AFB microscopy is to detect acid fast bacilli in clinical specimens and cultures Both viable and non viable bacilli will stain and be counted The results of examination of stained smears are reported in a standardized way so that results can be compared and used in a manner that is relevant to patient care The commonly used scoring systems are published by WHO IUATLD and CDC USA Reporting must be according to the WHO IUATLD system Principle With Auramine O stain organisms fluoresce bright yellow non specific debris stains pale yellow and the background is almost black With Auramine Rhodamine stain organisms fluoresce yellow red in an almost black background With Acridine Orange organisms fluoresce red orange in a black background The Ziehl Neelsen stains acid fast organisms red and the background debris stains blue All mycobacteria are acid fast and other genera such as Nocardia and Corynebacterium may be partially acid fast so microscopy cannot be used to determine individual species including M tuberculosis A positive smear is approximately 10 bacilli per ml or greater Procedure Materials Light bright field microscope for Ziehl Neelsen stain Flu
25. Subculture the MTB strain onto several LJ slants Incubate the tubes at 37 C 1 and observe growth visually Use colonies showing good confluent and pure growth within 10 15 days of first appearance Younger or older cultures may not give reliable results Remove growth from the slant by carefully scraping the colonies off the slant with a sterile loop or sterile wooden applicator stick Take extreme precaution not to scrape off any culture medium which gives false turbidity measurement Transfer growth into a screw cap tube containing 4 ml of sterile 7H9 broth and glass beads 6 10 beads 2 mm diameter which help to break up clumps Tube A Vortex Tube A for at least 1 2 minutes making sure the suspension is well dispensed and very turbid Turbidity should be greater than McFarland 1 standard Letthe suspension stand undisturbed for 20 minutes Using a transfer pipette carefully transfer the supernatant from Tube A to another sterile screw cap glass tube Tube B Avoid pipetting any sediment Let Tube B stand undisturbed for 15 minutes Carefully transfer the supernatant from Tube B into another screw cap glass tube Tube C without taking any sediment Adjust the turbidity of the suspension in Tube C to a McFarland No 0 5 standard by adding more 7H9 broth Mix well If the suspension is too turbid transfer some of the suspension to another sterile tube
26. Switzerland 2008 16 4 3 Monitoring of Sputum Processing Required Careful attention to technique when processing specimens is essential to preventing cross contamination from a heavily acid fast positive specimen to other possibly negative samples Running positive and negative controls on a regular basis assesses techniques and assures that all aspects of the culture process manual and instrumented from sputum processing to isolation are performing properly 16 4 3 1 Quality Monitoring of Sputum Processing Frequency Once per week or with each patient batch Controls Place controls at the end of the batch of patient tests with the positive control before the negative control Process both control specimens in the same manner as patient specimens and perform routine microscopy MGIT and LJ culture a Positive control 4 ml 7H9 Broth or a known negative sputum specimen inoculated with 700 ul of a 10 dilution of a McFarland No 0 5 suspension of M tuberculosis H37Rv or H37Ra Suspension should be prepared using 10 15 day old growth on LJ Refer to section 16 2 1 3 1 for details b Negative control 4 ml 7H9 broth Acceptable results a Positive control i Positive fluorescent smear ii Growth in MGIT TTD should be comparable with similar tests iii Growth on LJ culture colony counts should be comparable with similar tests b Negative control i Negative fluorescent smear ii No growth in MGIT o
27. acid alcohol decolorizer and methylene blue counterstain Acid fast organisms stain red while the background of debris stains blue The ZN stain confirms the acid fast property of mycobacteria Procedure Materials Tuberculocidal disinfectant Waste receptacles including splash proof receptacle for liquids Discard bucket with biohazard bag insert containing appropriate disinfectant Paper towel soaked in appropriate disinfectant icroscope slides frosted at one end new and clean Pencil for labeling slides Study labels Hot plate or slide warmer Bunsen burner or spirit lamp Sterile transfer pipettes with graduations marking volume individually wrapped Sterile loop or disposable applicator stick 25 Mycobacteriology Laboratory Manual 26 Mycobacteriology Laboratory Manual Ziehl Neelsen stain carbol fucshin 396 acid alcohol methylene blue Auramine stain auramine 0 5 1 acid alcohol 0 596 potassium permanganate or Auramine Rhodamine auramine rhodamine 0 5 196 acid alcohol 0 596 potassium permanganate or Acridine orange stain acridine orange solution in barbitone buffer 2N H SO acid alcohol decolorizer Staining sink Staining rack Slide drying rack Forceps Timer Vortex mixer Distilled water Wash bottle Forms 8 1 The 1 Appendix E Reagent Media QC Form Appendix C Daily AFB Staining QC Form Preparation of Smea
28. b Perform annual competency assessments of all technologists to control for technician related deviations Documentation a Document alllab trainings re trainings and competency assessments on the laboratory specific form b If any corrective measures are necessary prepare an Appendix K form to document the activities 16 5 Quality Improvement Continuous efforts to improve the overall quality of the laboratory by improving service function workflow personnel and customer satisfaction are necessary and require the input and support of all personnel Regular staff meetings should be held to share and discuss quality issues found with QC and OM to elicit suggestions for improvement and encourage input from staff on any issues of concern The Sponsor requires the documentation of efforts to improve the quality of the lab When Quality Control tests fail to give the proper results and or when deviations from baseline data are observed the use of a standardized form such as Appendix K must document the action taken to correct the problem The form includes the following components Form Number number assigned to each form to allow easy filing and retrieval Often the date is incorporated into this number Ex Form Number 01JAN11 a Description of the problem improvement The date and a short description are written here Ex Monthly TID QC exercise not performed in January 2011 Problem discovered 30JANT 1 Inve
29. c Review settings and maintenance records for centrifuge 97 Mycobacteriology Laboratory Manual 98 Mycobacteriology Laboratory Manual Documentation a Record data on the Monthly Data Monitors Forms Appendix 1 or other lab specific comparable document b If any corrective measures are necessary prepare an Appendix K form to document the activities Technologist Assessment All parameters described above should remain similar among different technologists responsible for processing specimens Tracking lab personnel along with their specimen responsibilities is helpful in the event that an unusual episode of contamination or cross contamination occurs or when overall positivity rates and other parameters deviate from the normal range 1 Procedure a Record the names of technologists performing procedures on the laboratory s processing worksheet b Record the lab accession numbers of specimens processed in each batch Results When conducting reviews on identified deviations in the parameters above check for any correlation with a specific technologist s which could indicate possible deviations from standard processing protocols Corrective Actions a If reviews implicate a specific staff member re train the technologist as appropriate Ideally review each step of the specimen processing SOP with the technologist s on a frequent quarterly basis and especially before each change in rotation
30. controll Negative control Pass Yes No Technician Initials lol e N O e 31 strain tested positive control Strain tested negative control Reviewed by Date Signature Comments Date format dd mmm yyyy 107 Mycobacteriology Laboratory Manual 108 Mycobacteriology Laboratory Manual APPENDIX D EQUIPMENT TEMPERATURE RECORD FORMS Equip Number Location room or equivalent 70 C 1 Temperature Range circle one 0 C 80 C 1 0 C Thermometer Note If emperature is out of range inform the supervisor and record any action taken below Month Year Day Time Taken Initials Temp 2 Time Taken Initials Action Adjustments Maintenance Notes CO 2 O o e o O 31 Date Reviewed by Signature Comments
31. o each MGIT tube Do not use MGIT 960 growth supplement or PZA supplement 6 Aseptically pipette 100 ul of the appropriately diluted drug into the corresponding tube Refer to the table above for appropriate volumes and working solution concentrations of the second line drugs 7 Itis important to add the correct drug to the corresponding tube 8 Do not add drugs to the MGIT GC tube 12 4 Inoculum for MGIT DST Carry out all work with positive MGIT tubes in biosafety cabinet using full PPE Bacterial density of the inoculum is critical to the correct performance of the susceptibility test thus the following instructions must be adhered to strictly Cultures must not be used to set up DST if more than 5 days have elapsed after signaling positive 12 4 1 Using an Inoculum from Positive MGIT Culture 1 Positive MGIT cultures must have pure growth of M tuberculosis ZN positive BAP negative MPT MTB 64 antigen test positive Section 10 Liquid Culture Mycobacteria Growth ndicator Tube MGIT and Section 13 Rapid Identification of M tuberculosis Complex Using an Immunochromatographic Assay in order to be tested for drug susceptibility 2 DST must not be set up on the same day a MGIT tube signals positive 3 If the culture is worked up one or two days after signaling positive it can be used directly to inoculate the DST MGIT tubes 4 fthe culture is used to set up DST between three and five days after signalin
32. 10 6 below In addition refer to Flow Chart 1 for further instructions and notify the Sponsor of this occurrence e Printouts of results of all negative tubes must be kept in the study binder e Autoclave all MGIT tubes prior to disposal 10 5 Dealing with Positive Tubes The positive tube indicator is the leftmost of the three drawer indicators It is marked with a plus sign and illuminates red to inform you that one or more positive tubes are present in ube is identified he drawer The indicator remains lit until all positive tubes are removed through the Remove Positive Tubes operation In addition an optional audible alarm sounds when a newly positive Functions Instructions Remove positives Press silence alarm to mute the audible alarm if alarm is activated Open the drawer with the illuminated RED positive light Press the remove positive tubes soft key All positive tubes will be indicated by flashing GREEN and RED lights Remove one tube at a time Scan the positive tube s barcode label by placing the ube in the alignment block in front of the scanner with the barcode label facing the scanner Rotate the tube if necessary The GREEN and RED lights will extinguish 6 7 Place the tube into a rack or carrier to transport after removal 8 Repeat steps 4 to 7 to remove additional positive tubes placing them in sequential order in the rack 9 When all positive tubes ha
33. 103 Mycobacteriology Laboratory Manual 104 APPENDIX C DAILY QC STAINING FORMS L Appendix Acridine Orange Daily QC Staining Form Year eet Note If QC is out of range inform the supervisor and record any action taken below Acridine Orange 2N H SO Acid Alcohol Batch or Lot Batch or Lot Batch or Lot Expiration Expiration Date Expiration Date Date Positive Positive Negative Negative Control control slide control slide control slide Slides date of date of date of date of preparation expiration preparation expiration Results Neg Confirmation Day Required Scanty 1 2 3 QC Pass Yes No Technician Initials Positive control Negative control O O I CO ND O N O e O 31 strain tested Strain tested negative positive control control Reviewed by Date Signature Comments Mycobacteriology Laboratory Manual Confirmation required by another technician or prepare another smear stain and read Date format dd mmm yyyy is csse ecce Years iaa ies Note If QC is out of range inform the supervi
34. 2 Login of Sputum Specimens 4 Place 100uL of specimen either MGIT culture or bacterial suspension from LJ slant see Section 13 2 2 above into the specimen well of the test device Change pipette tips between specimens 5 Start timer for 15 minutes 6 Examine the reading area of the test device after 15 minutes and record test results Do not interpret test after 60 minutes 13 4 Interpretation of Results The following pictures are specific to the BD TBc ID kit However the TAUNS Capilia TB test is interpreted in the same manner 69 Mycobacteriology Laboratory Manual 70 Mycobacteriology Laboratory Manual Table 13 Interpretation of Results C Reading Area H L T Specimen Area iv iv lv 1 2 3 Positive Pink purple to red lines form on the reading areas labeled Test T and Control C of the device Negative A pink purple to red line forms on the reading area labeled C of the device but not T Invalid If no line is observed on the reading area labeled C technical errors or product damage has occurred In this case the test should be considered invalid and repeated using a new device NOTES 1 6 If the rapid ID test is negative but the AFB smear and morphological characteristics of the isolate are consistent with MTB re incubate the tube at 37 C 1 C and repeat rapid ID test after 48 hours If MTB is now detected mark the result as positive for MTB comple
35. 2 Place the tubes in the following sequence in the 2 tube AST set carrier from left to right GC PZA 3 Aseptically add 0 8mL of BACTEC MGIT PZA supplement to each PZA tube 4 Aseptically pipette 100uL of the 8000ug ml MGIT PZA solution to the appropriately labeled MGIT tube 5 Do not add drugs to the MGIT GC tube 12 3 3 Preparation of Second Line Drugs NOTES f second line drugs are entered into the MGIT instrument using one of the instrument s three possible undefined drug set configurations a separate drug free GC tube must be prepared for use in a separate carrier from the SIRE tubes Alternatively second line drugs can be added behind a set of SIRE drugs using the 8 tube AST carrier thus requiring only one GC tube f needed blank MGIT tubes non inoculated drug free can be used to fill empty slots in a carrier 1 Label 7 mL MGIT tubes for each test isolate with a study label that includes identifying information described in Section 6 2 Login of Sputum Specimens 2 addition label tubes with the appropriate second line drug name or abbreviation e g AMK amikacin CAP capreomycin OFL ofloxacin etc 3 If a separate control will be used label a GC tube Growth Control 4 Load tubes in the carrier set in the same order consistently ensuring that the GC tube is always in the left most position 5 Aseptically add 0 8 mL of BACTEC MGIT SIRE Supplement or appropriate OADC Enrichment
36. 3 serial 10 fold dilutions of the adjusted solution 107 107 and 107 in 7H9 broth Prepare 20 50 cryotubes for each dilution Label with strain type dilution and expiration date six months after preparation Aliquot 1 5 ml of each dilution to the appropriate cryotubes and freeze at 70 C 10 up to six months Inoculation and Incubation Remove one aliquot of each dilution 107 10 and 107 from the freezer While thawing bring LJ medium to room temperature With a micropipettor and sterile aerosol resistant tips ART tips mix the working solution with the pipettor 2 3 times to ensure even distribution of MTB Inoculate LJ tubes with 200 ul of each dilution Incubate at 37 C 1 C reading weekly for 21 30 days Compare counts with the laboratory s established reference range 16 2 1 2 2 QC for 7H9 Broth Sterility Check Frequency Each new batch of prepared medium Controls Number of tubes equivalent to 1 396 of total volume prepared Acceptable Results No growth in any tube after 7 days of incubation Corrective Actions a If all tubes are contaminated notify supervisor immediately discard entire batch and prepare new media b If one tube is contaminated repeat exercise with at least 10 tubes c If gt 1 tube is contaminated upon repeat notify supervisor immediately and discard entire batch d Investigate and resolve problems then prepare new media Documentation
37. 7 mL MGIT tubes for each test isolate with a study label that includes identifying information described in Section 6 2 Login of Sputum Specimens In addition label tubes with one of each of the following GC Growth Control STR streptomycin INH isoniazid RIF rifampicin EMB ethambutol 2 Place the tubes in the STR INH RIF EMB ollowing sequence in the 5 tube AST set carrier from left to right GC 3 Aseptically add 0 8 mL of BACTEC MGIT SIRE Supplement provided in the SIRE kit to each SIRE tube It is important to use the supplement supplied with the kit 4 Aseptically pipette 100 ul of the appropriately reconstituted drug into the corresponding MGIT tube e g add 100 u etc 5 t is important to add 6 Do not add drugs to the MGIT GC tube 12 3 2 Preparation of PZA he correct drug to the corresponding tube of the 83 ug mL MGIT STR solution to the MGIT tube labeled STR When preparing tubes it is important to use the PZA media tubes and PZA Supplement supplied with the PZA kit Do not use the conventional MGIT tubes or the SIRE supplement as the pH is different for both PZA medium and supplement 1 Label two 7mL MGIT PZA media tubes for each test isolate with a study label that includes identifying information described in Section 6 2 Login of Sputum Specimens In addition label tubes with one of each of the following GC Growth Control or PZA pyrazinamide
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39. CFU ml a McFarland No 1 0 standard equals approximately 3 x10 CFU The standard is compared visually or using a densitometer spectrophotometer to a suspension of bacteria in sterile 7H9 broth and adjusted by adding more broth if too heavy or more bacteria if too light However the latter must be done after making a single cell suspension i e breaking up clumps with beads allowing clumps to settle and transferring the supernatant to a new tube An inoculum that is too heavy or too light will adversely affect the results so it is important to adjust inoculums as closely as possible to the standard Preparation of Barium Sulfate Turbidity Standards McFarland standards are commercially available and prepared from suspensions of latex particles which lengthen the shelf life and stability of the standard Alternatively they can be made by mixing specified amounts of barium chloride and sulfuric acid This forms a barium sulfate precipitate which causes turbidity in the solution 1 To prepare a McFarland No 1 standard mix 0 1 ml of 1 17596 barium chloride dihydrate BaCl e2H O with 9 9 ml of 1 sulfuric acid H SO 2 2 For a McFarland No 0 5 standard mix 0 05 ml of 1 17596 barium chloride dihydrate BaCl e2H O with 9 95 ml of 1 sulfuric acid H SO 3 Seal the capped tubes with wax parafilm or some other means of preventing evaporation 4 Label the standards with the McFarland number date of preparation and date o
40. Comments section Flow Chart 4 Invalid x200 Errors from MGIT DST NOTE All work must be done in the biological safety cabinet Prepare new MGIT culture from primary culture tube Vortex tube well let sit 5 10 minutes Make a 1 100 dilution of the culture tube using saline or 7H9 broth Supplement a new MGIT tube with 0 8 ml MGIT Growth Supplement without PANTA Inoculate 0 5 ml of the 1 100 diluted sample into the new MGIT tube Cap tube tightly mix well by gently inverting 3 4 times Enter into instrument and monitor until tube turns positive When tube turns positive screen for purity with ZN and BAP If purity verified re incubate tube until it is 3 5 days old record GU value on lab worksheet Prepare new control and drug containing media Remove drugs from freezer let thaw in refrigerator while labeling tubes and adding supplement Aseptically add 0 8 ml SIRE or PZA supplement to each GC and drug tube as appropriate Aseptically add 0 1 ml 100 ul of each drug to its appropriately labeled tube Set up new DST Vortex 3 5 day old culture tube well Let tube sit 5 10 minutes to settle big clumps Use the supernatant broth without disturbing the sediment Using transfer pipette aseptically add 0 5 ml of MGIT culture broth to each drug tube DO NOT DILUTE culture Recap tubes tightly Mix by inverting gently 3 4 times Inoculate GC tube s with 0 5 ml of a
41. Indicator Tube Mycobacteria other than TB N acetyl L cysteine Sodium Hydroxide Note to File Nontuberculous mycobacteria Antibiotic supplement for MGIT tubes Mycobacteriology Laboratory Manual vi Mycobacteriology Laboratory Manual PCR PPE PZA OADC QA QC QM RIF SIRE SOP STR TB TIP TNTC TSA TTD UV WHO ZN Polymerase chain reaction Personal protective equipment Pyrazinamide Oleic acid albumin dextrose and catalase Quality Assurance Quality Control Quality Improvement Quality Management Rifampicin Streptomycin isoniazid rifampicin ethambutol Standard Operating Procedure Streptomycin Tuberculosis Time in protocol from the MGIT printout Too numerous to count Trypticase Soy Agar Time to detection Ultraviolet World Health Organization Ziehl Neelsen stain 1 INTRODUCTION The diagnosis treatment and monitoring of tuberculosis are conducted in a wide range of laboratory facilities worldwide using a variety of methods equipment and capacity Clinical trials for new tuberculosis drugs base efficacy on microbiologic endpoints such as sputum culture conversion SCC of Mycobacterium tuberculosis MTB To ensure high quality results and comparability of data from all participating laboratories a comprehensive laboratory manual that standardizes key laboratory procedures was developed and implemented for a multi country randomized placebo controlled Phase II
42. MGIT tube let stand for about 1 minute Aseptically transfer entire volume of broth into a 50 ml disposable centrifuge tube Add an equal quantity of 496 NaOH solution for a final concentration of 296 NaOH Mix well and let stand for 15 20 minutes mixing and inverting the tube periodically Add phosphate buffer pH 6 8 to the 50 ml mark and mix well 6 Centrifuge at 3 000 xg for 15 20 minutes 7 Pour off the supernatant fluid 8 Resuspend the sediment in 0 5 ml of phosphate buffer and mix well 9 Inoculate 0 5 ml into a fresh MGIT tube supplemented with MGIT growth supplement PANTA according to Section 10 1 above and reincubate following instructions in Section 10 2 and 10 3 10 11 Reporting Results of MGIT Cultures Mycobacterial growth must be reported immediate following table to report the result of the MGIT culture on Appendix B y on the laboratory worksheet Use the MGIT ZN BAP Time to Result TTD Manne Result Result ID Result Appendix B Result tenes esult Positive Positive No complex detected Positive for M Original TTD from growth by rapid ID test tuberculosis complex MGIT printout HAIN Genolype D positive for and final TTD from Mycobacterium CM complex record ID Appendix M if or HAIN Genolype method applicable tests Positive Positive Growth complex detected Positive for M Original TTD fro
43. N AQ Affix a completed study purum specimen Only tick N A for V2 or V3 specimen hbe here Sputum specimen Received E Processed ate ime ate ime i dd mmm yyyy 24hr clock dd mmm yyyy 24hr clock ml tick all boxes that apply Liquid Saliva O Blood O Purulent O Viscou O Sputum induced Yes _ Technician initials Sputum collected by Patient O Site O Date Comments AFB microscopy from sputum specimen Type of smear oe pn damine E oor a Technician tick one box PENS Oates n 5 initials Acridine Orange 0 mmm yyyy Smear Result ps Pong tick one box Negative Rare Few Many TNTC required Comments MGIT 960 system culture of sputum Pate of inoculation Original TTD Days Hours Final Time Days Hours from MGIT i Date of MGIT result printout TTD origina dd mmm yyyy or revised Negative for MTB complex Positive for MTB complex MGIT Culture Result Positive for MTB complex and contaminated tick one box No TB growth but positive Contaminated for other mycobacteria O Unknown ZN Result ba cgi E a RE C ontamingted and or MOTT tick one box tick one N A N A box Negative MTB complex Identification of AFB Positive for MTB complex N A TBc ID mm ID Test Method Capilia Teen piel etie O Other Did specimen require re decontamination Yes ON Tech initials date SP equ 5 9 dd mmm y
44. NALC powder 696 NaOH 2 996 Na citrate Phosphate buffer pH 6 8 or components if made in house 19 Mycobacteriology Laboratory Manual 20 Mycobacteriology Laboratory Manual Forms 7 1 Analytical balance Precision balance Weigh boats Vortex mixer Clean rack for 50 ml centrifuge tubes Refrigerated centrifuge with covered bucket inserts for 50 ml conical tubes Timer 2 ml disposable serological pipette sterile individually wrapped Pipette aid Drummond Scientific Portable Pipet Aid XP or similar Sterile transfer pipettes with graduations marking volume individually wrapped Magnetic stirring bar and stirrer pH meter or appropriately sensitive pH paper Slide warmer hot plate Study labels Pencil for labeling slides Permanent marker Shaker icroscope slides frosted one end new and clean GIT tubes LJ tubes Paper towels e Laboratory Specimen Requisition Form this is a site specific form used for routine laboratory requests Specimen Processing Worksheet Workbook this is a site specific form used for routine laboratory tests Appendix D Equipment Temperature Record or use site specific form if available and equivalent Appendix B Study Source Document Worksheet Preparation of NaOH NALC Na Citrate Solution and Phosphate Buffer Advance Preparation of Solutions 696 Sodium Hydroxide 2 996 Sodium Citrate 1 To
45. Sputum Induced by circling Yes or No 9 After the specimen is collected complete the following Laboratory Specimen Requisition Form all relevant fields must be completed Appendix A must contain the following site name and number screening ID number and or subject ID number subject initials visit number date and time of collection specimen number 1 2 or N A for V2 or V3 whether sputum was induced or not who the specimen was collected by either patient or site staff total volume time sputum was at room temperature study label date time of dispatch to laboratory mode of transport name of person collecting or receiving if patient collected sample the specimen name and signature of person completing the form transport courier information to be completed by driver or site personnel can complete this section if a courier is used 10 The laboratory section will be completed by laboratory staff when they receive the specimen 5 2 Storage of Sputum Specimens 1 Check that the tube is tightly capped properly labeled and that the screening and or subject ID on the tube matches the screening and or subject ID on the requisition and Appendix A forms 2 Refrigerate specimens at 2 8 C until ready for transport to the laboratory Refrigeration reduces the growth of contaminants in the specimen If a refrigerator is
46. Supplement TD Drag TID Drug ech Number Number days result days result initia s hours S R hours S R Date strain Olher strains Supervisor Review tested if tested date initials Drug Information Drug Lot Received Expiration Quantity Date Manufacturer Drug Number Date Date Received Opened BD Streptomycin BD Isoniazid BD Rifampicin BD Ethambutol BD PZA Date format dd mmm yyyy 131 Mycobacteriology Laboratory Manual 132 QC poss MGIT SIRE ther Strains Yes No Drug Tested Media Lot Supplement Date Tested TID D TID D Tech Tech initials TU iu yw Number Number days result days result initials hours S R hours S R ES MTB strain Other strains Supervisor Review tested if tested date initials Drug Information Drug Amikacin Capreomycin Gatifloxacin Kanamycin Levofloxacin Moxifloxacin Ofloxacin Source Brand Drug Lot Number Received Date Expiration Date Quantity Date Opened Date format dd mmm yyyy Mycobacteriology Laboratory Manual 133 Kuo0j amp Joqe 1 KAKAjUuuJ pp e1eq
47. a Note to File to explain the Investigate and resolve any discrepancies errors and send a copy of the NTF Report DST following routine study procedures to the site 13 RAPID IDENTIFICATION OF M TUBERCULOSIS COMPLEX USING AN IMMUNOCHROMATOGRAPHIC ASSAY Purpose To rapidly 1 h and accurately detect Mycobacterium tuberculosis complex MTB in MGIT and 1 AFB positive cultures without special instruments or equipment To differentiate MTB from mycobacteria other than tuberculosis MOTT for the effective treatment of the disease To ensure consistency across all participating sites only the Becton Dickinson BD MGIT TBc Identification Test TBc ID or the Tauns Capilia TB Neo Test Capilia will be used Definitive identification will be performed at every timepoint from all AFB positive MGIT and LJ cultures Principle A rapid immunochromatographic assay will be used to differentiate MTB and MOTT BD s MGIT TBc ID and Tauns Capilia TB are both lateral flow immunochromatographic assays The BD assay detects MPT64 antigen while Capilia detects MPB64 antigen a mycobacterial protein that is specifically secreted from MTB cells during culture When a sample is added to the test device MPT64 MPB64 antigen binds to anti MPT64 MPB64 antibodies conjugated to colloidal gold particles present on the test strip forming an antigen antibody complex This antigen antibody complex then migrates across the test strip to the rea
48. as necessary Documentation of this delegation must be captured in the study documentation Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 2 SAMPLE SPECIMEN TIMETABLE Table 2 1 Sample schedule of Microbiological Assessments for All Treatment Groups Intensive Treatment Period Visit Number 112314 516171819 10 11112113 14 15 16 17 18 19 20 21 22 Day Week Current diagnosis of MDR TB Sputum AFB smear culture MGIT culture Confirmation of MTB isolate identification 1st 2ndHine drug susceptibility testing Short long term storage a Two sputum samples will be tested at each scheduled visit except for Visits 2 and 3 Day 1 and Day 1 on these days only one sputum sample will be collected by the site staff When collected by the patient at home early in the morning and preferably prior patients this specimen will be collec using induction if the patient is unab b Definitive identification of MTB wil required for injectable s and fluoroquinolone s will be performed with MG d Short term storage for MTB iso performed by subculturing to LJ slan storage of MTB isolates recovered rom positive cultures MGIT and or LJ glycerol at 70 to 80 for six mon hs after the conclusion of the study un wo specimens are collected the first specimen will be
49. be incubated to test for sterility and 1 396 mus tested for performance characteristics ability to support a certain amount of growth in a speci time of incubation Baseline criteria for optimal growth with standardized inocula and incuba periods should be established from results of several well prepared batches be ied ion The time to detection of growth and number and size of colonies within a specified time of incubation should be critically evaluated If a newly prepared batch of medium when tes ed yields results outside the established range it must be considered unsatisfactory Colony counts 2096 above or below the established range may be considered unacceptable however it is preferable to establish lab specific criteria that is determine mean CFU and standard deviation of several good prepared batches Required procedures for Sterility and Performance QC of each media type are detailed below Media can be released for routine laboratory use after passing all QC checks for sterility and performance 79 Mycobacteriology Laboratory Manual 80 Mycobacteriology Laboratory Manual 16 2 1 2 1 QC Protocol for LJ Medium Sterility Check 2 ES Frequency Each new batch of prepared medium Controls a 1 396 of LJ tubes from a batch for example for a batch of 100 tubes select 2 tubes b Incubate for 14 days at 37 C 1 Acceptable Results No growth on any tube Visual in
50. been completed to signify that the data has been reviewed and is accurate GIT printouts and AST Reports can also be filed in this section behind the corresponding Appendix B form QC Forms f the lab has a separate filing system for these documents it is acceptable to maintain that system However either a note to file explaining the location of these forms should be placed under the QC Forms tab of the binder or a copy of the forms containing information related to study specimens should be filed under the QC Forms tab of the binder particularly at the conclusion of the trial Examples of documents that may be filed in the binder include but are not limited to edia QC forms Equipment maintenance records Proficiency testing results onthly data monitoring forms worksheets Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual Equipment Temperature Logs f the lab has a separate filing system for these logs it is acceptable to maintain that system However either a note to file explaining the location of these forms should be placed under the Equipment Temperature Logs tab of the binder or a copy of the forms used during the time period of the study should be filed under the Equipment Temperature Logs tab of the binder particularly at the conclusion of the trial Shipping Records All records for the shipment of specimens to the central laboratory must be maintained i
51. by site staff Patient sputum collection instructions are documented elsewhere Principle Sputum specimens will be collected according to the visit schedules in Section 2 Sample Specimen Timetable On visits 2 and 3 only one sputum specimen will be collected from the patient Thereafter two sputum specimens will be collected at each timepoint The first specimen considered to be the morning specimen collected prior to the morning meal and any dosing will be labeled Sputum Specimen 1 The second specimen considered spot specimen will be labeled Sputum Specimen 2 Sputum Specimen 1 This specimen will be collected by the patient at home unless s he is hospitalized The patient will use a special sputum collection container that minimizes contamination and leakage and eliminates the need to transfer sputum from a container to a 50 ml tube The container has the following parts 50 ml centrifuge tube and cap Built in funnel Outer container and lined cap Sealed label If the specimen is collected by the site a sterile 50 ml centrifuge tube will be used In either case the specimen will be sent by the site to the laboratory Sputum Specimen 2 This specimen will be collected by the site staff in a sterile 50 ml centrifuge tube PROCEDURE Materials Sterile disposable single use screw capped conical centrifuge tubes 50 ml Sponsor provided Sputum Collection Kit Permanent marker
52. count can be considered final However if the count increases substantially e g 1 at week 2 2 at week 3 the following week continue to read the culture weekly until growth stabilizes 4 Use the following standardized reporting scheme to report growth from the solid culture on the laboratory worksheet and Appendix B Laboratory ZN ID Growth Resort Eh RESI Study Report Solid Culture None growth N A N A Negative for MTB complex 1 9 colonies Record actual pos TB growth 1 9 colonies record ID number result and test method 10 100 colonies 1 pos TB growth 10 100 colonies record ID result and test method 100 200 colonies 24 pos growth more than 100 colonies record ID result and test method gt 200 colonies too numerous 34 pos TB growth innumerable or confluent to count or confluent record ID result and test method Other mycobacterial growth Positive for other neg complex growth but mycobacteria positive for other mycobacteria record ID result and test method Contaminated Contaminated N A N A Contaminated ZN growth in presence of Positive for pos MIB Positive for complex and contamination and contaminated record ID result and contaminated test method 1 If growth appears to be or resembles MOTT and the MPT MTB 64 antigen test is indeterminate due to the presence of contaminants presumptively ID the culture by looking for t
53. entered tubes Start at front of drawer and remove in sequential order placing in same order in rack GREEN drawer indicator light extinguishes the barcode scanner turns off and the lt ok gt icon appears on the display screen steps for other drawers indicating negative tubes Remove negatives single tube each negative tube removed must be scanned by the barcode scanner Follow steps 1 2 as above Stations with negative tubes light up with flashing GREEN lights and the barcode As each negative tube is removed the barcode label should be scanned prior to the tube in a rack in the sequential order of removal Continue to remove desired tubes and scan their barcode labels Follow steps 67 above scanner turns on removing the next tube The instrument will beep indicating that the correct tube was scanned The LED at the empty station extinguishes Print Unloaded Negative Tubes Report Press print report soft key and select unloaded negative tubes gt soft key When report finishes printing compare unloaded tubes with report Resolve any discrepancies If tubes match report press the ok soft key When you press the ole soft key the information contained in the report is removed from the d atabase NOTES e Visually inspect all negative tubes prior to disposal Any tubes that appear to have growth must be processed according to Section
54. for Second line Drugs There is no means for designating the drug name or concentrations of second line drugs in the MGIT system Therefore a site specific procedure must be developed for documenting second line drug testing in MGIT There are two options to perform second line DST 1 Manual interpretation Use AST set carrier and enter in the instrument as Undefined drugs In this case interpretation of results is done manually using the GU values from the unloaded AST set report Instrument interpretation Use AST set carrier and enter in the instrument as a first line drug protocol Each second line drug is coded with the name of one of the first line drugs The instrument will interpret results automatically when the test is complete For both manual and instrument interpretation the following procedures for correcting the MGIT unloaded AST reports must be written and performed by all applicable staff Instructions for preparing the antibiotic stock solutions and dilutions needed to achieve the working solutions AST carrier s used Configuration of the drugs in the AST carrier set s Approved abbreviations for the full drug names Process for clarifying the drug names on the MGIT printout Cross out each undefined or first line drug name using one line through the printed drug name and add the applicable 2nd line drug name or approved abbreviation next to it Cross out each concentration list
55. for clinical trials for the treatment of multidrug resistant tuberculosis MDR TB The laboratory manual references best practices from the Clinical and Laboratory Standards Institute CLSI the Foundation for Innovative New Diagnostics FIND MGIT Procedure Manual and laboratory procedures from the National Institutes of Health TB Research Unit as well as biosafety recommendations from the World Health Organization WHO and the US Centers for Disease Control and Prevention CDC Each procedure lists the materials equipment and forms required for the test along with the applicable quality control procedures The underlying goal of the laboratory manual is to maximize recovery of MTB even in the presence of contaminants to provide an accurate picture of the microbiological performance of the investigational compound Thus recovery efforts for MGIT cultures are highlighted in several flow charts designed to address scenarios such as mixed cultures contamination and early positive cultures positive MGIT signal with negative confirmatory tests In addition quality and standardized reporting is highlighted throughout the manual to ensure consistent reporting across laboratories Finally as clinical trials are highly regulated quality control QC and quality assurance QA are of utmost importance in the laboratory The manual provides detailed QA QC procedures as well as comprehensive appendices to record these data 1 1 Study D
56. indicator on the drawer in addition to indicating positive MGIT growth tubes in the drawer also indicates the presence of one or more completed AST sets The station indicators illuminate in the same pattern as with individual MGIT tubes However with AST tube sets ALL the stations for the set illuminate or flash The leftmost station is ALWAYS the assigned location of the set s Growth Control tube Additional details are found in the BACTEC MGIT 960 System s AST manual which should be stored within easy access of the MGIT system The operator of the MGIT must be familiar with this manual Procedure Materials Discard bucket with biohazard bag and appropriate tuberculocidal disinfectant e 7ml MGIT tubes e 7ml MGIT PZA medium tubes BD SIRE MGIT kit reagents BD Pyrazinamide MGIT kit reagents Second line drug powders depending upon local standard of care recommendations Amikacin Sigma catalog 1774 or 2324 Capreomycin Sigma catalog CA142 Gatifloxacin Sigma catalog G7298 Kanamycin Sigma catalog K1876 or K4000 Levofloxacin Sigma catalog 28266 Moxifloxacin Sigma catalog 32477 Ofloxacin Sigma catalog 08757 BD BACTEC MGIT SIRE and PZA supplement MGIT OADC Enrichment MGIT supplement for second line drugs BD catalog 245116 AST carrier sets Sterile distilled deionized water NaOH for dissolving Gatifloxacin and Ofloxacin if applicable McFarland standards 0
57. mycobacterial growth is confluent and 3 drug susceptibility tests are more difficult to perform using egg based media because some drugs must be adjusted to account for their loss by heating or by interaction with certain components of the egg As with all media preparation attention must be given to purity of chemical components including quality of eggs preparing and sterilizing medium and glassware exposure of final product to excessive heat or sunlight and method and length of storage All lab prepared media must be tested for sterility and performance characteristics before being used Procedure Materials e LJ medium e Sterile transfer pipettes with graduations marking volume individually packaged e Tuberculocidal disinfectant e Discard bucket with biohazard bag insert containing appropriate disinfectant e Sterile loop or disposable applicator stick e Ziehl Neelsen stain carbol fuchsin 3 acid alcohol methylene blue Parafilm icroscope slides frosted at one end new and clean Paper towel soaked in disinfectant Sterile distilled water Permanent marker Pencil for labeling slides e Study labels Incubator Mycobacteriology Laboratory Manual 48 Mycobacteriology Laboratory Manual Forms e ab specific forms for reading recording LJ results e Laboratory Specimen Requisition Form this is a site specific form used for routine laboratory requests Appendix B Study Source Document Workshe
58. not available specimens can be held in coolers with ice packs NOTE Refrigerators in which sputum specimens are stored must be monitored with daily temperature readings using an internal detached thermometer The acceptable range is 2 8 C Record the temperature daily on the appropriate Equipment Temperature Record Form Appendix D 5 3 Transport of Sputum Specimens 1 Specimens should be delivered to the laboratory the same day as collected if the study site is in the same city as the mycobacteriology laboratory Spu or early afternoon so Spu on processing the same d Spu may be stored in the si um specimens col um specimens co lected in the morning should reach the laboratory in the morning hat they can be processed the same day lected in the late afternoon should either arrive to the laboratory he same day for processing the next day or early the following morning for um specimens co lected at the very end of the day when transport is not available 5 refrigerator overnight but must be delivered to the laboratory early the following morning 13 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 2 If specimens are transported longer distances they must be delivered as soon as possible but no later than 48 hours from time of collection These specimens must be stored in the refrigerator and transported on ice Arrange a pick up time
59. number of contaminated cultures reported in that month Calculate the percentage 96 contaminated number contaminated number reported x 100 96 contamination NOTE Using this method the cultures processed in any given month may not correlate exactly with the cultures reported in that same month but statistics over time will provide consistent indicators for the overall performance of lab procedures Repeat Step 1 for LJ cultures Repeat steps 1 and 2 separating patient collected specimens and hospital clinic collected specimens for both MGIT and LJ cultures Record the 96 contaminated for each medium and compare results with data from the previous month and the 5 average Calculate the on going cumulative mean rate for each medium 16 4 6 AFB Smear Review for Technical Competency Strongly recommended Periodic smear review is recommended to assess and improve technician proficiency and assure consistency among examiners 1 Frequency Once a month 2 Acceptable results a Negative smears should be resulted as negative by all technicians b Positive smears should not vary by more than one quantification level Corrective actions a Review discrepant results with technician s and re examine smears that are not in agreement b Provide additional training as indicated to improve performance 4 Documentation Save results in QC binder and technicians personnel files if appropriate 95
60. or fails to grow M tuberculosis The culture must be identified as a pure growth of M tuberculosis ZN positive MPT MTB 64 antigen or molecular test positive Section 11 Solid Culture Lowenstein Jensen LJ Media and Section 13 Rapid Identification of M tuberculosis Complex Using a Chromatographic Immunoassay Add 4 ml of Middlebrook 7H9 Broth or BBL MGIT broth to a 16 5 x 128 mm sterile tube with cap containing 6 10 2 mm glass beads or use tube the same size as lab s McFarland standards Using a sterile loop or applicator stick scrape as many colonies as possible from growth no more than 14 days old Do not remove any solid medium Suspend the colonies in the Middlebrook 7H9 broth Vortex the suspension for 2 3 min to break up the larger clumps The suspension should exceed a 1 0 McFarland standard in turbidity Let the suspension stand for 30 min without disturbing Sediment should settle to the bottom of the tube Transfer the supernatant fluid to another 16 5 x 128 mm sterile tube with cap avoid transferring any of the sediment and let the suspension stand for another 15 min Transfer the supernatant fluid it should be smooth free of any clumps to a third 16 5 x 128 mm sterile tube Using 7H9 broth adjust the suspension to a 0 5 McFarland standard Do not adjust below or above a 0 5 McFarland Standard Dilute 1 ml of the adjusted suspension in 4 ml of sterile saline 1 5 dilution using a new 16 5 x 128 mm ste
61. organisms This exercise assesses consistency of technical performance in preparing MTB suspensions according to a turbidity standard diluting MTB suspensions and pipetting skills 1 Frequency Once per month and performed by alternating technicians if appropriate 2 Controls One dilution of M tuberculosis H37Rv or H37Ra in 7H9 broth It is highly recommended that a frozen stock no older than six months be used to prepare the dilution 93 Mycobacteriology Laboratory Manual 94 Mycobacteriology Laboratory Manual 3 Acceptable results a The TTD must fall within the 6 10 day range as referenced in the FIND MGIT Procedure Manual b Month to month consistency in the lab s established TTD range for the 1 500 dilution Corrective actions TTD results will be closely monitored by comparing each monthly TTD result to the laboratory s own ongoing range of results and to a global range established by the Sponsor laboratory network If expected results vary by more than 2 standard deviations within the laboratory s own established range a Review data and all procedures with technician performing the exercise and repeat test under observation if necessary b If TTD is significantly decreased check culture for contamination and if contaminated repeat exercise with fresh culture c If TTD is significantly increased check viability of culture and age of culture if using frozen stocks Documentation a Reco
62. plastic bag for any leaks or cracks in the outer container If a leak is seen do not open the plastic bag Autoclave the container and bag and discard Record this occurrence in the laboratory Comments section on Appendix A and in the Lab Specimen Logbook Registry It is important to notify the site about the reason for any leaks If a container is cracked or not closed properly site staff can re educate patients and or the courier Open the plastic biohazard bag remove the sputum container and verify the specimen information on the study label against the Laboratory Specimen Requisition Form and Appendix A Do not remove the white cap or the plastic label from the sputum collection container unless specimen receipt is performed in a biosafety cabinet The individual receiving the specimens records their name signature and date time of receipt as well as a description of the specimens on the Laboratory Section of Appendix A 15 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 2 If the laboratory finds a mislabeled or mismatched specimen and requisition the following procedures must be followed For a specimen with an ID on the tube and a mismatched incomplete or missing requisition The laboratory will contact the site to obtain any needed information before the specimen is processed Specimens without requisitions are unacceptable and will not be processed until a requisition fo
63. test on Appendix M 39 Mycobacteriology Laboratory Manual 40 Mycobacteriology Laboratory Manual 6 ii In addition subculture the MGIT broth to a LJ slant Section 11 Solid Culture Lowenstein Jensen LJ Media and incubate both the LJ and MGIT at 37 1 in an incubator Record the LJ subculture date on Appendix M iii Notify the Sponsor immediately for further assistance on how to report this culture result and document the date of notification on Appendix M In rare cases non acid fast bacteria may be seen on a ZN negative smear while the BAP has no growth at 72 hours of incubation Re incubate the BAP for an additional 24 hours and repeat the ZN smear from the MGIT If the same organism is again identified on the smear and verified by a second technician and the 96 hour BAP remains no growth note the presence and type of non acid fast organisms on the laboratory worksheet In addition consult the lab supervisor and the Sponsor immediately for further assistance on how to report this culture result 10 8 Drug Susceptibility Testing from MGIT Cultures 1 MTB growth from the MGIT tube will be used for drug susceptibility testing at specified timepoints following the guidelines outlined in Section 12 Drug Susceptibility Testing MGIT System If a MGIT culture is resulted as Positive for MTB complex and contaminated a DST is required see Section 2 Sample Specimen Timetable and the primary LJ cu
64. the visit The laboratory director or delegate must countersign for each visitor Visitors include but are not limited to auditors CRAs Sponsor representatives and site staff Appendix A Specimen Transfer Forms File the original form in the laboratory binder s under the Specimen Transfer Form tab by Subject ID and visit number Upon completion of each form copy and send it to the Clinical Research Coordinator CRC for inclusion in the subject s binder Insert additional tabs to separate patients by Subject IDs especially when the lab receives a large volume of specimens Alternatively file the Appendix A form in front of the appropriate Appendix B form according to subject ID and visit interval Appendix B Study Source Document Worksheets File the original form in the laboratory binder s consecutively by Subject ID and sequentially by visit interval Insert tabs to separate patients by Subject IDs especially when the lab receives a large volume of specimens Update information from lab source registers and worksheets onto the form in a timely manner Remove the form from the binder only when necessary to make a copy with updated material for the CRC the technician or supervisor must sign their initials and the date in he margin next to new information that is being sent to the site Supervisor must sign and date the form in the designated spaces when all tests for he specimen have
65. which will allow the specimens to be delivered on a week day unless the laboratory is open on Saturdays and delivery can be made on Saturdays Notify lab if a Saturday delivery is planned Before transporting from the clinic a designated staff member of the study team must verify for each transport box that The total number of sputum tubes containers in the box corresponds to the number of Laboratory Specimen Requisition and Appendix A forms one of each form per specimen The screening ID number or subject ID number on the container tube corresponds to hat on the Laboratory Specimen Requisition and Appendix A forms The Laboratory Specimen Requisition and Appendix A forms contain all the requested information for each patient The Dispatch Section of Appendix A must be completed and signed by the individual who collected the specimen or the person responsible for handing off the specimens to he transport service When this verification is complete put the specimens in the transport container with ice packs For transport on site or within the city each specimen should be in a sealed plastic bag If bags are not available check to ensure that caps are tightly closed and place containers or tubes in a rack to keep upright during transport For longer distances each specimen must be packaged in a separate sealed plastic bag before placing in a transport cooler with ice cold packs Place the Laboratory Specimen Requisition and A
66. with personnel 4 Documentation a Record data on the Monthly Data Monitors Forms Appendix or other comparable lab specific document b If any corrective measures are necessary prepare an Appendix K form to document the activities Isolation Rate of MTB and MOTT ile Procedure a Count the number of MTB isolates and calculate the percentage using the total MGIT cultures reported in a given month MTB culture total culture positive x 100 Count the number of MOTT isolates and calculate the percentage using the total MGIT cultures reported in a given month MOTT culture total culture positive x 100 Repeat steps a and b for cultures d Record isolation rates for both species and compare results with data from the previous month and lab s average e Calculate the cumulative mean positivity rate for each mycobacterial species Results Isolation of MTB complex organisms in relation to other mycobacterial species should remain fairly constant over time If there is a sudden increase in isolation of MOTT it may be due to increase in numbers of follow up specimens from subjects colonized with MOTT a common observation However this may also indicate the presence of an environmental contaminant or cross contamination event Corrective Actions If there is a significant increase in MOTT isolation seemingly unrelated to individual patient colonization thoroughly review all procedures e
67. 1 Visit 2 has a positive culture from one specimen e Visit 3 has a positive culture from one specimen ep 7 Microbiology Lab Manual Microbiology Lab Manual Subj ID 1002 Visit 4 has positive cultures from two specimens 1 ana Visit 5 has positive cultures from two specimens 1 CO and 2 Cryobox 1 Cryobox 2 Subj ID 1001 Subj ID 1002 Subj ID 1001 Subj ID 1002 V2 V4 5 5 2 V2 v3 v3 V4 V5 V5 aliquot 1 aliquot 2 aliquot 1 aliquot 2 1 2 1 2 aliquot 3 aliquot 4 aliquot 3 aliquot 4 1 2 1 2 4 Record on the Freezer Storage Log for M tuberculosis Isolates Form Appendix J the screening and or subject ID visit number specimen if applicable aliquot if applicable total volume date cryotube is prepared and lab accession number Also include the freezer number if applicable rack number box number and place position numbers on the freezer storage log If a rack is not used record the location where the box is stored in the freezer 5 Record on Appendix B that isolate was banked for long term storage 15 SHIPPING OF FROZEN ISOLATES Purpose This section provides instructions on the proper preparation and shipping of MTB isolates Materials Cryoboxes plastic size must be compatible with dimensions of lab specific cryovial
68. 5 and 1 0 Pipet Aid p1000 and p200 pipettes or equivalent and sterile aerosol resistant tips Sterile graduated serological pipettes 5 and 10 ml Sterile graduated transfer pipettes individually wrapped Sterile saline Blood agar plates Middlebrook 7H9 broth for preparing inoculum from positive LJ cultures e 16 5 x 128 mm sterile disposable tubes or tubes of the same size diameter as the lab s McFarland standards Sterile disposable loop or applicator stick Capped sterile tubes containing 2 mm glass beads for preparing inoculum from LJ BBL Middlebrook 7H9 broth Vortex Analytical balance and weigh boats Graduated cylinder Erlenmeyer flask or media bottles Sterile syringes 10 or 20 ml Syringe filters 0 22 pore size e g Millex GS 2 ml sterile cryotubes with screw top polyethylene or polypropylene BD Bactec AST Carriers Permanent marker Study labels Forms Lab specific forms for recording DST results Appendix F DST QC Form or use site specific form if available and equivalent Appendix B Study Source Document 12 1 Preparing AST Carriers The susceptibility test may be configured in a variety of formats Since different profiles of non MGIT drugs will be tested check the BACTEC MGIT 960 User s Manual AST Instructions and Appendix A for AST carrier sizes and optional configurations When setting up the AST tube sets you must use the correct size
69. 60 Instrument overview The BACTEC MGIT 960 instrument is capable of monitoring a total of 960 7 ml MGIT tubes The tubes are arranged in three continuously incubated drawers labeled A B and C each of which holds up to 320 tubes Each drawer contains an apparatus consisting of Tube rack rack in the drawer that holds the MGIT tubes Stations individual wells in the rack into which tubes are inserted The detector assembly sits below the rack and has 16 detectors one for each row of stations The assembly moves from left to right and back taking test readings for each of the 20 station columns and the calibrator tube Drawer status indicators three lamps located on the front of each drawer one indicates a positive one indicates a negative and one indicates a station error Barcode scanner located at the front of the instrument it is used to scan tube labels for Specimen identification The scanner turns on automatically LCD display and keypad presents all the information needed to monitor the instrument and station status to enter and remove tubes set up the instrument print reports and perform routine instrument maintenance Additional details are found in the BACTEC MGIT 960 System s User s Manual Chapters 3 and 4 which should be stored within easy access of the MGIT system The operator of the MGIT must be familiar with this manual Summary of how the MGIT instrument works The instrum
70. ABLE OF CONTENTS u 1 2 ST OF ABBREVIATIONS AND ACRONYMS INTRODUCTION SAMPLE SPECIMEN TIMETABLE SPECIMEN FLOWCHART LABORATORY BIOSAFETY AND INFECTION CONTROL SPECIMEN COLLECTION STORAGE AND TRANSPORT RECEIPT AND LOGIN OF SPUTUM SPECIMENS IN THE LABORATORY PROCESSING SPUTUM FOR SMEAR MICROSCOPY AND QUALITATIVE CULTURE ACID FAST BACILLI MICROSCOPY AFB PREPARATION AND STAINING ACID FAST BACILLI MICROSCOPY AFB EXAMINATION LIQUID CULTURE MYCOBACTERIA GROWTH INDICATOR TUBE MGIT Flow Chart 1 General Algorithm MGIT 960 Cultures Flow Chart 2 Contaminated MGIT Cultures Flow Chart 2A Suspected MOTT Cultures Flow Chart 3 MGIT Early Positive Cultures ZN BAP negative 1 SOLID CULTURE LOWENSTEIN JENSEN LJ MEDIA 2 DRUG SUSCEPTIBILITY TESTING MGIT SYSTEM Flow Chart 4 Invalid x200 Errors from MGIT DST Flow Chart 5 Invalid x400 Errors from MGIT DST 13 RAPID IDENTIFICATION OF M TUBERCULOSIS COMPLEX USING AN IMMUNOCHROMATOGRAPHIC ASSAY 14 LONG TERM STORAGE OF ISOLATES 15 SHIPPING OF FROZEN ISOLATES 16 QUALITY ASSURANCE REFERENCES 25 29 32 43 44 45 46 47 65 66 67 72 75 77 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E APPENDIX F APPENDIX G APPENDIX H APPENDIX I APPENDIX J APPENDIX K APPENDIX L APPENDIX M APPENDIX N SPECIME
71. DST Quality Control Follow instructions in Section 12 5 Growth Control Tube Preparation and Inoculation for the drugs being tested Important considerations when preparing the DST QC are Proper reconstitution of lyophilized drugs Proper preparation of drug powder suspensions Proper dilution of the QC organism for the Growth Control and drug tubes Monitor QC drugs until signaled complete by the instrument and interpret the same as for patient isolates 16 4 Quality Monitoring Activities Monitoring of critical practices that may affect the outcome of clinical trial results is required for the duration of the study These practices include 1 Temperature monitoring 2 Equipment maintenance 3 Regular monitoring of specimen processing 4 MGIT time to detection TTD monthly exercise 5 Contamination rate assessment 6 Supervisor review of QC activities on a monthly basis In addition though not required the following quality monitoring activities are strongly recommended and will serve to strengthen laboratory results 1 Review of AFB examination competency 2 Analysis of laboratory data 16 4 1 Temperature Monitoring Required 16 4 1 1 Thermometers Internal thermometers must be used for all equipment monitoring All thermometers used in assessing equipment temperatures must be calibrated against a standardized certified thermometer US Bureau of Standards or equivalent before putting into use and annu
72. Guideline Third Edition NCCLS Document GP26 A3 Wayne PA 2004 9 Ezzella J et al Guidelines on Good Clinical Laboratory Practice Bridging Operations between Research and Clinical Research Laboratories Journal of Pharmaceutical and Biomedical Analysis 2008 46 1 18 29 10 International Union Against Tuberculosis and Lung Disease Priorities for Tuberculosis Bacteriology Services in Low Income Countries 2nd Edition Paris France 2007 Isenberg HD Editor Clinical Microbiology Procedure Handbook Vol 1 2nd Edition American ociety for Microbiology Publisher Washington D C 2004 12 Kent PT Kubica GP Public Health Microbiology a Guide for the Level Ill 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 National Institutes of Health DAIDS Guidelines for Good Clinical Laboratory Practice Standards HIV Clinical Research Support Contract 1 50022 Bethesda Maryland 2008 4 Siddigi SH and R sch Gerdes S MGIT Procedure Manual Foundation for Innovative Diagnos tics July 2006 5 World Health Organization Good Clinical Laboratory Practice GLCP Geneva Switzerland 2009 6 World Health Organization Laboratory Biosafety Manual Third Edition Geneva Switzerland 2004 7 World Health Organization Maintenance Manual for Laboratory Equipmen
73. L N3WIO3dS avis hate aia Y a e a S NE a RS pp JosiAsedng gt 1403 VIVO ATHLNOW 1 XIaN3ddv u1uou y sainyjnd uodn sjequunu AON PO Bny Aine eun Kow yow SPs 4 010 jue218d 010 4 1001 4 4 jue21ed 241 MESS pei e o ld 040 sjou yoo enin enn e1nj n Jpeuis 4 BINN 5 pp eioq 40 1 Wesem 138 139 Kuo0j amp Joqe 1 ujuoul y gt uodn peseq sjequinu AON PO das
74. MGIT and to detect positive samples rapidly To make a semi quantitative assessment of the bacterial load by determining the time taken for culture tubes to signal positive time to detection TTD in the BACTEC MGIT 960 system The MGIT culture result along with confirmatory ZN BAP and rapid ID tests are the primary indicators of the presence of viable MTB in the sputum Principle Mycobacteria multiply in a nutrient rich medium while contaminating bacteria are inhibited by the addition of a cocktail of antibiotics Growth of bacteria including mycobacteria is indicated by fluorescence which increases proportionally as oxygen decreases in the tube The instrument detects this fluorescence in the medium using a UV light and complex computer algorithms Sputum specimens are processed Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture and inoculated into 7ml MGIT tubes which are supplemented with OADC Growth Supplement and a cocktail of antibiotics PANTA The MGIT tubes contain a fluorescent compound embedded in the base of the tube which is sensitive to the presence of oxygen dissolved in the broth Initially the large amount of oxygen quenches the emissions from the compound and little fluorescence is detected Bacteria present in the concentrated sputum specimens metabolize oxygen in the culture medium allowing the fluorescence to be detected Blood samples are not suitable for the MGIT system BACTEC MGIT 9
75. N TRANSFER FORM STUDY SOURCE DOCUMENT WORKSHEET DAILY QC STAINING FORMS EQUIPMENT TEMPERATURE RECORD FORMS NEW REAGENTS MEDIA QC FORMS DST QC INVENTORY FORMS MTB IDENTIFICATION QC FORM ICA SLIDE TEST MPT64 MTB 64 WEEKLY MGIT L CULTURE QC FORM MONTHLY DATA MONITORING FORMS STORAGE LOG FORMS CONTINUOUS QUALITY IMPROVEMENT FORM MGIT TTD WORKSHEET EARLY MGIT POSITIVES EARLY CONTAMINATED CULTURES TRACKING WORKSHEET LABORATORY VISITOR LOG 101 102 104 108 131 135 136 137 142 144 145 146 147 LIST OF ABBREVIATIONS AND ACRONYMS AFB AST BAP BD BSC BSL COA CDC CFR CFU CLSI CRA CRC DST EMB FIND GC GU ICA INH IUATLD LJ MDR TB MTB MGIT MOTT NALC NaOH NTF NTM PANTA Acid fast bacilli Antimycobacterial susceptibility testing see DST Blood agar pla D Becton Dickinson Biological Safety Cabinet Biosafety level Certificate of analysis US Centers for Disease Control and Prevention US Code of Federal Regulations Colony forming unit Clinical Laboratory Standards Institute Clinical Research Associate Clinical Research Coordinator Drug susceptibility testing see AST Ethambutol Foundation for Innovative New Diagnostics Growth control Growth unit Immunochromatographic assay Isoniazid International Union Against Tuberculosis and Lung Disease L wenstein Jensen Multidrug resistant tuberculosis Mycobacterium tuberculosis complex Mycobacteria Growth
76. ach new batch of prepared medium Controls 1 396 of total number of plates prepared tested with diluted suspension of E coli or Staphylococcus aureus e g 296 of a 100 ml batch of medium is 4 tubes in total See procedure below 3 Acceptable results Growth of typical colonies within 48 hours 4 Corrective Actions a If no growth is seen repeat performance check b If colonies are not typical of the bacteria used or the growth is mixed repeat performance check with a fresh culture of the stock strain c If performance still not as expected upon repeat testing notify supervisor immediately and discard entire batch d Investigate and resolve problems then prepare new media 5 Documentation Record results on the Reagent Media QC Form Appendix E If QC results are not acceptable prepare an Appendix K form to document the corrective action Preparation of Bacterial Suspension and Working Dilutions 1 The QC strain of Escherichia coli or Staphylococcus aureus can be a clinical isolate from the microbiology laboratory or reference strain To maintain a frozen stock of the control bacteria a Prepare a suspension from 24 hour growth from BAP in Tryptic Soy broth with 1596 glycerol or other appropriate storage medium to the density of a McFarland No 1 standard b Aliquot 1 5 ml volume into labeled cryovials C Freeze at 70 to 80 10 for up to twelve months To prepare a fresh suspension T
77. age NOTES 1 If ZN is not performed because culture is negative mark N A for this test on Appendix B 2 In the unlikely event that a culture result cannot be obtained e g broken or lost tube report the solid culture result as Unknown and provide a brief explanation in the 1 Comments field of Appendix B In addition notify the Sponsor of any cultures reported as Unknown 12 DRUG SUSCEPTIBILITY TESTING MGIT SYSTEM Purpose Multidrug resistant MDR TB is caused by strains of MTB resistant to at least isoniazid and rifampicin and effective patient management requires optimized treatment regimens These complex regimens can include a fluoroquinolone injectable drugs either an aminoglycoside or a cyclic peptide compounds from other drug classes as well as any remaining first line susceptible drugs Therefore reliable drug susceptibility testing DST of these anti TB drugs is crucial for the management of MDR TB and for preventing emergence of additional drug resistance in these patients DST will be performed on isolates from newly enrolled patients and at regular timepoints throughout the trial As two specimens are collected at each timepoint DST will be performed on the first positive culture from the set only not both All first line drugs streptomycin isoniazid rifampicin ethambutol SIRE and pyrazinamide PZA will be tested in the MGIT 960 system Additionally the fluoroquinolones gatifloxacin levo
78. ally thereafter either in house or through a commercially available service Identify thermometers by a numbering system and maintain documentation of their calibration in a lab worksheet or logbook Table 16 3 Equipment Temperature Ranges Equipment Temperature Ranges Refrigerators 2 8 Freezer Ultralow 70 to 80 10 Incubators 37 1 Refrigerated centrifuge 412 960 37 1 Room lemperature varies a Temperature of the refrigerator where sputum specimens are stored until they are sent to the laboratory must be monitored daily b Internal instrument temperature reading should be x 1 5 C of manual reading c Range determined by requirements of reagents or instrument housed in the particular room MGIT room 19 30 room containing unrefrigerated centrifuge 20 16 4 1 2 Temperature Reading Recording 1 Frequency a Daily reading recording of temperatures is required preferably in the early morning before work commences Twice daily readings are recommended as equipment may malfunction during working hours which may not be detected until the following day b Weekends holidays may be excluded if no staff are available to monitor the equipment during those times check temperatures immediately the beginning of the next working day 89 Mycobacteriology Laboratory Manual 90 Mycobacteriology Laboratory Manual 2 Acceptable results Temperatures are wi
79. amination solution since mycobacteria can be killed off if exposed to NaOH longer than 20 minutes Transfer tubes in a 50 ml tube rack to the centrifuge Place tubes in centrifuge buckets ensuring they are equally balanced and the biosafety covers lids are properly placed on each of the buckets 23 Mycobacteriology Laboratory Manual 24 Mycobacteriology Laboratory Manual 15 Centrifuge at 3 000 xg note this is not 3 000 rpm for 15 min at 4 12 C The 15 minute centrifugation period should begin only after 3000 xg speed has been achieved 6 Once centrifugation is complete remove the centrifuge buckets without delay and carry to the BSC prior to opening the biosafety covers 17 Carefully decant the supernatant into a splash proof container containing a tuberculocidal disinfectant taking care not to disturb the sediment at the bottom of the tube 8 Add sterile phosphate buffer pH 6 8 from individual 50 ml aliquots to the pellet to a final volume of 2 5 ml and resuspend the sediment using a pipette gently aspirating and expelling buffer If necessary compare the volume in the tube to a reference 50ml tube with a pre measured 2 5 ml volume of water Use a new pipette for each specimen 19 Use the suspended pellet for culture in the MGIT BACTEC 960 TB System Section 10 Liquid Culture Mycobacteria Growth Indicator Tube MGIT and culture on LJ media Section 11 Solid Culture Lowenstein Jensen LJ Me
80. ance is required for all equipment in his list In addition if large equipment is moved relocated it must be re certified or serviced prior using Table 16 5 Minimum Equipment Maintenance Schedule Equipment Maintenance Semi annually Annually Autoclave Maintenance by autoclave technician recommended required Micropipettors Pipette Aid Calibration recommended required Analytical balance Calibration recommended required Biosafety cabinet Certification required Centrifuge Calibration of speed timer temperature recommended required Inspection cleaning and lubrication by service recommended required Microscopes provider Clean condensers fans and blower motor verify recommended required Air conditioners mechanics and check filter Clean condensers verify door gaskets recommended required Freezer 70 to 80 Clean fan and blower motor routine maintenance recommended required Refrigerators Clean condensers and fans routine maintenance recommended required Check door gasket seal heating and cooling recommended required Incubator elements electronic components routine maintenance Calibration tube check thorough cleaning required of relevant components For further details on equipment maintenance and cleaning refer to Maintenance Manual for Laboratory Equipment 2nd Edition World Health Organization Geneva
81. and Incubation il De 4 Remove one aliquot of the 10 and 107 dilutions from the freezer While thawing dilutions bring broth to room temperature If not already aliquoted into tubes aseptically dispense 4 5 ml 7H9 broth into appropriate number of sterile disposable tubes e g 16 5 x 128 mm With a micropipettor and ART tips mix the working solution with the pipettor 2 3 times to ensure even distribution of and inoculate tubes with 500 ul of each dilution Vortex to mix Incubate tubes with caps loosened at 37 1 reading weekly for 7 14 days Observe all tubes for evidence of growth and note the approximate number of days for characteristic growth to appear 16 2 1 2 3 QC Protocol for Blood Agar Medium Sterility Check Frequency Each new batch of prepared medium Controls 1 396 of total number of plates prepared incubated at 35 37 C for 72 hrs Acceptable results No growth on any plate Corrective Actions a If growth is seen on any plate repeat sterility check using 10 additional plates b If gt 1 plate is contaminated upon repeat testing notify supervisor immediately and discard entire batch c Investigate and resolve problems then prepare new media Documentation Record results on the Reagent Media QC Form Appendix E If contamination is seen prepare an Appendix K form to document the corrective action Performance Check 2 Frequency E
82. and adjust the turbidity to a McFarland No 0 5 standard Tube C is the stock suspension for QC testing This suspension may be frozen in small aliquots 1 5 ml in cryotubes at 70 to 80 C 10 C The frozen suspensions may be used for up to six months Once thawed do not refreeze 13 Ideally ensure that new frozen stock passes testing indicated below before using in routine QC procedures Preparation of Dilutions ile 2 Remove one aliquot of the stock suspension from the freezer and allow thaw Alternatively prepare a fresh 0 5 McFarland suspension using colonies within 10 15 days of first appearance from LJ media Carefully adjust the inoculum with a spectrophotometer or visually with a Wickerham card Dilute the stock suspension freshly prepared or frozen 1 5 by transferring 1 0 ml of suspension to 4 0 ml of sterile water or saline Mix well Tube 1 Dilute 1 10 by adding 0 5 ml of suspension from Tube 1 into 4 5 ml of sterile water or saline Tube 2 Mix well and then dilute 1 10 again by adding 0 5 ml from Tube 2 to 4 5 ml of sterile water or saline Tube 3 Mix well The final dilution of Tube 3 is 1 500 85 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual Inoculation and Incubation 1 Supplement MGIT medium with Growth Supplement and PANTA as specified in Section 10 Liquid Culture Mycobacteria Growth Indicator Tube MGIT
83. ata Maintenance of Documents 1 1 1 Source Documents All information recorded for the purposes of the study regardless of form or the media on which it may be recorded is considered source data Source data are contained in source documents i e the original records where results and observations are entered Items that constitute source documents include but are not limited to Manually collected data such as lab requisitions lab registers and laboratory worksheets AFB smear log DST worksheets QC records etc Data generated from automated instruments MGIT unloaded positive negative and DST reports Specimen Transfer Form Appendix A Study Source Document Appendix B QA QC records All original source documents must be maintained do not destroy or discard these records 1 1 2 Notes to Files A Note to File NTF is a document used to describe an error i e protocol lab manual deviation or provide clarification for a result The Sponsor an individual institution company or organization Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual that takes the responsibility to initiate manage or finance a clinical trial must have a standard template with the following three sections Description of the issue s Steps taken to mitigate the issue s Resolution of the issue s NTFs must be filed in the study lab binder 1 1 3 Appendix K Forms An Appe
84. ative Negative Control control slide control slide control slide Slides date of date of date of date of preparation expiration preparation expiration Results Neg Confirmation Day Required Scanty 1 2 3 QC Pass Yes No Technician Initials Positive control Negative control e C O eo 31 strain tested Strain tested negative positive control control Reviewed by Date Signature Comments Confirmation required by another technician or prepare another smear stain and read Date format dd mmm yyyy Mycobacteriology Laboratory Manual Note If QC is out of range inform supervisor and record any action taken below ZN Stain Batch Lot Expiration Date Acid Alcohol Decolorizer Batch or Lot Expiration Date Methylene Blue Batch or Lot Expiration Date Positive Control Slides date of preparation Positive control slide date of expiration Negative control slide date of preparation Negative control slide date of expiration Results Posi tive Negative Da Y
85. ch subject are printed out and filed by patient and visit interval in the lab binder Paper printouts must identify the screening and or subject ID number date of printout and clear page numbering Each page of the printout must be initialed and dated by the lab director or designee The following information must be captured on the last page of the printout either written in ink or by means of a pre printed label Total number of pages in the printout Date and signature of the lab director or designee A statement that reads confirm that the contents of this printout fully reflect the data captured for this subject as of today Each time modifications are made to data that has previously been printed the appropriate page section must be printed again and the lab director or designee must initial and date the change The previous printout must remain in the laboratory binder do not discard The lab director or designee must also inform the site coordinator data entry person and site monitor of the modifications This notification should be documented by the appropriate laboratory staff The Microbiology Lab Worksheets binder provided by the Sponsor must contain at least seven tabs listed below If additional binders are required please notify your CRA Appendix N Laboratory Visitor Log Each time someone visits the lab to discuss a trial s he must sign the visitor log Appendix N with the date and purpose of
86. cohol and let stand for 2 min Wash off the acid alcohol with distilled water Flood slides with 0 596 potassium permanganate for 1 2 min Do not allow potassium permanganate to act over 2 min or it might quench the fluorescence of acid fast bacilli Wash off the stain with distilled water Allow slides to air dry in the slide rack DO NOT BLOT Protect smears from light and examine immediately using the fluorescent microscope If unable to read right away place slides in covered box 8 3 2 Acridine Orange Procedure d Place slides on staining rack so they are at least 1 cm apart flood with 2N H SO and let stand for 10 min Wash off the 2N 2504 with distilled water Flood slides with acridine orange working solution and let stand for 10 min Wash off the stain with distilled water and tilt slide to drain Flood the slide with 296 acid alcohol and let stand for no more than 3 min Wash off the acid alcohol with distilled water Allow slides to air dry in the slide rack DO NOT BLOT Protect smears from light and examine immediately using the fluorescent microscope If unable to read right away place slides in covered box 27 Mycobacteriology Laboratory Manual 28 Mycobacteriology Laboratory Manual 8 4 Procedure for ZN Staining 1 Place slides on staining rack so they are at least 1 cm apart and flood with carbol fuchsin 2 Heatthe slide to steaming with the flame from a Bunsen burner An electric heat
87. cteriology Laboratory Manual Lot Batch Results Number 2 sie Run Bom Quantity pr Days MIB QC pass Prepared ateo Prepared 95967 growth e Y SC Yes No Broth Tech Initials Batch Tech Initials Control Initials roti UG le strain Unino Monthly Supervisor Siral culated onio sted Review date initials New Lot Batch Media Component Brand Lot Received Expiry Quantity Date Manufacturer Number Date Date Received Opened Middlebrook 7H9 basal medium Glycerol ADC or OADC supplement Date format dd mmm yyyy 127 Mycobacteriology Laboratory Manual 128 Date Lot Batch Blood Media Expiry Date Results Results Date Source Prepared Date of pm Tested Positive Seali Tech hang Meta Number Tech Batch P Tech Initials Growth Control Initials Initials Strain tested Unino Monthly Supervisor for culated Review performance plates Initials Date New Lot Batch Media C Lot Received Expiry Quantity Date emponen Manufacturer Number Date Date Received Opened TSA base Date format dd mmm yyyy Mycobacteriology Laborato
88. ction area where it is captured by a second specific MPT64 MPB64 antibody fixed to the membrane If MPT64 MPB64 antigen is present in the sample a color reaction is produced by the labeled colloidal gold particles and is visualized as a pink or purple to red line An internal positive control is included to validate proper test performance The test will detect the following species of MTB complex M tuberculosis M bovis M africanum and M microti These tests have been shown to be highly sensitive 9596 and specific 9596 in a number of studies conducted in clinical settings Procedure Materials TBc ID Test device or Capilia TB Test device Capilia extraction buffer commercially available or in house prepared TBc ID extraction buffer NaCl Tween 80 Clean cylinder and media flask or bottle Analytical balance Weigh boats Distilled water Vortex mixer Timer e 200 uL micropipette e 200 sterile aerosol resistant tips 67 Mycobacteriology Laboratory Manual 68 Mycobacteriology Laboratory Manual Sterile 2 mL cryovials 10 uL sterile disposable loops Waste receptacle with biohazard bag and tuberculocidal disinfectant Permanent marker Forms Lab specific forms for recording ID test results Appendix G Identification QC Form or use site specific form if available and equivalent Appendix B Study Source Document 13 1 Preparation of TBc ID Ext
89. ction checks must be performed DAILY every morning preferably before unloading or oading of tubes Functions Instructions External indicator lamp verification Close all drawers Press the maintenance soft key Press the test indicators soft key Press test drawer indicators soft key All three external indicator lamps on all three drawers should light as well as instrument Alarm indicator Record this function check on Maintenance Log Drawer indicator lamp verification Open drawer A Press the test green LEDs soft key he green LEDs at all the stations should light If any does not you should block e station s ess test green LEDs soft key again to extinguish the green LEDs ess test red LEDs soft key he red LEDs at all the stations should light If any does not you should block ation s Press the test red LEDs soft key again to extinguish the red LEDs Repeat steps 1 5 for Drawers B and C Record this function check on MGIT Maintenance Log gt 0 lt gt a Temperature verification Verity that the drawer temperature is currently within 1 5 C of the manual reading for Record all manual and instrument drawer temperatures on the Maintenance log Check the temperature readings on the internal thermometers located in each drawer From the main status screen press the t
90. d all personnel should exit the containment room and allow the BSC to run for at least 30 minutes before entering the room Outside the BSC but within the lab all staff should leave the room for at least 30 minutes to allow the aerosols to settle The person cleaning the spill should wear a respirator when disinfecting the area If the lab is a biosafety level 3 BSL3 laboratory all staff should leave the room and the person disinfecting the spill can enter immediately wearing a respirator without waiting for the aerosols to settle 4 3 Recommended Personal Protective Equipment Staff working in the containment lab must wear protective laboratory clothing such as a solid front or wrap around gown If scrub suits are worn protective gowns should be worn on top The scrub suits should be changed daily The protective gown must have long sleeves with snug knit cuffs Gloves must be worn and must be long enough to overlap the sleeves of the gown Hair covers caps and shoe covers are recommended All outer protective clothing must be removed when leaving the containment laboratory Respiratory protective devices are highly recommended while working in the containment lab Any respirator conforming to the National Institute for Occupational Safety and Health NIOSH N 95 rating European Committee for Standardization FFP2 rating or equivalent is acceptable Respirator protection is more than just wearing a mask Attentio
91. d beads and may be aggregated side by side and end to end to form cords especially when grown in liquid culture MGIT The AFB appear bright red against the background material counterstained blue 9 3 Reporting Smear Results If QC smear results are acceptable patient smear results can be reported Smear results of con and centrated sputum specimens and positive cultures are reported on the laboratory worksheet transferred to Appendix B 9 4 Internal Quality Control Ap Ositive and negative control smear must be included in each batch of patient tests and with new lots of reagents See Section 16 Quality Assurance for further details 9 5 Study Data Reporting Record the type of smear and the smear results of both concentrated sputum specimens and positive cultures on Appendix B Fluorescent Stain Refer to the study reporting scheme in the table in Section 9 1 above ZN Stain Smear results will be recorded as positive for acid fast bacilli or negative no AFB seen 9 6 Detecting Source of False AFB Smear Results Condition Cause Corrective Action Old used microscope slides retain material from previous smears Use only new slides AFB transferred from a positive smear to a negative smear Use a staining rack and keep slides from touching each other do not use staining jars False Food particles Request another specimen positive Precipitated stains U
92. dia The same pipette can then be used to prepare the smear for AFB microscopy Section 8 AFB Microscopy Preparation and Staining Inoculate culture media and prepare smear for AFB stain immediately after suspending the pellet using the same pipette 20 When finished dispose of the pipette into the biohazard discard bucket 21 It is recommended to store any remaining sediment at 2 8 at least until the sputum smear is confirmed as acceptable Autoclave sputum sediment before discarding 7 4 Internal Quality Control and Data Monitors 1 Record preparation information for each new batch of digestion decontamination reagents such as lot numbers expiry dates pH of buffer etc See Section 16 3 for more details 2 Include one negative control sample to control for cross contamination and one positive control sample to verify accuracy of methods weekly with a batch of specimens These samples must be included near the end of the batch and handled as patient specimens being processed for smear microscopy and culture MGIT and LJ media See Section 16 4 3 1 for more details 7 5 Study Data Reporting Record the date and time of sputum processing as well as the character quality of the sputum on Appendix B 8 ACID FAST BACILLI MICROSCOPY AFB PREPARATION AND STAINING Purpose The purpose of AFB microscopy is to detect acid fast bacilli AFB by microscopic examination of clinical specimens and cultures Both living and d
93. diately and investigate potential causes for failure c After investigation is complete and QC is acceptable repeat patient tests and report results Documentation Record results for new extraction buffer on Appendix E Record results for new kits MTB Identification QC Form Appendix If QC results are not acceptable prepare an Appendix K form to document the corrective action Procedure 1 2 Follow procedures outlined in Section 13 to test controls Perform QC tests immediately before testing the patient batch and resolve issues if applicable 87 Mycobacteriology Laboratory Manual 88 Mycobacteriology Laboratory Manual 16 3 4 Drug Susceptibility Testing Quality Control Frequency a Upon receipt of a new lot or new shipment of MGIT 960 SIRE kit PZA kit or PZA tubes b Upon receipt of a new lot new shipment or newly prepared batch of stock solutions of antibiotic powders for second line drugs c Weekly or with each batch of patient testing Controls A pan susceptible M tuberculosis H37Rv or H37Ra strain Acceptable results Susceptible results for all drugs within the defined time protocol e g SIRE and second line drugs within 4 13 days PZA within 4 21 days Corrective Actions If the control results show any resistance do not report patient results for that particular drug s a Thoroughly review cause for unacceptable results and repeat QC test if acceptable
94. dicators 3 Remove the carrier starting with the completed set closest to the front of the drawer and scan its barcode label The LEDs at this station extinguish 4 Repeat steps 2 3 to remove additional AST sets 5 Place completed AST sets in the AST tube rack Printing an Unloaded AST 1 Press the printer soft key to access report selection Set report 2 Press unloaded AST sets report soft key 3 Match the AST sets with the printed report and resolve any discrepancies NOTES Remember to load tubes into the carrier set each time in the same order i e with the same drug sequence growth control undefined drug 1 undefined drug 2 etc For further information please see instructions in the BD MGIT DST manual appendix A of the manual lists all possible AST set configurations 12 10 Interpretation of DST Results for Second Line Drugs For all set configurations except the undefined drug protocol when the growth unit GU of the GC tube reaches 2400 within the timed protocol the instrument marks the AST set complete and interprets the results For undefined drugs only GUs are recorded on the unloaded set report however susceptibility is not interpreted by the instrument The interpretation must be made manually for the undefined option according to the following criteria S Susceptible the GU of the drug tube is less than 100 R Resistant the GU of the drug tube is 100 or mo
95. e n the Comments section of Appendix B i If the ID test is now positive for MTB this culture is resulted as Positive for MTB complex and contaminated ii If the ID test is still negative perform the GenoType Mycobacterium CM or MTBDRoplus tests following the package insert instructions 1 If either test is positive for complex the culture is resulted as Positive for complex and contaminated 2 Ifthe GenoType Mycobacterium CM testis positive for a non tuberculous mycobacteria or if the MTBDRplus test is negative for MTB complex the culture is resulted as No M tuberculosis complex growth but positive for other mycobacteria b If the ZN from the BAP is negative use the results from the rapid ID test to result the MGIT culture i If rapid test is positive for MTB the culture is Positive for M tuberculosis and contaminated ii If rapid ID test is negative for MTB follow the steps in 3a above for reincubation and retesting ZN Result BAP Result TID Positive growth Any 4 Perform a MPT MTB 64 antigen test to confirm the presence of MTB If MTB is confirmed record the culture result as Positive for M tuberculosis complex If the test is negative or invalid reincubate for 48 hours and retest with the MPT MPB 64 antigen test Document the reincubation and repeat testing in the Comments section of Append
96. ead viable and non viable bacilli will stain and be counted A semi quantitative grading system is used to report the number of AFB observed in stained smears All sputum smears are prepared from decontaminated and concentrated specimens Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture These smears are stained with fluorescent stains either auramine O or auramine rhodamine The Ziehl Neelsen stain can be used to confirm fluorescent smear results but these results will not be reported The Ziehl Neelsen stain is used to confirm the presence of AFB in positive cultures MGIT LJ If an alternative fluorescent stain such as acridine orange is used microscopic examination of the sputum smear must conform to the reading and reporting procedures described in Section 9 Acid fast Bacilli Microscopy AFB Examination Principle For the fluorochrome stain the principle of stain decolorizer and counterstain is the same as for Ziehl Neelsen staining With auramine O stain organisms fluoresce bright yellow non specific debris stains pale yellow and the background is almost black With auramine rhodaminestain organisms fluoresce yellow red in an almost black background Fluorochrome stain is more sensitive than Ziehl Neelsen because the smear can be examined under a lower power thus more fields can be read in the same amount of time and the bacilli stand out brightly The Ziehl Neelsen method uses a carbol fuchsin stain
97. econstitute PZA drug vial with 2 5 ml of sterile distilled deionized water PZA Kit to make a stock solution of 8000 pg ml Store lyophilized drugs at 2 8 C upon receipt and reconstitute prior to use Once reconstituted aliquot any leftover drug solutions and freeze at 70 to 80 C 10 up to 6 months or up to he date of original expiry whichever comes first Once thawed discard any leftover drug and do not store or refreeze 12 2 2 Second Line Drug Stock Preparation Source of drugs All second line drugs will be obtained in chemically pure form from Sigma or the appropriate pharmaceutical company Acceptable drug powders are labeled with the generic name its assay potency usually expressed in micrograms ug of drug per mg of powder and its expiration date The powders are stored as recommended by the manufacturer When removed from the freezer it must come to room temperature before it is opened to avoid condensation of water Check the Certificate of Analysis COA provided by Sigma for each drug lot number If the COA is not included with the antibiotic shipment it can be downloaded by lot number from the Sigma web site Contact the supplier or manufacturer if any value is missing or cannot be clearly determined from the COA If potency information is not included on the COA it may be determined from the formula below Weighing drugs All antimicrobial agents are assayed for standard units of acti
98. ed for the first line drug and replace it with the appropriate concentration for the actual drug tested If undefined protocol is used simply write in the concentration tested for each drug Initial and date the corrected MGIT report after all drug names and concentrations have been updated Entering and removing AST carrier sets is the same as described above in Section 12 7 with one extra step of changing the AST set definitions if applicable Open desired drawer in MGIT instrument and follow instructions below 61 Mycobacteriology Laboratory Manual 62 Mycobacteriology Laboratory Manual Function Directions Entering new 2nd line 1 Follow steps 1 4 in Entering New AST sets above sets 2 Press the lt change AST set definition gt soft key 3 Use the UP ARROW or DOWN ARROW key to scroll through the available definitions for the scanned carrier size As you scroll the default set is indicated by a check mark in the main body of the display 4 Press the lt okay gt soft key to select the highlighted set The number of undefined drugs must correlate with the number of drugs being tested 5 Follow steps 5 7 in Entering New AST sets above Removing completed AST 1 Open the desired drawer Press the remove completed AST sets gt soft sets indicated a red on the drawer 2 The first completed AST set stations illuminate with FLASHING GREEN in
99. eekly for 8 intervals Cultures can be read on the bench as long as the caps are NOT loosened To observe fine growth a strong direct light from the angle poise lamp must be shone onto the slant surface M tuberculosis usually grows as a buff colored dry colony which is very distinctive 11 2 Working up Growth on Primary Solid Culture 1 To check for AFB and purity select 2 to 3 colonies of representative growth using a sterile loop or applicator stick make a smear and perform a ZN stain Section 8 Acid fast Bacilli Microscopy AFB Preparation and Staining and Section 9 Acid fast Bacilli Microscopy AFB Examination NOTE Contamination of may be obvious and a ZN smear may either not be necessary or not practical e g when the entire slant dissolves in these situations mark N A on Appendix B If contamination is only present on a small surface area of the slant continue to incubate to allow MTB to grow If a ZN smear is performed the results should be recorded on the lab worksheet and Appendix B Note the colony morphology and pigmentation on solid medium and describe these features in the laboratory worksheet workbook Also assess time for growth to appear on medium Typical growth characteristics along with Ziehl Neelsen staining properties are suggestive of M tuberculosis complex 3 At all visits growth that is AFB positive requires confirmation as M tuberculosis complex using the MPT MTB 64 antigen tes
100. ejnuru pupys Je PUD 0 jo sjom 10uejur S 499 J ouoopp YOZ 446 An puiejbui 10 9 10 papasu SD 10 IDeA aduUO suoioejuisip eipudoiddp puo jouelur bugos jueqiosqp 568 eui ui peiors jueudinbe ad psg woy 4551 uope enpisei jubj2ejurstp ouoojp YOZ YIM Pappu j eor jueqiosqp uli Wd WY Alp e puns 1 YIM AjuBnoioui se pyins yom Jeu o jueuidinb3 5 1ueuudinb3 91 92 Mycobacteriology Laboratory Manual 16 4 2 2 Equipment Maintenance Routine in house maintenance must be carried out at the time intervals recommended by the manufacturer of the equipment and a schedule developed for performing the applicable maintenance Table 16 5 Maintenance may be performed by in house technical personnel and or a vendor service contract Maintain documentation of all routine maintenance in a lab worksheet or logbook A suggested maintenance schedule performed in house or by outside technical personnel as applicable is shown below Semi annual or annual mainten
101. emperature soft key to access drawer emperature readings each of the drawers The instrument air fil 1 Remove the face er maintenance must be performed at least monthly To clean the filter plate by grasping it along the bottom edge at the finger holes Gently but irmly pull straight out 2 Remove the filter and wash in an antimicrobial disinfectant 3 Dry thoroughly with paper towels and replace in the instrument 4 The cut out in the faceplate should surround the on off switch Firmly press in towards the instrument The faceplate will snap into place 5 Record this maintenance on the MGIT Maintenance Log on the day it was performed 10 1 Inoculation of MGIT Tubes Prior to use examine all MGIT tubes for evidence of contamination or damage as these are unsuitable for use Inoculation of MGIT tubes must be done inside the biosafety cabinet with full PPE 1 While the specimens are being processed Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture prepare the antibiotic supplement PANTA for the MGIT tubes Reconstitute MGIT PANTA with 15 ml of MGIT Growth Supplement This mixture is stable for 5 days if stored at 2 8 C If the remaining mixture will be stored label it with the contents the date of preparation date of expiry and preparer initials 2 Label the side of each MGIT tube using the study label that contains identifying information described in Secti
102. ent continuously reads all the tubes A row of LEDs below the tubes illuminates activating their fluorescent sensors Photo detectors take the readings A test cycle of all drawers is completed every 60 minutes Positive cultures are immediately flagged by an indicator light on the front of the drawer and an optional audible alarm and are displayed on the LCD screen When positive tubes are identified they should be removed from the instrument for confirmation of results and for isolation and detection of the organism Definition of a Growth Unit GU The Growth Unit is an algorithmic measure of sensor fluorescence derived from the raw fluorescencevoltagesignalproducedbyopticalintegrationofa MGITtubeinthe960instrument The MGIT 960 takes a reading every hour on the hour An Instrument Positive tube is signaled automatically via internal algorithms when the GU reaches or exceeds the cut off value of 75 units This value is identified as a True Positive and is confirmed by further tests such as ZN staining and contamination checks see Sampling Tubes for Further Analysis section below If the MGIT signals the tube as positive and records a GU of 0 or higher within 5 hours of incubation growth has occurred very rapidly and exploded past the 75 unit cut off Represented graphically this growth curve would be very steep compared to the gradual curve generated by the True Positive If explosive growth has occurred the software rec
103. ent version of the document Editor Kelly W Stinson Clinical Manager Microbiology Otsuka Pharmaceutical Development amp Commercialization Inc Kathleen Eisenach University of Arkansas for Medical Sciences Susan Kayes OPDC Makoto Matsumoto Otsuka Pharmaceutical Co Ltd Japan OPCJ Salman Siddiqi Consulting Mycobacteriologist Suguru Nakashima Hiroyuki Hashizume OPCJ Juliano Timm OPDC Anne Morrissey OPDC Marra Mendoza Otsuka Manila Research Center OMRC Princy Mathai OPDC Acknowledgement The Otsuka global microbiology program could not have been implemented without the hard work dedication and input of the laboratory directors managers and staff from each trial laboratory and the in country microbiology consultants Lab Directors Managers Jun Yue Shanghai Pulmonary Hospital Clinical Laboratory Shanghai China Zhao Yanlin Tuberculosis Reference Laboratory of China Beijing China Mona Abdelmonis Khalifa El Seka El Baidaa Abbassia Chest Hospital Cairo Egypt Tiina Kummik Tartu University Clinics United Laboratories Dep of Mycobacteriology Tartu Estonia Klavdia Levina North Estonian Medical Centre Diagnostica clinics Laboratory Mycobacteriology Section Tallinn Estonia Akio Aono Japan Anti Tuberculosis Association Fukujuji Hospital Tokyo Japan Motohisa Tomita Kinki chuo Chest Medical Center National Hospital Organization Osaka Japan Sungweon Ryoo Molecular mycobact
104. entification Results Record results including all repeat tests as positive for MTB complex negative for MTB complex or invalid test on the laboratory worksheet 13 6 Internal Quality Control A positive and a negative control must be tested with each new lot or new shipment of kits received and with each new batch of extraction buffer prepared Similarly these controls must be run weekly or along with each batch of patient isolates when tests are set up less frequently Refer to Section 16 Quality Assurance for details 13 7 Study Data Reporting 1 If identification is not performed because culture is negative mark N A in the appropriate section on Appendix B 2 If the test confirms the presence of complex Record the Identification of AFB result as Positive for MTB complex in the MGIT or LJ culture section on Appendix B Record the test method Capilia or TBc ID and any comments in the MGIT or LJ culture section on Appendix B 3 If the rapid ID test is negative for MTB complex Proceed with instructions for molecular testing in Flowchart 2A If MTB is detected Record the Identification of AFB result as Positive for MTB complex in the MGIT or LJ culture section on Appendix B Specify the ID test used to confirm MTB HAIN GenoType Mycobacterium CM or GenoType MTBDRplus If MTB is not detected record the Identification of AFB result as Negative for MTB complex in the MGIT or LJ cul
105. eriology Unit Korean Institute of Tuberculosis Seoul Korea Chang Ki Kim Molecular mycobacteriology Unit Korean Institute of Tuberculosis Seoul Korea Nackmoon Sung Clinical Research Center CRC National Masan TB Hospital Masan Korea Girts Skenders State Agency Infectology Center of Latvia Clinic of Tuberculosis and Lung Diseases Riga Latvia Jurgita Kazlauskaite Public Institution Republican Siauliai Hospital Subsidiary Hospital of Tuberculosis and Lung Diseases Siauliai Lithuania Sandra Svambarytes Public Institution Republican Siauliai Hospital Subsidiary Hospital of Tuberculosis and Lung Diseases Siauliai Lithuania Front page photo credit Edwin Cortero Tuyay Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual Edita Pimkina Infectious Diseases and Tuberculosis Hospital Affiliate of Public institution Vilnius University Hospital Vilnius Lithuania Elena Romancenco Public Medical Sanitary Institution PMSI Institute of Phtysiopneumology Chiril Draganiuc Blufstein Karina Vidarte Blufstein Labo Laboratorio Clinico Chisinau Moldova ratorio Clinico Lima Peru Grace Egos Tropical Disease Foundation Inc Manila Philippines in memoriam Thelma Tupasi Tropical Disease Foundation Inc Manila Philippines Henry Evasco Tropical Disease Foundation Inc Manila Philippines Mara Gibson Clinical Laboratory Services Johannesburg South Africa Saloshini Natalie Ramsamy C
106. es 1 Thaw individual cryovials to room temperature 2 Dilute as appropriate in sterile distilled water to achieve the correct working concentration and use without delay Test concentrations for each drug and the volumes added to MGIT tubes are listed in the table below 3 Discard excess drug and never re freeze 55 Mycobacteriology Laboratory Manual 56 Mycobacteriology Laboratory Manual Table 12 1 Drug Concentrations for DST in MGIT DS Concentration of drug in Volume added to MGIT ELLA working solution pg ml tubes for test pl STR 83 00 1 0 MGIT INH 8 3 00 0 1 MGIT RIF 83 00 1 0 MGIT EMB 415 00 5 0 PZA 8000 00 100 Amikacin 83 00 1 0 Capreomycin 208 00 2 5 Gatifloxacin Kanamycin Levofloxacin 166 00 2 0 Moxifloxacin 20 75 00 0 25 Ofloxacin 166 00 2 0 Enviomycin 1 World Health Organization Policy Guidance on Drug Susceptibility Testing DST of second line anti tuberculosis drugs Geneva WHO 2008 WHO HTM TB 2008 392 2 WHO has not recommended a critical concentration for the testing this drug must submit a written rationale for the cri testing performed testing of this drug in the MGI 960 instrument Laboratories ical concentration used for testing as well as any validation 12 3 Preparation of Tubes for Susceptibility Testing 12 3 1 Preparation of SIRE 1 Label five
107. et Appendix E Reagent Media QC Form e Appendix J Storage Log for M tuberculosis Isolates on Media 11 1 Inoculation and Incubation of Solid Cultures Inoculation of slants must be done inside the biosafety cabinet using full PPE 1 Label LJ tube using the study specific labels that contain identifying information as described in Section 6 2 Login of Sputum Specimens Remove any excess water in the slant using a sterile transfer pipette Inoculate the tube with 200 ul of the sample either well mixed processed sputum Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture or positive MGIT tube Section 10 Liquid Culture Mycobacteria Growth Indicator Tube MGIT using a sterile graduated disposable pipette Spread inoculum evenly over entire surface of medium Take care to minimize aerosol generation when sampling from positive MGIT tubes as this broth will contain large numbers of M tuberculosis Replace cap and ensure there are no droplets around the rim of the tube Wipe off the outside of the tube with a paper towel soaked in tuberculocidal disinfectant Leave tube in slanted position with cap loosened until inoculum is absorbed about a week then tighten cap securely and incubate in upright position at 37 C 1 C Alternatively the tube can be incubated immediately in an upright position with cap loose for the first week of incubation Examine and record results for the cultures w
108. f expiration 6 months from preparation date 5 Store standards in the dark at 20 25 C 6 Discard after 6 months or sooner if any volume is lost 7 Before each use vortex standard vigorously mixing the fine white precipitate of barium sulfate in the tube to achieve a uniform turbid appearance Replace standard if large particles appear deally the accuracy of the density of a prepared McFarland standard should be checked by using a densitometer or spectrophotometer with a 1 cm light path assuring parameters correspond to he guidelines in the table below Table 16 2 Properties of McFarland Standards McFarland Standard No 0 5 1 0 Barium chloride 0 05 ml 0 1 ml 1 0 Sulfuric acid 9 95 ml 9 9 ml Transmittance 74 3 55 6 Absorbance 0 132 0 257 at wavelength of 600 nm Adjusting Turbidity for Inocula 1 Use tubes of the same size diameter for preparing isolate suspensions and dilutions as the size of the McFarland standard tube 2 If available use a spectrophotometer or similar instrument to adjust the turbidities 3 Use a Wickerham card as a visual guide for adjusting the turbidities when instrumentation is unavailable 16 2 1 2 Laboratory Prepared Media Lab prepared media must be thoroughly quality controlled It is recommended to make small batches of media e g every four weeks or more frequently to ensure freshness and quality 1 396 of each new batch of medium must
109. f fields appropriate for the degree of positivity e g the higher the smear positivity the fewer fields counted Average the count for the number of fields and record on the lab worksheet the appropriate result according to the table Table 9 1 Grading Scale for Fluorescent Stain What you see 200x What you see 400x What to report No AFB in one length No AFB in one length No AFB observed 1 4 AFB in one length 1 2 AFB in one length Confirmation required 5 49 AFB in one length 3 24 AFB in one length Scanty 3 24 AFB in one field 1 6 AFB in one field 1 25 250 AFB in field 7 60 AFB in one field 2 gt 250 AFB in one field gt 60 AFB in one field 3 The number of AF8 indicates how infectious the patient is It is important to record exactly what you see onfirmation required by another technician or prepare another smear stain and read 9 2 Examination of Ziehl Neelsen Stain 1 Using a bright field microscope Ziehl Neelsen smears are examined with the 100X oil objective 10X eye piece for a total of 1000X magnification Take care not to touch the slide with the tip of the dropper when dispensing oil Always wipe oil from the oil immersion lens after each AFB positive smear is read AFB will have similar morphology as fluorescence stained bacilli They are variable in shape from very short rods to long filaments Often they are bent contain heavily staine
110. floxacin moxifloxacin ofloxacin and injectable drugs amikacin capreomycin kanamycin used in local standard of care will be tested in the MGIT 960 system unless the MGIT method is unreliable for a specific second line drug in MGIT DST of these second line drugs is well established and results are reliable and reproducible Therefore they are the only DST results required for testing positive cultures from patients If a fluoroquinolone or injectable cannot be tested in MGIT it is acceptable to perform DST using the proportion method on solid medium Each lab performing solid media testing must submit their approved SOP for this procedure and demonstrate satisfactory performance in routine quality control testing of reference strains While the study protocol does not require testing reporting of other second third line drugs the investigator and or lab manager may request DST of these drugs as necessary to appropriately manage the study patients These results will be reported on a separate lab specific form not on Appendix B The MGIT system is recommended for testing second line drugs because 1 the system yields results faster than the growth based methods using a solid medium 2 testing must be carried out according to the recommended published procedures in order for the system to be operational providing some degree of standardization and 3 the instrument automatically detects presence of growth and completion of test Fur
111. g safety cleaning specimen processing culture media etc using supervisor observation whenever necessary Documentation a Record data on the Monthly Data Monitors Forms Appendix I or other lab specific comparable document b If any corrective measures are necessary prepare an Appendix K form to document the activities Time to Detection 15 Procedure a Calculate the time to detection for MGIT cultures from smear positive and smear negative specimens that are determined to be MTB b Repeat step a for LJ cultures c Record results for both media and compare results with data from the previous month and lab s average d Calculate the cumulative average TTDs for each medium type Results If the monthly average TTD is 2096 of the lab s established average without an increase in contamination rate or isolation of rapidly growing mycobacteria this likely indicates that processing has improved If the monthly average TTD gt 20 of the lab s established range processing of specimens may be too harsh or centrifuge speed time may be less than optimal Corrective Actions a If there is a significant increase in TTD unrelated to visit interval review processing procedures and discuss with personnel using supervisor observation if necessary b Although an improvement in processing is generally considered beneficial significant decreases in TTD may also prompt review of applicable procedures
112. g positive dilute 1 ml of MGIT broth in 4 ml of sterile saline 1 5 dilution 57 Mycobacteriology Laboratory Manual 58 Mycobacteriology Laboratory Manual 5 NOTE If a laboratory routinely experiences x200 errors when performing DST in the local patient population the 3 5 day culture can be used undiluted as a first step as described in Flow Chart 4 Monitor this technique very closely to ensure that an excess of x400 errors is not subsequently produced If the culture has been positive longer than five days subculture into a fresh MGIT tube Vortex MGIT broth well to mix thoroughly Leave 5 10 minutes to allow any large clumps to settle Supplement a new MGIT tube with 0 8 ml Growth Supplement without PANTA Remove inoculum from the supernatant broth and make a 1 100 dilution of the positive MGIT tube into sterile saline or 7H9 broth Mix tube well by inverting gently several times Inoculate new MGIT tube with 0 5 ml of the 1 100 diluted specimen Cap tube tightly and mix well by inverting gently several times Enter tube into MGIT 960 instrument and monitor until it becomes positive Use new tube for DST tests from one to five days of positivity as described above 12 4 2 Using an Inoculum from Positive LJ Culture Carry out all work with positive LJ slants in biosafety cabinet using full PPE 1 DST can be performed with growth from positive LJ slants if the MGIT tube is contaminated
113. gative controls for MGIT and LJ cultures Internal quality assessment to improve microscopy results Positive and negative controls for new staining reagents for AFB smears Positive and negative controls for AFB smears Reference strain for drug susceptibility testing MGIT Time to detection QC for MTB reference strain Sterility and performance testing of culture media U 7H9 Broth BAP MGIT Maintenance See Section 10 Liquid Culture MGIT Positive negative and reagent controls for identification kits Complete Monthly Data Monitors form Reference strain testing for new lots of MGIT SIRE PZA kits and other anti TB drugs or drug kits MGIT QC Report See Section 10 liquid Culture MGIT Positive negative and reagent controls for new lots of identification kits Mycobacteriology Laboratory Manual 78 Mycobacteriology Laboratory Manual 16 2 Quality Control Activities 16 2 1 Media QC 16 2 1 1 McFarland Turbidity Standards Many QC procedures involve preparing dilutions from a standardized suspension of organisms using a turbidity standard so that the number of bacteria present will be within a given range According to CLSI guideline M24 A each McFarland turbidity standard is equivalent to the turbidity of a bacterial concentration For example a McFarland No 0 5 turbidity standard is equal to the turbidity of approximately 1 5 x 108
114. global laboratory initiative advancing TB diagnosis Mycobacteriology Laboratory Manual First Edition April 2014 A publication of the Global Laboratory Initiative a Working Group of the Stop TB Partnership Stop Partnership This document was developed to ensure high quality results and comparability of data from a network of international tuberculosis laboratories handling sputum specimens for Otsuka Pharmaceutical Development and Commercialization OPDC sponsored multidrug resistant tuberculosis MDR TB clinical trials for the development of Deltyba delamanid It consists of standardized procedures related to sputum collection handling analyses and reporting with a focus on testing that has the greatest impact on microbiology endpoints for MDR TB clinical trials sputum culture conversion in liquid and solid media A specialized Otsuka team including a microbiology expert with extensive experience in mycobacteriology laboratory based research in international settings a senior clinical research associate CRA with prior experience in international TB research and a senior laboratory technologist with clinical experience working in several international TB laboratories drafted the first version of this manual Input from multiple lab directors managers and consultants in the Otsuka trials network as well as review of data generated from the clinical trials themselves prompted several revisions incorporated in this curr
115. icable manufacturer s recommendations as well as any appropriate quality control and assurance activities described in Section 16 Quality Assurance All DST reporting guidelines outlined below are applicable to DST on solid media 12 12 Reporting of DST Results for All Drugs Record Susceptible Resistant or Test Failed on the internal lab worksheet book and the lab requisition form if applicable Also keep the MGIT printouts for all DST results with the patient records 12 13 Internal Quality Control It is extremely important to perform quality control on the drug sensitivity testing procedure This testing must be performed For each new batch of reagents MGIT drug kits other drugs media etc Weekly in a DST run when patient tests are run weekly With each batch of patient isolates when DST is performed less frequently Using a pan susceptible strain such as H37Rv which is sensitive to all of the test drugs Record these results on Appendix F DST QC Form See Section 16 Quality Assurance NOTE If the laboratory chooses to include other strains such as clinical isolates with resistance to one or more of the second line drugs for quality control of drug susceptibility testing either upon receipt of new drug lots or at regular intervals these results should be recorded on Appendix F DST QC form 12 14 Study Data Reporting 1 If a specimen was not positive for or the visit interval d
116. id not require DST testing mark Not Applicable or Not Required by Protocol on Appendix B 2 Report susceptibility testing results on Appendix B for the specimen tested as follows Mark each drug result as Susceptible S Resistant R Test Failed TF or Not Tested ND Record the testing method date result was obtained and the critical concentration of each drug tested f any discrepant results are seen or retesting is performed note this occurrence in the DST Comments section 3 If the other specimen 1 or 2 for the same visit was also positive for MTB mark the non tested specimen as Not Applicable or Not Required by Protocol on the associated Appendix B 4 For INH RIF any fluoroquinolone or any injectable drug only if a subsequent test result is inconsistent with previous results for a patient especially if the drug profile changes from 63 Mycobacteriology Laboratory Manual 64 Mycobacteriology Laboratory Manual R to S review data and repeat the test If the repeat result is discordant with the 1st test result for the current visit repeat the test a 3rd time and record this as the final result on Appendix B If a specimen was positive for MTB complex at a time point requiring DST but the isolate was unavailable for testing e g culture contaminated and could not be purified report susceptibility results for the individual drugs as ND and add an explanation in the DST
117. imen Number Date of Collection Date Stored Tech Initials Date Discarded Tech Initials Supervisor Review Date Initials Sputum specimen number should be 1 or 2 or N A if from visits 2 or 3 Date format dd mmm yyyy 143 Mycobacteriology Laboratory Manual 144 Mycobacteriology Laboratory Manual APPENDIX K CONTINUOUS QUALITY IMPROVEMENT FORM Ql Number iocis Date Form Prepared Type of Issue O Clerical error Equipment problem O Procedural error O QC failure Supply problem Ii Other list ete SECTION I SUMMARY OF ISSUE Date of occurrence Describe the problem issue preventable error etc Has this same issue occurred previously 0 Yes No Unknown If Yes include QI form number SECTION Il INVESTIGATION Describe the root cause of the problem SECTION IIl CORRECTIVE ACTION FOR THIS INCIDENT Describe corrective action taken to resolve this issue Expected for resolution Did this incident impact any patient results O Yes O No N A If Yes describe Was retesting necessary C Yes No N A If Yes of re test Results acceptable O Yes Comments Attach docume
118. in which the sputum will be collected If able instruct the patient to stand Give the patient a glass of water bottled or boiled to rinse the mouth free of food particles Instruct the patient to rinse twice Instruct the patient to produce sputum after rinsing mouth as described above Use a demonstrator glass and tube container to show the patient the procedure if this will help Take a deep breath Hold breath for a moment Cough deeply and vigorously at the same time the breath is coming out Release sputum into the labeled tube container by holding it to the lower lip and gently releasing the specimen Close the tube container tightly with the screw on lid without touching the inside of the lid Avoid spills or soiling the outside of the container f the patient cannot cough spontaneously instruct the patient to take several deep breaths and hold the breath momentarily Repeating this several times may induce coughing f a patient is unable to spontaneously expectorate a sputum sample an attempt will be made induce sputum production by aerosol inhalation Contact the study physician to conduct his procedure Sputum induction will be performed according to the standard procedure at the site The Laboratory Specimen Requisition Form and the Appendix A Specimen Transfer Form must also accompany the appropriately labeled induced sputum to the lab Clearly mark that the sputum has been induced under the section labeled
119. inal stock concentration with sterile distilled water For example if the recommended solvent for Ofloxacin is 0 1 N NaOH 1 Add weighed drug to a sterile tube e g 50 ml Falcon tube 2 Prepare 0 1 N 0 1 M NaOH For example dissolve 4 g NaOH in 1 liter sterile distilled water 3 Add 0 1 NaOH solution drop wise shaking gently after each addition Add NaOH just until drug dissolves completely and the solution is clear 4 Finish diluting drug up to the appropriate volume calculated below with sterile distilled water Formula for volume of solvent Actual weight x Potency Desired concentration Volume Example To prepare a stock solution containing 10 000 ug ml of kanamycin with powder that has a potency of 750 ug mg 170 to 200 mg of powder should be accurately weighed If the actual weight is 180 0 mg the volume of solvent needed is as follows Volume 180 0 mg x 750 ug mg 213 5 ml 10 000 ug ml All stock solutions must be made at least 1 000 ug ml or ten fold higher than the drug s working concentration see table below and sterilized by membrane filtration 0 22 um pore size unless otherwise recommended by the manufacturer Dispense small volumes of sterile stock solutions into sterile cryovials carefully seal and store for up to 12 months at 70 to 80 C 10 C or up to the date of the original drug powder expiry whichever comes first Preparing working solutions Before inoculating MGIT tub
120. incubated this date represents the date when the final culture result was obtained and final TTD calculated from Appendix M 2 Final date of MGIT culture completion on Appendix B should be the date all testing in the GIT section is complete i e ZN BAP and ID 3 If ZN and ID are not performed because culture is negative mark N A for each of hese tests on Appendix B 4 If the MGIT culture is determined to be Positive for M tuberculosis complex and the original ZN was negative culture was re incubated and subsequent ZN was positive for acid ast bacilli two TTDs will be reported 1 the original TTD from the MGIT printout and 2 the revised final TTD as reported on Appendix M 5 Positive for M tuberculosis complex and contaminated culture is re decontaminated perform ID DST etc record the MGIT culture result the TTD and the date of result rom the original sputum culture on the lab worksheet and Appendix B It should also be noted on Appendix B that the MGIT tube was decontaminated and re cultured If unable to successfully recover MTB from a Positive for M tuberculosis complex and contaminated culture document this fact in the MGIT Comments field on Appendix B 6 Ifthe MGIT culture is determined to be Contaminated record both the original TTD from the MGIT printout and the final TTD as recorded on Appendix M if reincubation was performed 7 the MGIT culture is determi
121. ined in the certification process Clinical centrifuges must be equipped with biosafety canisters buckets with aerosol containment lids Microcentrifuges should be operated inside BSC using tubes with caps having O rings Recommended Biosafety Practices Take caution when performing aerosol generating procedures such as centrifugation vortexing mixing pipetting pouring and inoculation of media For example Delay opening caps until aerosols have settled Open centrifuge canisters only inside the BSC Use pipettes that are easy to control Eject micropipette tips down inside discard bucket Heat fix slides on a warmer in the BSC heating them at 65 75 C for at least two hours before handling outside the BSC Heat fixed smears may contain viable tubercle bacilli but they are not easily aerosolized if dried on a slide warmer for two hours Inside the BSC keep arms parallel to the work surface work in the center and minimize arm movements once beginning work do not move hands out of the hood until work is completed These precautions will minimize interruption of airflow inside the BSC Keep the amount of equipment inside the BSC to a minimum so as to not interfere with the airflow pattern Disinfect the BSC and all work surfaces with a tuberculocidal disinfectant capable of killing MTB before and after every procedure The most common tuberculocidal disinfectants are bleach hypochlorite a
122. ing block may also be used Apply only enough additional heat to keep the slide steaming for 5 minutes Do not let the stain boil or dry Add additional stain if necessary 3 Wash off the stain with distilled water 4 Flood slides with 396 acid alcohol 5 Let stand for 2 3 min more acid alcohol should be used if the smear is heavily stained 6 Wash off the acid alcohol with distilled water and tilt the slides to drain 7 Flood the slides with methylene blue and let stand for 1 2 minute 8 Wash off the methylene blue with distilled water 9 Tilt the slides to drain 10 Allow slides to air dry in the slide rack DO NOT BLOT NOTES Heat fixing slide warmer or flame does not always kill mycobacteria exercise care when handling slides Slides must not touch each other when placed on staining rack to prevent transfer of material from one slide to another Fluorochrome stained smears lose fluorescence with time and exposure to light Keep smears covered and examine within 24 hours of staining 8 5 Internal Quality Control 1 Record lot numbers expiry dates etc for the staining reagents on the Daily AFB Staining QC Form Appendix C 2 Positive and negative control slides must be included with every batch of patient slides and when new lots of stains are received Record these results on the Reagent Media QC Form Appendix E See Section 16 3 1 Acid Fast Stains for more details 8 6 Storage of Slides 1
123. itials date Method OTBcID O Capila 777 dd mmm yyyy Final date of U culture completion Tech Initial dd mmm yyyy Comments Drug susceptibility testing S Sensitive R Resistant TF Test Failed ND Not Tested Not Applicable or Not Required per Protocol SO eel aia ll c ERES es Isoniazid MGI 0 1 oO Rifampicin MG 1 0 0 Pyrazinamide MG 100 oO Ethambutol MG 5 0 0 Streptomycin MG 1 0 00010 Amikacin MG 1 0 MG 2 5 0010 Ofloxacin MG 2 0 oO Moxifloxacin MG 0 25 Levofloxacin MG 2 0 o Gatifloxacin oO Enviomycin Sulfate 0 Ist line DST Tech 2nd line DST Tech Initials Date Initials Date dd mmm yyyy dd mmm yyyy Comments Shipping Was were isolate s shipped Yes No O 0 10 20 date of shipment Only tick N A for V2 or dd mmm yyyy For V2 and V3 shipping only Aliquot 1 Aliquot 2 Date of shipment Date of shipment dd mmm yyyy dd mmm yyyy Comments Supervisor signature Date nae My signature confirms that all data have been reviewed for accuracy and that all pre filled information on this form is accurate for this specimen
124. ive GC pass P 4 Reagent Date of P Tech Initial Yes N Batch Initials Negative Yes No Stain Control Control strain Strain Monthly tested tested Supervisor positive negative Review control control date initials New Lot Batch Staining Reagents Brand Received Expiry Quantity Date Component Manufacturer Lot Number Date Date Received Opened Fuchsin Phenol Ethanol HCL concentrated Methylene blue Date format dd mmm yyyy Mycobacteriology Laboratory Manual 121 Kuo0j amp ioqe 1 2 AKKAju1uuu pp 0594 sjputut eyop Maroy eAuisod Josisadng Aqujuow gemes ities rie JequinN JequinN ee JequinN Toss estem nuoro evum m Fpa ssod DO eayisog T en g joyooyy 09105 122 Reagent poe Quantity pH for Put into Tech 9 Reagent E
125. ive for other mycobacteria original TTD from MGIT printout If test remains invalid decontaminate MGIT repeat rapid ID test and notify Sponsor representative Suspected MOTT from Flow Chart 2 ZN Positive Growth Rapid ID test Negative or invalid Action Reincubate for 48 hours and retest with Rapid ID test If MTB positive from rapid ID test Report ZN positive BAP contaminated Positive for MTB and Contaminated original TTD from MGIT printout and final TTD from Appendix M if applicable If MTB negative or invalid from rapid ID test Action Perform HAIN Genolype Mycobacterium CM or HAIN GenoType MTBDRplus If MTB positive on Mycobacterium CM or GenoType MTBDRplus test Report ZN positive BAP contaminated Positive for MTB and contaminated original TTD from MGIT printout and final TTD from Appendix M if applicable gt If positive for MOTT on Mycobacterium CM test or MTB negative on GenoType MTBDRplus Report ZN positive BAP contaminated No TB growth but positive for other mycobacteria original TTD from MGIT printout and final TTD from Appendix M if applicable If test remains invalid decontaminate MGIT repeat rapid ID test and notify Sponsor representative 45 Mycobacteriology Laboratory Manual 46 Mycobacteriology Laboratory Manual Flow Chart 3 MGIT Early Positive Culture
126. ix B i If the ID test is now positive for this culture is resulted as Positive for MTB complex ii If the ID test is still negative perform the GenoType Mycobacterium CM or GenoType MTBDRplus tests following the package insert instructions 1 If either test is positive for MTB complex the culture is resulted as Positive for complex 2 Ifthe GenoType Mycobacterium CM testis positive for a non tuberculous mycobacteria or if the MTBDRplus test is negative for MTB complex the culture is resulted as No M tuberculosis complex growth but positive for other mycobacteria BAP Result TID growth Any ZN Result Negative 5 Although rare this situation does occur and requires further investigation Re incubate the MGIT tube for at least another 3 days start counting from the date of the original positive signal or until the MGIT signals positive again Repeat the ZN smear and BAP taking note of the original TTD and date of positive MGIT result Record the results from the initial subsequent testing including original date of positivity and TTD results from each ZN BAP test whether the culture was reincubated location of incubation total number of days reincubated and TTD from MGIT printout if applicable date of MGIT result at 42 days if applicable and final results of this culture on Appendix M Early MGIT Positive Early Contaminated Cultures Worksheet a
127. ix E New Reagent Media QC Appendix J Freezer Storage Log for Isolates 14 1 Preparation of Media Note The following instructions are for preparing media using Difco dehydrated base Follow specific instructions if another manufacturer s base medium is used and adjust quantities if a smaller batch is needed 1 Suspend 4 7 g 7H9 Broth Base in 900 ml distilled water Mix thoroughly 2 Add 2 ml of glycerol rinse pipette a few times with broth to dislodge all material 3 Heat gently if necessary to dissolve the medium completely 4 Autoclave at 121 C for 10 minutes Cool to 45 in a water bath 209 Oy wm Aseptically add 100 ml of ADC or OADC enrichment Mix gently but thoroughly Label container with media name date prepared expiry date batch lot number and initials Test media for sterility and performance See Section16 2 1 2 2 Store at 2 8 C up to 3 months protect from any direct light Discard any prepared media that shows signs of contamination discoloration or evaporation 14 2 Freezing MTB Isolates Freezing of MTB Isolates must be performed in a BSC using full PPE 1 Label cryotube with study label containing screening and or subject ID visit number aliquot or sputum specimen date cryotube is prepared and lab accession number See example below 2 Dispense 1 0 1 2 ml of 7H9 broth into cryotube using a sterile transfer pipette 3 Check the label on the LJ tube and the c
128. l strain Strain Monthly tested tested Supervisor positive negative Review control control date initials New Lot Batch Staining Reagents Brand Received Expiry Quantity Date Component Manufacturer Lot Number Date Date Received Opened Acridine Orange Sodium carbonate Sulfuric acid Benzalkonium chloride Sodium barbital Tricine Ethanol HCL concentrated Date format dd mmm yyyy Confirmation required by another technician or prepare another smear stain and read Mycobacteriology Laboratory Manual 116 Mycobacteriology Laboratory Manual Lot Batch Results Neg Confirmation Number of Quantity Date Tested Required Scanty 1 2 3 QC pass Prepared d Batch Prepared Tech Initials pM Negative Yes No Stain Control Control strain Strain Monthly tested tested Supervisor positive negative Review control control date initials New Lot Batch Staining Reagents Brand Received Expiry Quantity Date Manvfacturer Lot Number Date Date Received Opened Auramine Phenol Ethanol HCL concentrated Potassium permanganate Date format dd mmm yyyy Confirmation required by another technician or pre are another smear stain and read 117
129. learly labeled with a study label that includes identifying information as described in Section 6 2 Login of Sputum Specimens 3 Login these data for each specimen on the Storage Log for M tuberculosis Isolates on Media Appendix J 4 Use these LJ subcultures to prepare the frozen isolates for long term storage i e for 6 months after the conclusion of the study Refer to Section 14 Long Term Storage of MTB Isolates 5 After one year of storage LJ subcultures can be designated for disposal provided that all necessary testing has been completed and all frozen stock aliquots have been appropriately stored NOTE Approval to discard these LJ subcultures must be given by the Sponsor prior to disposal 11 6 Quality Control of Media Records of batch numbers dates of preparation expiration dates and QC results of all media should be documented on the Reagent Media QC Form Appendix E when media is put into use Refer to Section 16 Quality Assurance for details on sterility and performance testing for both laboratory prepared and commercially prepared media 11 7 Study Data Reporting Use the reporting scheme in Section 11 4 above to record results of LJ culture growth Record date of inoculation date of result ZN result and identification of species in the LJ Culture of Sputum section on Appendix B In addition check the appropriate box on pg 1 of Appendix B to confirm whether the isolate was archived for short term stor
130. lture is unavailable for testing one attempt should be made to recover M tuberculosis see Section 10 10 below 10 9 Subculture and Storage of MGIT Cultures 1 Vortex the MGIT tube well and remove tube cap Using a disposable pipette remove a small aliquot 200 ul of broth and inoculate 1 2 LJ tube s incubate at 37 C 1 Section 11 Solid Culture Lowenstein Jensen LJ Media Growth from this LJ subculture can be used for drug susceptibility testing if this test is unsuccessful using the broth This U subculture is used for the short term and long term storage of isolates Section 11 Solid Culture Lowenstein Jensen LJ and Section 14 Long Term Storage of MTB Isolates Store all instrument positive MGIT tubes including cultures growing MTB or MOTT for at least 3 months after the culture is finalized at room temperature away from direct light 10 10 Decontamination of Contaminated MGIT Culture Decontamination of a MGIT tube should be performed in the following situations When the culture result is Positive for MTB and contaminated and the specimen is from a timepoint that requires DST and the corresponding primary LJ culture is not positive for MTB e f the antigen test result is indeterminate owing to the presence of contaminants To prepare the short term LJ storage and subsequent long term frozen isolate if the corresponding primary LJ culture is not positive for MTB Vortex contaminated
131. m by rapid ID test tuberculosis complex printout HAIN Genolype and contaminated and final TTD from Mycobacterium CM D positive for MTB Appendix M if or HAIN Genolype complex record ID applicable tests method Positive Positive Negative MTB not detected by o M tuberculosis Original TTD from or growth HAIN Genolype complex growth but MGIT printout MITBDRplus test or positive for other and final TTD from MOTT detected by mycobacteria ID Appendix M if HAIN Genolype negative for MTB applicable Mycobacterium CM test complex record ID method Positive Negative Growth Contaminated Original TTD from MGIT printout and final TTD from Appendix M if applicable Negative N A N A N A Negative for M Original TTD from tuberculosis complex MG final T printout and a TID of 42 days O hours Refer to Section 10 14 Study Data Reporting for additional instructions for recording the TTD NOTE If MGIT culture signals negative but tube appears turbid when removed from the instrument process according to the guidelines in Section 10 6 and Flow Chart 1 notifying the Sponsor of this occurrence 10 12 Quality Control of MGIT Media 1 Record lot number expiry dates etc for new MGIT medium and supplement PANTA on the Reagent Media QC form Appendix E 2 Quality control procedures for MGIT media and for the evaluation of TTD reproducibilit
132. n Section 16 2 1 3 1 below for new lots shipments of MGIT culture medium using an M tuberculosis strain Refer to the MGIT culture media package insert for further instructions on QC procedures with non tuberculous mycobacterial strains QC of Commercial Blood Agar Plates Contamination can sometimes occur during transit of commercial BAP media Monitor each lot closely and if contamination issues are suspected follow the procedure as recommended in Section 16 2 1 2 3 above 16 2 1 3 1 QC Protocol for BACTEC MGIT 960 Culture Medium and MGIT 960 Growth Supplement Kit Frequency Each new lot or new shipment of 960 media or growth supplement Controls Dilutions of M tuberculosis H37Rv or H37Ra in 7H9 broth Acceptable results MGIT tube will fluoresce positive TTD in 6 to 10 days Corrective actions a If the TTD is not within the specified range repeat the test b If test still does not give satisfactory results upon repeat notify supervisor immediately and check the viability of the inoculum age of the culture if stored frozen and other procedures i If all procedures are within established specifications contact Technical Services at BD Diagnostic Systems for assistance and withhold use of lot Documentation Record results on the Reagent Media QC Form Appendix E If QC results are not acceptable prepare an Appendix K form to document the corrective action Preparation of Culture Suspension
133. n appropriately diluted MGIT culture broth as per routine procedure i e 1 10 dilution for PZA 1 100 dilution for SIRE and second line drugs Enter tubes into the instrument and monitor until instrument signals as complete DST Acceptable DST Not Acceptable Instrument signals positive in appropriate timeframe Machine gives another error Check tubes visually for any signs of contamination Mark result as TF test failed on ZN subculture tube s as applicable appendix B and write explanatory Compare DST profile with previous test results from note in the Comments section this patient if available Write a Note to File to explain the Investigate and resolve any discrepancies errors and send a copy of the NTF Report DST following routine study procedures to the site 65 Mycobacteriology Laboratory Manual 66 Mycobacteriology Laboratory Manual Flow Chart 5 Invalid x400 Errors from MGIT DST NOTE All work must be done in the biological safety cabinet DST Acceptable DST Not Acceptable Instrument signals positive in appropriate timeframe Machine gives another error Check tubes visually for any signs of contamination Mark result as TF test failed on ZN subculture tube s as applicable appendix B and write explanatory Compare DST profile with previous test results from note in the comments section this patient if available Write
134. n must be given to Selecting the appropriate respirator for the individual Conducting fit testing Training personnel on the use fit checking and storage of the respirator NOTE Laboratory infections are nearly always caused by poorly monitored BSCs or a BSC in which normal aerosol containment capability is compromised thereby permitting escape of droplet nuclei A respirator can serve as an additional barrier to reduce the likelihood that tubercle bacilli will enter the lung 4 4 Personnel Protection Training and Monitoring e All personnel working in the containment lab must have proper training on biosafety procedures use of personal protective equipment and how to monitor all equipment especially the BSC for proper operation Documentation of this training must be kept with personnel training records Staff should receive frequent re training and be monitored to ensure compliance e Staff should be participating in a TB screening program per the laboratory s national guidelines for preventing TB infection 5 SPECIMEN COLLECTION STORAGE AND TRANSPORT Purpose Proper collection and transport of sputum specimens is required to ensure quality laboratory results Adherence to procedural details will result in collecting adequate and quality sputum specimens for analysis in the mycobacteriology laboratory and maintaining correct identity of the specimen The procedure below details sputum collection at the site
135. n taken be Month Year Day Temp 1 CO 1 Time Taken Initials Temp 2 Gon 2 Time Taken Initials CO tank pressure Action Adjustments Maintenance Notes RI wlrm O O 31 Date Reviewed by Signature Comments Date format dd mmm yyyy Equip Number Location room or equivalent Temperature Range 4 12 Thermometer If emperature is out of range inform the supervisor and record any action taken below Month NOG sideris Day Temp 1 Time Taken Initials Temp 2 Time Taken Initials Action Maintenance Adjustments Notes 1 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 8 9 20 21 22 23 24 25 26 27 28 29 30 31 Date Reviewed by Signature Comments Date format dd mmm yyyy Mycobacteriology Laboratory Manual 114 Mycobacteriology Laboratory Manual L
136. n this section including copies of requisition forms and airway bills Trial Correspondence All trial related correspondence including emails and faxes should be maintained in this section 1 1 5 Document Storage Records must be kept in a secure location that maintains their integrity and confidentiality such as a locked room or file cabinet with access restricted to authorized personnel Storage conditions must also minimize the risk of environmental damage and the threat of accidental destruction such as from fire or flood Confidentiality of both study patients and non study patients must be maintained at all times 1 2 Study Data Retention of Documents The retention of all original study data shall be the responsibility of the Principal Investigator the individual responsible for conducting the clinical trial and who assumes accountability for the reatment of human subjects and the integrity of the trial data on behalf of the Sponsor but at all times shall remain the property of the Sponsor Laboratory study records including all source documents as described in Section 1 1 must be retained and be made available for review under appropriate circumstances until permission for heir disposal has been given by the Sponsor Records will be stored in the laboratory s facilities unless permission to transfer and store elsewhere is agreed upon between the laboratory and the Sponsor 1 3 Supervision of the Study A superviso
137. nature and date time of receipt as well as a description of the specimen on the Laboratory Section of Appendix A Follow steps 2 4 5 6 and 7 above Note Induced sputum may appear watery and resemble saliva due to the method of collection These specimens are acceptable for testing Specimens that are visibly contaminated with food particles should only be processed if the patient is not accessible to provide another specimen within 48 hours of the scheduled visit Call the site immediately to instruct the patient to try again with a new pre labeled specimen container Record this information in the Comments section on the Appendix A form 6 2 Login of Sputum Specimens Specimens Laboratory Specimen Requisition Forms and Appendix A forms are transferred to the mycobacteriology lab if received in a separate receiving area Specimens must be stored at 2 8 C if not processed immediately Assign a unique laboratory accession number to each specimen note that sputum specimen 311 and sputum specimen 2 must have separate accession numbers This may take place in the general lab receiving area or in the mycobacteriology lab Record patient and specimen information in the lab database or specimen log book according to the laboratory s standard operating procedure Note the study visit number for which the specimen has been collected to ensure the appropriate work up of a positive culture Place laboratory accession number on the st
138. nd phenol based disinfectants Diluted working bleach solutions must be prepared daily and stay at or above 0 5 96 chlorine undiluted commercial bleach is usually 4 5 96 Similarly phenol based disinfectants must be diluted daily preferably with deionized not hard water to 2 5 96 Both types of disinfectants are only effective if left in contact with the contaminated material for at least 15 minutes Check the manufacturer s recommendations for the specific disinfectant to be used Place all wastes containing MTB in a leak proof container or autoclavable plastic bag that contains disinfectant solution which can be sealed before being removed from the BSC and autoclaved Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual The autoclave should be monitored with a spore test at least monthly to ensure that sterility is achieved Avoid practices that can result in spills e g hand carrying tubes vials and bottles or improperly stacking racks or baskets All tubes plates and other containers should be transported on carts in protected racks or baskets Write a procedure for the appropriate handling of a spill both inside and outside the BSC The procedure should include Inside the BSC the BSC should continue to run and the appropriate disinfectant should be applied to the spill for at least 20 minutes after which the items can be transported to the autoclave If a liquid culture is spille
139. ndix K form is required when a QC test fails to give proper results It is also used to document any quality improvements made by the laboratory See Section 16 5 Quality Improvement for an example of how to complete this form In some cases a situation may require both an Appendix K and If there is any uncertainty about which form to complete discuss with the lab director and or the Sponsor 1 1 4 Document Filing and Access The timely filing of essential documents in the laboratory using an organized system of indexing and labeling greatly assists in the successful management of the study Since laboratory documents are subject to review by CRAs auditors and regulatory inspectors they must be stored in a manner that facilitates easy retrieval and review while protecting the confidentiality of all patients study and non study For those laboratories using computerized systems that are not 21 CFR Part 11 compliant to capture and retain source documents electronically the following procedure must be followed The lab director or designee must verify in writing the data that the system captures including location documentation of each data point and how the system is controlled e g only authorized individuals have access to the system and an audit trail is maintained If quality control records are stored electronically these data must also be printed and stored appropriately The lab must ensure that all data for ea
140. ned to be Negative for M tuberculosis Complex record the TIP value reported on the MGIT unloaded negatives printout as the original TTD on Appendix B The final TTD should be written as 42 days O hours on Appendix B 8 In the unlikely event that a culture result cannot be obtained e g broken or lost MGIT ube MGIT instrument malfunction etc report the MGIT culture result as Unknown and provide a brief explanation in the MGIT Comments field on Appendix B In addition notify he Sponsor of any cultures reported as Unknown Flow Chart 1 General Algorithm MGIT 960 Cultures Negative Signal At the end of the 42 day protocol Action Observe tube visually before discarding If any suspicion of growth do ZN amp BAP Otherwise report as negative for MTB Positive Action ZN amp BAP Signal True Positive ZN Positive amp BAP Negative Rapid ID test Positive Action Short and long term storage DST if required Report ZN positive BAP negative Positive for MTB original TTD from MGIT printout and revised TTD from Appendix M if applicable Suspected MOTT ZN Positive amp BAP Negative Rapid ID test Negative Action Refer to Flow chart 2A Positive and Contaminated ZN Positive amp BAP Growth contamination Action Refer to Flow chart 2 Contaminated ZN
141. nel as well as a hazard to others who may be exposed to infectious aerosols in the laboratory Tubercle bacilli may be present in sputum gastric lavage fluids cerebrospinal fluid urine and in a variety of tissues MTB bacilli may survive in heat fixed smears and may be aerosolized during the processing of specimens and inoculation of culture media Although clinical specimens from TB cases contain a low infective dose of MTB all Specimens from suspected or known TB cases must be considered potentially infectious and handled with appropriate precautions Furthermore MTB cultures and drug susceptibility testing procedures generate high concentrations of organisms that pose an increased risk of aerosol production Thus all aforementioned procedures necessitate special biosafety containment and practices Principle Regardless of the level of risk for spread of TB infection standards are recommended for all laboratories handling specimens from suspected or known TB cases These standards are endorsed by WHO US CDC and US National Institute of Health NIH In addition Good Clinical Laboratory Practice GCLP standards a set of regulations that guide laboratories conducting clinical trials require a laboratory design that ensures the safety of personnel and the quality of work In summary these standards require Administrative controls including good lab practices SOPs and accident management plans Engineering controls such as a co
142. nt the total number of positive MGIT cultures Use these values to calculate the MGIT culture positivity from smear negative specimens smear negative total culture positive reported in the month x 100 Repeat Steps a and b for cultures Record isolation rates and compare results with data from the previous month and the lab s average Calculate the cumulative mean positivity rate for each medium Results a 9096 of smear positive specimens should be culture positive b 5096 of culture positive specimens may be from smear negative specimens c Rates should be relatively consistent for each medium Due to the higher sensitivity of liquid medium the isolation rate on may be slightly lower especially if smears show very few AFB Corrective Actions a If there is a significant decrease in overall culture positivity isolation rate for both media types review decontamination procedures If there is a significant decrease in only one media type check the growth performance following media QC guidelines above If the LJ isolation rate differs by gt 20 of the MGIT isolation rate for the same set of specimens perform QC procedures for the applicable lot of commercial or homemade following the guidelines in Section 16 2 1 2 1 QC Protocol for LJ Medium If there is a significant change in smear positivity negativity unrelated to the visit intervals examined review all microscopy procedures and discuss
143. ntation to this report SECTION IV PREVENTION Describe policies ractices etc to be implemented as a result of this investigation to prevent further occurrences if applicable Identify the individual s responsible for monitoring the effectiveness of these policies practices Dates Name Signature Title of Person Identifying Issue ese Dates sce Name Signature Title of Responsible Daf Name Signature Title of Supervisor sss Date format dd mmm yyyy 145 APPENDIX L MGIT TTD WORKSHEET MTB strain used Source of strain ATCC for dilutions WHO etc of subcultures ls this strain pan susceptible Yes No Type of suspension used frozen or fresh subculture TID Results TID days and hours Tech Supervisor Review Date Inoculated 1 500 Tube 3 Initials Date lnitials Estimated Concentrations Tube 3 log 10 Date format dd mmm yyyy Mycobacteriology Laboratory Manual 146 eunjno euj ipu days eui eq oj uorpes si nse1 jou aby ava 7 NZ
144. ntrolled ventilation system Use of personal protective apparel equipment appropriate for the task Waste management procedures General lab safety procedures including physical biohazard fire chemical and electrical safety Materials Equipment for all TB Laboratory Procedures Class Biosafety Cabinet BSC N95 FFP2 or equivalent respirators Lab gowns Disposable gloves Biohazard bags Tuberculocidal disinfectant Waste receptacles Centrifuge with biosafety canisters lids Autoclave Autoclave tape Spore test for autoclave 4 1 4 2 Recommended Facilities and Equipment The lab must be contained i e physically separated from other labs Access to the lab must be restricted preferably through an anteroom Controlled ventilation should be installed which maintains a directional airflow into the lab supported by a visual monitoring device showing that proper directional airflow is maintained at all times Air from the containment lab should not be re circulated to other areas in the building this environment can be achieved through high efficiency particulate air HEPA filtration within the ventilation system Class Il biosafety cabinets BSC vertical laminar flow which blows HEPA filtered air over work area UV lamp optional must be used for all manipulations of clinical specimens and positive cultures BSCs must be certified at least annually by personnel tra
145. ocation room or equivalent Temperature Range see below Thermometer Note If temperature is out of range inform the supervisor and record any action taken below Month Year Temp Time Taken Temp 2 Time Taken iw SS Min Max Initials Min Max Initials Action Adjustments Maintenance Notes COIN Ala RR wl rm O 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Date Reviewed by Signature Comments Date format dd mmm yyyy Based on requirements of reagents or instrument housed in the particular room e g MGIT room 19 30 room containing unrefrigerated centrifuge lt 20 APPENDIX E NEW REAGENTS MEDIA QC FORMS Appendix E New Reagents Media QC Form Acridine Orange lot Batch Results Neg Confirmation Number of gte PITY Quantity Date Tested Required Scanty 1 2 3 pass Prepared Reagent Date of Prepared Tech Initial 7 Yes N epared prepared Barche ech Initials Positive Negative Yes No Stain Control Contro
146. ocumented in the lab worksheets notebook Please refer to Flow Charts 1 3 for additional guidance 1 This tube is con ZN Result BAP Result TID Negative Growth gt 7 days aminated it can be discarded ZN Result BAP Result TID Negative Growth lt 7 days 2 Re incubate the MGIT tube for another 14 days and repeat the ZN smear and BAP This ensures that low numbers of M tuberculosis do not go undetected a If the subsequent ZN is positive follow step 3 below b the subsequent ZN is negati ve this tube is confirmed as contaminated and can be discarded Record the results from initial subsequent testing and reincubation of this culture on Appendix M Early MGIT Pos 3 Perform a ZN smear from the BAP the source of contamination In addition perform a M broth to confirm the presence of culture see Section 10 10 below if the contaminatio a If the ZN from the BAP is posi MGIT culture for 48 hours and reincubation and repeat testing i itive Early Contaminated Cultures Tracking Worksheet ZN Result BAP Result TID Positive Growth Any ive and the ID tes retest with the MPT n interferes w is negative 64 anti growth to determine if rapidly growing mycobacteria are PT MPB 64 an TB If necessary one attempt should be made to purify the igen test from the MGIT th the antigen test or MTB reincubate the gen test Document th
147. ode not available soft key 4 The display shows the default carrier set Check that the AST set definition is displayed for the AST set currently being entered 5 The station LEDs of all the assigned stations br the set illuminate GREEN Insert the tube set into the indicated stations ensuring that the Growth Control tube is located in the leftmost indicated station Make sure that all the tubes AND the carriers are fully seated in the drawer 6 Repeat Steps 1 5 for each of the AST sets you want to enter 7 When nished close the drawer and wait a moment while the drawer performs a quick scan of drawer contents If you have misplaced any AST sets the quick scan can detect this and put the affected set into error status Correct any errors before the MGIT 960 begins testing Removing completed AST 1 Open the desired drawer Press the remove completed AST sets gt soft key sets indicated by a red 2 The first completed AST set stations illuminate with FLASHING GREEN on the drawer indicators Remove the carrier starting with the completed set closest to the front of the drawer and scan its barcode label The LEDs at this station extinguish Repeat steps 2 3 to remove additional AST sets Place completed AST sets in the AST tube rack Press the printer soft key to access report selection Press lt unloaded AST sets report soft key Match the AST sets with the
148. om temperature However ensure the solution is brought to room temperature prior to using on subsequent specimens f more or less of this solution will be used in one day prepare the appropriate volume needed Discard any unused NaOH NALC solution at the end of the day Pour a smaller volume of this working solution into a 50 ml conical tube and use this smaller aliquot for digesting decontaminating specimens This technique reduces the potential for contaminating the larger stock bottle Table 7 1 Preparation of NaOH NALC Na citrate digestant solution Volume 6 NaOH 2 9 Na citrate Amount NALC to add needed ml grams 50 25 25 0 25 100 50 50 0 50 200 100 100 1 00 250 125 125 1 25 500 250 250 2 50 1000 500 500 5 00 7 2 Preparation of BSC and Special Microbiology Practices All processing must be done in a biological safety cabinet BSC using full PPE 1 Before processing the day s specimens clean BSC surfaces with 7096 ethanol and let stand 3 minutes prior to drying with a paper towel Prepare a splash proof waste receptacle with tuberculocidal disinfectant at the appropriate concentration and place inside BSC for disposal of liquids Place paper towels soaked in tuberculocidal disinfectant over the entire work surface inside the BSC Place a discard bucket with a biohazard bag containing tuberculocidal disinfectant inside BSC for disposal of contaminated materials Do n
149. on period A count of the number of negative cultures appears for each drawer in the summary region of the main screen menu next to the filled circle icon When no growth is detected after 42 days the negative tube indicator marked with a minus sign and located in the center of the three drawer indicators illuminates GREEN The indicator remains lit until all negative tubes are removed through the lt remove negative tubes gt operation Tubes can be removed via batch or single tube operation 35 Mycobacteriology Laboratory Manual 36 Mycobacteriology Laboratory Manual Functions Instructions Remove negatives batch removal of all final negative cultures from the instrument without having to scan the individual tube barcodes Open drawer where the CREEN light is on 2 Press remove negative batch gt soft key The barcode scanner turns off and 4 When all negatives are removed press the ok soft key When all negatives have been removed the instrument beeps 3 times the Close the drawer gently or press exit to continue with the next task Repeat Press lt remove negative tubes gt soft key tubes cannot be scanned Stations with negative tubes light up with flashing GREEN lights Remove all tubes in the flashing green stations prior to closing the drawer If negative tubes remain in the drawer atter it is de the instrument will register these tubes as newly
150. on 6 2 Login of Sputum Specimens Record the MGIT tube number from the barcode label on the lab MGIT worksheet 3 Once dissolved add 0 8 ml of the PANTA growth supplement mixture to each MGIT tube using a repeat pipettor if available or a micropipettor with sterile tip taking care not to contaminate the tubes 4 Using a sterile graduated transfer pipette add 0 5 ml of well mixed sputum sediment Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture to the MGIT tube Dispose of transfer pipette in biohazard bucket 5 Tightly recap the MGIT tube and mix well by gently inverting the tube several times 10 2 Entering Tubes in the Machine Enter tubes into the instrument as soon as possible Always scan the MGIT barcode first the instrument will assign the stations Functions Instructions Accession 1 Open the desired drawer Press tube entry soft key Place the tube in the alignment block in front of the scanner with the barcode label facing scanner Rotate the tube if necessary The instrument will beep once to indicate that the tube was scanned 4 the tube s label is damaged use a spare barcode label 5 The assigned station is shown in the main body of the display along with the scanned sequence number In addition the station LED of the assigned station for that tube will illuminate GREEN Carefully and completely place the tube in the appropriate position Repeat s
151. ords a T in the growth column Explosive growth usually means that the MGIT tube is contaminated Contamination will be confirmed by a negative ZN smear and growth on blood agar see the Sampling Tubes for Further Analysis section below The GU is not an indication of biomass within a culture tube A MGIT tube that has been flagged as positive usually contains a biomass of approximately 10 to 109 CFU ml However the tube can signal positive when the CFU is too low to yield a positive ZN smear Thus there is no direct correlation of biomass and GU at the time of instrument positivity Procedure Materials MGIT tubes 7 ml MGIT PANTA MGIT Growth Supplement 15 ml tube racks Sterile transfer pipettes with graduations marking volume individually wrapped Sterile serological pipettes 10 or 20 ml Pipette aid p1000 pipette or equivalent with sterile aerosol resistant tips Tuberculocidal disinfectant Discard bucket with biohazard bag insert containing appropriate disinfectant Study labels Permanent marker Mycobacteriology Laboratory Manual 34 Mycobacteriology Laboratory Manual Forms e Lab specific forms for recording MGIT data Appendix D Equipment Temperature Record or use site specific form if available and equivalent Appendix B Study Source Document Worksheet MGIT Maintenance Log this is a site specific form MGIT Daily Initiation Routine All fun
152. orescent microscope for fluorescent stain Immersion oil for Ziehl Neelsen stain Slide holder Slide storage box ens paper Lens cleaning solution Dark room for examination under fluorescence Forms Laboratory Specimen Requisition Form this is a site specific form used for routine laboratory requests Laboratory worksheet workbook this is a site specific form used for initial recording of smear results Appendix C Daily AFB Staining QC Form Appendix B Study Source Document Worksheet 29 Mycobacteriology Laboratory Manual 30 Mycobacteriology Laboratory Manual 9 1 Examination of Fluorescent Stain Examine fluorescent stained slides within 24 hours of staining Save all sputum smears in slide boxes 1 With a fluorescent microscope scan the entire smear with the 20X objective with 10X eyepiece for a total of 200X magnification Using 20X magnification one 2 cm length is equivalent to 30 fields which is sufficient to Occasionally use the 40X objective to see more detailed bacterial morphology Confirming morphology at higher magnification avoids a false positive report due to fluorescing debris Mycobacteria appear as rod shaped coccoid or filamentous bacilli They may be vary in shape and staining intensity and may occur singly in small groups containing a few bacilli or as 2 report a negative result 3 4 large clumps 5 Count bacilli in the number o
153. ot work with more specimens than can be placed in the centrifuge at one time When processing multiple batches on the same day clean the BSC with disinfectant turn on UV lights if available for 20 minutes and allow air to circulate in the BSC for 20 minutes between batches prior to repeating steps 1 4 Place specimen tubes at least one space apart in rack to aid in preventing contamination Work methodically with the tubes on one side and discard buckets close to the specimens to avoid spillage confusion of samples To reduce the risk of cross contamination ensure that reagent containers do not come in contact with the edge of the specimen container Remove only one cap at a time from the tubes 50 ml specimen tube culture tubes to avoid cross contamination and misplacing the caps on the wrong tubes Ensure that tubes bottles racks pipette aid etc that are removed from the safety cabinet are free from any droplets potential contaminants If necessary wipe the rack or tube with a paper towel soaked in tuberculocidal disinfectant prior to removal from the BSC In addition if any suspicion of droplets from a specimen is seen on gloves wipe gloves with a disinfectant soaked towel and change gloves prior to processing additional specimens Upon completion of work place paper towels in discard bucket Wipe all BSC surfaces with 70 ethanol let stand 3 minutes and wipe dry If BSC is turned off in the evening be sure to lea
154. oth 4 drops and inoculate the blood agar plate with 2 drops streaking with the pipette tip Use the same pipette to place 2 drops of broth on a slide Use wax pencil or slides with etched circles to help contain smear contents Dispose of transfer pipette into the biohazard discard bucket 3 Re enter the MGIT tube into the instrument within 5 hours of removal to ensure the original culture results are retained in the instrument database 4 Heat fix the slide for Ziehl Neelsen ZN staining as described in Section 8 Acid fast Bacilli Microscopy AFB Preparation and Staining Examine the ZN smear for the presence of acid fast bacteria AFB as described in Section 9 Acid fast Bacilli Microscopy AFB Examination 37 Mycobacteriology Laboratory Manual 38 Mycobacteriology Laboratory Manual If AFB are seen describe them as typical atypical and note whether cording is seen Record the description and smear result on the laboratory worksheet 5 Incubate the blood agar plate in the incubator at 35 1 for 72 hours if 35 C incubation is not available incubate at 37 C The lower temperature is preferred as it facilitates the growth of contaminants 10 7 Interpreting MGIT Results Below are the possible results that can be obtained from the ZN BAP testing Beneath each table are the subsequent steps to take to determine a final result for the MGIT culture All results and further testing steps should be d
155. owing drug resistant strain Please refer to Flow Chart 4 for further instructions X400 System detects indications of possible contaminated or overinoculated tube and does not provide an interpretation of the AST set results Check the tube for turbidity and subculture to a blood agar plate to rule out contamination of the specimen Please refer to Flow Chart 5 for further instructions Other conditions such as power failure NOTES he Observe all resistant tubes visually for evidence of contamination when first removed from he instrument Perform a ZN stain on any suspicious tube and subculture to a BAP In addition when drug resistance is observed and the patient s isolate has not been tested before or if the isolate was not previously resistant to the drug test tube s with ZN and BAP to ensure that growth is not due to contaminants or MOTT DST for SIRE and PZA should only be repeated once if the first test fails If a valid result cannot be obtained after the second attempt the test should be reported as TF test failed and a ote to File should be written to document the DST failure f DST results for isoniazid or rifampicin are inconsistent with previous results for the same patient review the results and QC and repeat the test If the repeat result is discrepant with he first result repeat the test a third time and record the third test result as the tiebreaker 12 9 Entering and Removing AST Sets
156. person 0 courier lab messenger other of person collecting receiving if patient collected specimen Name of person completing this form Signature Transport Section This section should be completed by the driver courier Specimens refrigerated or kept on ice tick one Yes L1 No O Unknown Driver name or name of courier Signature Laboratory Section This section should be completed by the laboratory technician receiving he specimen Date time specimen received 24h clock Specimens refrigerated or kept on ice upon receipt tick one Sample in good condition i e container is intact and properly labeled no leakage specimen at proper temperature tick one Lab accession number If no give details 15 study label missing Yes Name of technician Signature Date format dd mmm yyyy Comments 101 Mycobacteriology Laboratory Manual 102 Mycobacteriology Laboratory Manual APPENDIX B STUDY SOURCE DOCUMENT WORKSHEET Lab Accession Site Patient Initials Screening ID Subject ID Visit Sou Seed 10 20
157. platform to improve homogenization Adjust speed of rotator so that it mixes the specimen well while ensuring the entire sample is exposed to the digestion decontamination solution Start with an rpm of 60 increasing speed in small increments e g 5 rpm until a gentle but even mixing of the sample occurs usually between 60 and 80 rpm depending upon the instrument Do not agitate the tube too vigorously extensive aeration causes oxidation of NALC making it ineffective for liquefying sputum Once the ideal speed is identified use for processing of all sputum samples e f a shaker is unavailable vortex the tubes gently to mimic the shaking action 2 3 more times during the incubation period Repeat steps 6 8 for subsequent specimens at 30 sec or 1 minute intervals Make sure the specimen is completely liquefied If still mucoid after 10 minutes add 50 mg of NALC powder and vortex 15 30 sec When time has elapsed remove tube from shaker Add phosphate buffer pH 6 8 to the 50 ml mark on the centrifuge tube using pre dispensed 50 ml aliquots of buffer Mix by inversion or vortex Discard unused buffer Continue to add phosphate buffer to all specimens at 30 sec or 1 minute intervals as above so that each specimen is ONLY exposed to digestion decontamination solution for a maximum of 20 minutes It is very important that the phosphate buffer is added within 20 minutes of adding the digestion decont
158. ppendix A forms in an envelope and attach to the outside of the transport container If the courier service requests that the forms be enclosed then place envelope in a sealed leak proof bag and place the bag inside the transport container The name and signature of the transport driver date and time of pick up are recorded in the Transport Section of Appendix A If a courier service is being used this section can be filled in by the site study team member and the name of the courier and tracking number provided instead of the driver name and signature 6 RECEIPT AND LOGIN OF SPUTUM SPECIMENS IN THE LABORATORY Forms Laboratory Specimen Requisition Form this is a site specific form used for routine laboratory requests Laboratory Specimen Logbook Registry this is a site specific form used for routine laboratory requests If possible use a separate logbook for study patients and non study patients Study Labels Appendix A Specimen Transfer Form Appendix B Study Source Document Worksheet 6 1 Receipt of Sputum Specimens 6 1 1 Sputum Specimen 1 patient collected sputum 1 Patient collected specimens will arrive in a sealed plastic biohazard bag Study labels will be placed on the cap of the outer collection container and on the side of the plastic label Upon arrival at the laboratory specimen labels must be verified with the corresponding Laboratory Specimen Requisition Form and Appendix A Check the
159. prepare 1 L digestant decontaminant solution 6 NaOH dissolve 30 g NaOH in 500 ml distilled water 2 996 Na citrate dissolve 14 5 g Na citrate dihydrate in 500 ml distilled water Sterilize each reagent by autoclaving at 121 C for 15 minutes abel containers with the buffer name date prepared expiry date batch lot number and technician initials Store reagents refrigerated until needed 2 Alternatively the solutions may be mixed sterilized and stored in screw cap containers for later use For example e 4 L flask combine 1 L of 6 NaOH and 1 L of 2 9 sodium citrate Mix well and aliquot into 500 ml bottles Sterilize by autoclaving at 121 C for 15 minutes abel containers with the buffer name date prepared expiry date batch lot number and technician initials Store reagents refrigerated until needed Phosphate Buffer Solution 1 To prepare 1 5 L of phosphate buffer Combine 7 1 g disodium phosphate Na HPO 6 8 g monopotassium phosphate KH PO and 1 500 ml distilled water in a 2 or 4 L flask Stir with magnetic stirring bar on magnetic stirrer Check pH which should be 6 8 Adjust if necessary Add disodium phosphate to raise pH add monopotassium phosphate to lower pH Using a smaller flask or beaker dispense buffer into smaller volume containers for storage ideally 50 ml are dispensed into 60 ml sterile Nalgene tubes alternatively 100 200 ml sterile container
160. printed report and resolve any discrepancies w Printing an Unloaded AST Set report Cn NOTE If a station error has occurred the indicator will illuminate YELLOW Refer to the BACTEC MGIT 960 System s AST manual Chapters 4 and 6 for assistance in resolving this error 12 8 Interpretation of DST Results for SIRE and PZA The BACTEC MGIT 960 instrument continually monitors all tubes for increased fluorescence Analysis of fluorescence in the drug containing tubes compared to the fluorescence in the Growth Control tube is used to determine susceptibility results When the growth unit GU of the growth control reaches 400 within 4 13 days SIRE or 4 21 days PZA the GU values of the drug containing vials are evaluated 5 Susceptible the GU of the drug tube is less than 100 Resistant the GU of the drug tube is 100 or more Invalid tests are reported when certain conditions occur that may affect test results X Error or Indeterminate results reported when certain conditions occur that may affect the test If possible determine the cause of error In any case repeat testing with a pure culture of the isolate X200 System cannot detect sufficient indication of growth in the Growth Control tube in the specified protocol time and does not provide an interpretation of the AST set results Often a result of too little inoculum nonviable organisms or a slow gr
161. r and culture are expected to be processed on the day of receipt in the laboratory especially if on the following day s the lab is closed Specimens must be refrigerated if not processed within one hour 6 3 Study Data Reporting Record the following on Appendix B lab accession number site number screening ID number and or subject ID number patient initials visit number sputum specimen number 1 2 or N A date and time specimen was received specimen volume and type of specimen i e induced or not patient collected or site collected name of person collecting the specimen any comments PROCESSING SPUTUM FOR SMEAR MICROSCOPY AND QUALITATIVE CULTURE Purpose Processing sputum specimens has two objectives decontamination of bacteria other than mycobacteria and liquefaction of mucous and organic debris in the specimen Although there are several techniques available none of them are ideal meaning none of them will selectively destroy only contaminating flora and achieve complete liquefaction of the specimen A reasonable compromise is to destroy as much of the contaminating bacteria as possible while harming as few mycobacteria as possible All sputum specimens will be processed in this manner for preparation of AFB smears and liquid solid LJ cultures Principle N acetyl L cysteine NALC a mucolytic agent is used for rapid digestion which enables the decontaminating agent NaOH to be u
162. r must be assigned to oversee the trial laboratory staff and procedures to ensure the strict adherence to this laboratory manual At a minimum this person is responsible for the following oversight activities Ensuring relevant staff are trained on the manual guidelines and procedures and have read understood and will follow the laboratory manual Routine and timely review of Appendix B to verify completion and accuracy of all tests Monthly review of quality control quality monitoring and quality improvement activities including signing of all QC forms worksheets and confirmation of test results Semi annual review and follow up of study related proficiency testing results e Investigation of unacceptable or out of range QC results and proper documentation of corrective actions and improvement activities Adherence to and documentation of equipment cleaning and maintenance schedules as outlined in this manual Management of inventory for all study related consumables Adherence to biosafety and infection control principles and practices throughout the study Maintenance of patient confidentiality Organization of study documentation and provision of essential documents as applicable to CRAs auditors regulatory inspectors etc Coordination with study related staff including routine meetings to review documenta tion answer queries and resolve issues Supervisory roles may be delegated to qualified personnel
163. r on LJ culture Corrective actions If QC results are unexpected notify supervisor immediately and have technician repeat exercise with next processing batch growth is detected on either media from the negative control either cross contamination has occurred or there is a problem with sterility of the processing reagents Notify the Sponsor immediately if cross contamination of MTB is suspected i Observe the technician who processed these specimens for techniques known to contribute to contamination e g poor organization of tubes splashing when adding reagents opening caps too soon after vortexing etc ii Visually check all reagents used subculture to BAP if contamination is suspected iii Record patient samples processed at this time and monitor closely for expected results If no growth occurs on either media from the positive control check age of isolate used and diluting technique c If TTDs and or colony counts on LJ vary widely investigate possible cause for deviations Documentation Record the microscopy MGIT and LJ culture results on the Weekly MGIT LJ Culture QC Form Appendix If QC results are not acceptable prepare an Appendix K form to document the corrective action 16 4 4 MGIT 960 Time To Detection Monthly Exercise Required An important control measure of the MGIT culture system is to evaluate the consistency reproducibility of the TTD for known quantities of M tuberculosis
164. raction Buffer Buffer formula e 0 01 M e NaCl 0 145 M Tween 80 0 0196 To prepare 1 L of buffer 1 2 3 Using weigh boat and analytical balance weigh out 1 36 g of KH PO Weigh out 8 5 g of NaCl Dissolve powders in 500 mL distilled water in a volumetric flask Using a micropipette add 100 uL of Tween 80 pipette up and down repeatedly to dislodge all Tween material ix thoroughly and bring to a final volume of 1 L with distilled water using a cylinder Aliquot buffer into smaller volumes e g 250 ml before autoclaving Label containers with he buffer name date prepared expiry date batch lot number and initials of preparer Sterilize for 15 minutes at 121 C 15psi Store buffer at 2 8 for up to 6 months Before each use check buffer visually for signs of contamination or degradation and bring to room temperature 13 2 Specimen Preparation Specimen preparation and subsequent steps must be performed in a BSC using full PPE 13 2 1 From positive MGIT tubes 1 2 Ideally test AFB smear positive MGIT tubes within 5 days of instrument positivity Vortex the tightly capped MGIT tube for 30 seconds to ensure the suspension is well mixed 13 2 2 From positive LJ slants 1 Test 2 4 week old growth 2 Add 200uL of TBc ID extraction buffer or Capilia extraction buffer to a sterile cryovial 3 Using a sterile 10 uL loop scrape a loopful of several colonies and mix with b
165. rd results on the MGIT TTD Worksheet Appendix L b If QC results are not acceptable prepare an Appendix form to document the corrective actions c Each month upon test completion provide completed Appendix L form along with applicable MGIT printouts and any Appendix K forms to the Sponsor File copy of documents in laboratory QC binder Preparation of Culture Suspension Follow instructions in Section 16 2 1 3 1 QC Protocol for BACTEC MGIT 960 Culture Medium and MGIT 960 Growth Supplement Kit to prepare the culture suspension Preparation of 1 500 Dilution NOTE It is highly recommended to prepare the dilution from a frozen stock suspension 1 Remove one aliquot of the stock suspension from the freezer and thaw Alternatively prepare a fresh McFarland No 0 5 suspension using colonies within 10 15 days of firs appearance on LJ media Carefully adjust the inoculum with a spectrophotometer or visually with a Wickerham card 2 Dilute the stock suspension freshly prepared or frozen 1 5 by transferring 1 0 ml o suspension to 4 0 ml of sterile water or saline Mix well Tube 1 3 Dilute 1 10 two more times by adding 0 5 ml of suspension from Tube 1 into 4 5 ml o sterile water or saline Tube 2 Mix well and then again add 0 5 ml from Tube 2 to 4 5 m of sterile water or saline Tube 3 Tube 3 will be used for the inoculation and incubation of the MGIT tube This tube represents 1 500 dilution representing abo
166. re NOTES 1 Errors e g x200 and x400 are generated the same as for first line drugs depending upon he AST set configuration used for testing and necessitate repeating as described in Section 12 9 above 2 DST for second line drugs should only be repeated once if the first test fails If a valid result cannot be obtained after the second attempt report the test as TF provide a brief comment in the DST Comments section of Appendix B and write a Note to File to document the DST ailures 3 If DST results for any of the fluoroquinolones or injectable agents are inconsistent with previous results for the same patient review the results and QC and repeat the test If the repeat result is discrepant with the first result repeat the test a third time and record the third test result as he tiebreaker 12 11 DST for Enviomycin Gatifloxacin and Kanamycin WHO does not currently recommend critical concentrations for Gatifloxacin Kanamycin or Enviomycin in the MGIT system However some labs are testing these drugs as part of standard of care DST results will be accepted for Gatifloxacin and or Kanamycin in MGIT or solid media so long as the laboratory submits a written rationale for the critical concentration used for the drug as well as any validation testing performed e Enviomycin Ogawa media at the critical concentration of 20 ug ml The lab should follow their internal solid medium DST procedures any appl
167. regulatory authorities that the data produced are a true reflection of the results obtained during the study Quality assurance practices consist of Quality Control QC nternal QC monitoring of test performance media quality reagent activity DST accuracy etc External QC proficiency testing to assess technical competency test accuracy and reproducibility Quality Monitoring QM Monitoring of critical practices instrument function data entry contamination rates and culture positivity rates that may affect the outcome of research results Quality Improvement 01 Continuous effort to improve service function workflow customer satisfaction etc All quality monitoring practices require documentation of results corrective action and resolution of unacceptable results along with supervisor review Sample forms for documenting QC OM and procedures are included Appendices C N or if the laboratory has equivalent forms in use these may be used for documentation after review and approval from the Sponsor All QC results should be documented in the same manner as patient specimens MGIT and LJ culture results colony counts etc 16 1 Laboratory QC QM Schedule Table 16 1 Laboratory QC QM Schedule Daily or with each patient run Weekly or with each patient run Monthly New lot or batch Refrigerator freezer incubator rooms and centrifuge temperatures Positive and ne
168. repeat patient tests b If repeat results still unacceptable notify supervisor immediately prepare new drugs and or a new control as applicable to resolve issue c If unacceptable results persist with SIRE or PZA kit consult BD Technical Services for assistance d When QC results are acceptable repeat patient tests and report results Documentation Record results for new antibiotics and weekly testing on the DST QC Form Appendix F If QC results are not acceptable prepare an Appendix form to document the corrective action Inoculum Preparation The inoculum must consist of a pure culture of M tuberculosis H37Rv or H37Ra and be tested following the same procedures as for patient isolates The inoculum can be prepared from one of three options 1 A freshly grown culture of MTB in MGIT medium following the guidelines in Section 12 4 Inoculum for MGIT DST A fresh pure subculture of MTB growing on LJ medium following the guidelines in Section 12 4 2 Using an Inoculum from Positive LJ Culture A suspension of MTB prepared according to steps 1 12 in Section16 2 1 3 1 QC Protocol for BACTEC MGIT 960 Culture Medium and MGIT 960 Growth Supplement above a Remove one aliquot of the organism suspension from the freezer and allow to thaw b Dilute 1 ml of the suspension in sterile saline or 7H9 broth 1 5 dilution c Use this adjusted suspension as the inoculum for drug tests and growth control Set up of
169. ribed above If examining a positive MGIT culture vortex tube well unscrew MGIT tube cap and sample an aliquot of broth using a disposable pipette Place 100 ul 2 drops of broth onto the slide spreading it to cover an area approximately 1 x 2 cm Dispose of transfer pipette into the biohazard discard bucket If examining colonies on solid medium dispense 100 ul of distilled water on a glass slide with a transfer pipette Using a sterile loop or disposable applicator stick transfer 2 to 3 colonies to the water and gently mix to make a smooth thin suspension Dispose of applicator stick into the biohazard discard bucket Air dry smear Place the slides on a hot plate or slide warmer at a temperature between 65 to 75 for at least 2 hours longer time is preferable to heat fix the samples Ideally remove smears from BSC after heat fixation for staining 8 3 Procedure for Fluorescent Staining 8 3 1 or Auramine Rhodamine Procedure NOTE The procedures below should be strictly followed unless a commercial staining kit is used in which case the manufacturer s instructions should be followed exactly as written in the package insert d Place slides on staining rack so they are at least 1 cm apart and flood with auramine or auramine rhodamine stain and let stand for 20 min Rinse the stain away with distilled water and tilt slide to drain Water must be chlorine free Flood the slide with 0 596 acid al
170. rile tube This diluted inoculum is used for preparation of the MGIT DST tubes 12 5 Growth Control Tube Preparation and Inoculation 12 5 1 For preparation of SIRE and second line Drug GC Tube s ils Vortex original MGIT tube diluted MGIT culture or diluted inoculum from LJ and mix well let suspension settle for 5 10 minutes Aseptically pipette 0 1 ml of the organism suspension into 10 ml of sterile saline to prepare the 1 100 GC suspension 196 growth control Mix the GC suspension thoroughly by gently inverting 3 4 times Inoculate 0 5 ml of the 1 100 GC suspension into all MGIT tubes labeled GC for that specimen using a micropipettor and sterile aerosol resistant tips 12 5 2 For preparation of PZA GC Tube ili Vortex original MGIT tube diluted MGIT culture or diluted inoculum from LJ as applicable to mix well let suspension settle for 5 10 minutes Aseptically pipette 0 5 ml of the organism suspension into 4 5 ml of sterile saline to prepare the 1 10 Growth Control suspension Mix the Growth Control suspension thoroughly by gently inverting 3 4 times Inoculate 0 5 ml of the 1 10 Growth Control suspension into the PZA MGIT tube labeled GC for that specimen using a micropipettor and sterile aerosol resistant tips NOTE It is important to use an appropriately prepared 1 10 dilution for the GC tube for PZA DST to ensure accurate results and avoid PZA AST set errors 12 6 Inoculation of Tubes Containing Tes
171. rm is received The laboratory will contact the site For unlabeled specimens There are no exceptions to this policy The laboratory will not process unlabeled specimens and specimens must always be labeled at the time of collection Notify the site when an unlabeled specimen is received The specimen will remain in the laboratory and must be labeled by someone from the site who will sign a statement taking responsibility for the labeling This statement must be filed with the Appendix A form in the study laboratory binder The fact that the specimen was received unlabeled and subsequently labeled will be entered in the laboratory Comments section of the Appendix A form and in the lab specimen logbook or database if available 3 Collection containers must be opened in a biosafety cabinet Only open one specimen container at a time to prevent mislabeling Remove the plastic label from the container by tearing along the perforations Unscrew the funnel portion from the container Remove the 50 ml centrifuge tube and close tightly using the cap provided e Immediately prepare study label with the information in Section 6 2 Login of Sputum Specimens and place on the 50 ml tube Container funnel and white cap must be autoclaved prior to disposal 4 Sputum specimens must be 2 3 ml in volume If the specimen is less than 2ml discuss the volume with the lab director and determine if the quali
172. rol control date initials New Lot Batch Staining Reagents Brand Received Expiry Quantity Date Manufacturer Lot Number Date Date Received Opened Auramine O Phenol Ethanol HCL concentrated Potassium permanganate Date format dd mmm yyyy Confirmation required by another technician or pre are another smear stain and read 119 Kuo0j amp Joqe 1 peal pue uies ajedaud 10 ueiiuupe q uoneuunuo sjpuiut ejop eAuisod JosiAJedns Aujuow urpis uiouis Nie peise pees voe JequinN paisa JequinN mas noon maa CUE E z 1 peunbey ejpubBupuueg uinisspjog Jeuo v 120 Lot Batch Results Number of Date E Quantity Date Tested Positive Negat
173. rs from Processed Sputum slides must remain in the biological safety cabinet until they have dried Label the frosted end of the slide in pencil with the laboratory accession number screening ID number and or subject ID number visit number sputum specimen number 1 or 2 unless specimen is from V2 or V3 and date Working in a biological safety cabinet vortex the decontaminated sediment see Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture to mix thoroughly Use a transfer pipette to place 100 ul 2 drops of well mixed resuspended pellet from the digested decontaminated specimen onto the slide spreading over an area approximately 1x2 cm Air dry the smear Place the slides on a hot plate or slide warmer at a temperature between 65 to 75 for at least 2 hours longer time is preferable to heat fix the samples Do not expose slides to UV light Work systematically through the samples with slides on one side and the discard bucket in close proximity often best at back of cabinet Remember to open only one specimen tube at a time Dispose of the transfer pipette into the biohazard discard bucket Ideally remove smears from BSC after heat fixation for staining 8 2 Preparation of Smears from Positive Cultures 1 2 Label frosted end of slide in pencil with laboratory accession number screening and or subject ID number sputum specimen number if applicable and date Work in a BSC as desc
174. ry Manual Appearance Date Brand lot Received Expiration Quantity Not TP Date Tech QC pass Manufacturer Number Date Date Received Acceptable Acceptable Use Initials Yes No Comments corrective qued date initials Date format dd mmm yyyy Explain in comments section 129 Mycobacteriology Laboratory Manual 130 Batch Results Number of P Quantity Date Tested Lot Number Positive Negative QC pass Prepared b initials Batcht Prepared Tech Initials TBcID Kit Positive Negative Yes No Buffer Control Control 2Strain 1 HE tested Monthly Supervisor fee negative Review date initials control New Lot Batch Extraction Buffer Brand Lot Received Expiry Quantity Date Manufacturer Number Date Date Received Opened Monopotassium phosphate Sodium chloride Tween 80 Date format dd mmm yyyy Mycobacteriology Laboratory Manual APPENDIX F DST QC INVENTORY FORMS Results nek QC pass SIRE or PZA MTB Other Strains Yes No Drug Tested gt Date Tested RE Pza Media Lot
175. ryotube to ensure they match before transferring growth from the slant to cryotube 4 Using a sterile loop disposable sterile applicator stick or sterile cotton swab wetted with sterile saline or broth transfer several sweeps of M tuberculosis growth from LJ subculture containing fresh pure growth Section 10 9 Subculture and Storage of MGIT Cultures and emulsify in broth e Use colonies showing good confluent and pure growth within 10 15 days of first appearance Older cultures will not provide reliable long term viability Density of suspension should be greater than a 1 0 McFarland standard Do not scrape off any culture medium this will give false turbidity 5 Tighten cap and thoroughly mix suspension using a vortex 6 Place cryotube in cryobox and freeze box in a rack or specified location in a 70 to 80 C 10 freezer NOTES 1 If a frozen isolate is used in the lab for any reason excluding shipping to central laboratory a replacement cryotube must be prepared and frozen 2 Freezer boxes must be organized to easily identify the isolates by patient and by sputum specimen number 1 or 2 in order to retrieve these isolates for further testing or shipping 3 Cryoboxes may contain isolates from more than one patient but the second aliquot from each visit sputum specimen 1 2 should be stored in a separate cryobox that exactly mirrors the first cryobox Consider the following example Subj ID 100
176. s ZN BAP negative 42 days ZN Positive BAP Negative Action Follow Flow Chart 1 ZN Positive BAP Growth Action Follow Flow Chart 2 ZN Negative BAP Growth Report ZN negative BAP contaminated culture contaminated original TTD from MGIT printout and final TTD from Appendix M 11 SOLID CULTURE LOWENSTEIN JENSEN LJ MEDIA Purpose The purpose of this procedure is to isolate and semi quantify growth of M tuberculosis on LJ medium Slants will be inoculated with decontaminated and concentrated sputum specimens Section 7 Processing Sputum for Smear Microscopy and Qualitative Culture Slants will also be inoculated from each positive MGIT tube Once good growth is obtained these positive slants will be stored in a cool dark place to archive the positive M tuberculosis isolates Principle Many different solid media are available for cultivating mycobacteria Most are variations of egg potato base or albumin agar base media There is no general consensus on which medium is best for routine isolation The advantages of egg based media such as LJ are 1 it is easy and economical to prepare 2 it is associated with lower contamination rates and 3 isolated colonies with characteristic colony morphology for MTB can be observed Disadvantages are 1 when contamination occurs it often involves total surface of medium 2 if contamination is slight it is not evident when
177. s when labeled and wrapped with Parafilm ape Biohazard bags Absorbent material Dry Ice to be provided by shipping vendor Shipping container and boxes to be provided by shipping vendor Parafilm Forms Shipping requisition form Shipping labels Airway bills Dangerous goods form Appendix B Study Source Document Worksheet Appendix J Freezer Storage Log for MTB Isolates 15 1 Isolate Shipping Schedule When shipping isolates it is recommended that duplicate tubes for each aliquot specimen be shipped in separate shipments to prevent total loss of a particular visit isolate An aliquot of each isolate shipped should be maintained at the lab 15 2 Packaging of Isolates NOTE Isolates must be packaged by lab staff with certification in the packaging and shipping of Category A infectious substances affecting humans as defined by the International Air Transport Association IATA 1 Ensure that isolates have been frozen at 70 to 80 C 10 C for a minimum of 48 hours prior to shipping 2 Wrap top of cryovials with Parafilm ensuring they fit comfortably into the cryobox without damaging the orange label 3 Follow specific instructions provided by the shipping vendor for shipping frozen isolates 4 Fill out a separate shipping requisition form for each shipment of isolates 5 File a copy of the requisition form s in the laboratory binder Mycobacteriology Laboratory Manual 76
178. s can be used abel containers with the buffer name date prepared expiry date batch lot number and technician initials Cap the tubes and sterilize in the autoclave These smaller aliquots will be used to wash the digested decontaminated specimen and resuspend the pellet 2 Alternatively stock solutions of disodium phosphate and monopotassium phosphate can be made in large volumes sterilized and stored separately When ready to use mix equal volumes in a sterile container and check pH using aseptic technique Then proceed to dispense 50 ml into 60 ml sterile Nalgene tubes as described above Daily Preparation of Solutions NaOH NALC Na citrate 1 To prepare NALC NaOH working solution Just before use determine how much reagent will be needed for the day s work e f solutions are stored separately mix equal volumes of the NaOH and Na citrate solutions in a sterile flask using a sterile cylinder 100 ml of each The resulting solution contains 3 NaOH Using the table add the appropriate amount of 0 596 NALC to the NaOH Na Citrate solution For example if a total of 200 ml NaOH Na Citrate solution is needed add 1 g NALC see table below to the flask Mix gently do not shake vigorously 21 Mycobacteriology Laboratory Manual 22 Mycobacteriology Laboratory Manual Store NaOH NALC solution at 2 8 between processing batches as NALC loses mucolytic activity on standing at ro
179. se only fresh stains without precipitates or contaminating organisms AFB transferred oil on the immersion Always wipe oil from the oil immersion lens after lens or through contaminated oil bottles each AFB positive smear is read Change oil bottles whenever there is evidence of contamination Smears that are too thick or slides that Proper digestion of specimen Avoid making thick are not clean causing material to be smears washed off during staining Smear area is too large making the Apply smear to a 1 x 2 cm area smear too thin Nonstaining or poorly staining Examine fluorescent stained smears within 24 hours Protect smear from UV light direct sunlight over False under heating during smear fixation store staining negative reagents in dark bottles high chlorine content in rinse water will affect fluorescence stain During staining drain off excess rinse water between steps to avoiding diluting staining reagents Incorrect slide warmer temperature Set temperature at 65 75 C and monitor on a daily weekly basis Incomplete slide reading Search smear in a uniform manner and read suggested number of fields 31 Mycobacteriology Laboratory Manual 32 Mycobacteriology Laboratory Manual 10 LIQUID CULTURE MYCOBACTERIA GROWTH INDICATOR TUBE MGIT Purpose To amplify the number of Mycobacterium tuberculosis MTB organisms in a sample using a liquid culture media
180. sed at lower final concentration in sputum NALC loses activity rapidly in solution so it is made fresh daily Sodium citrate exerts a stabilizing effect on the NALC by chelating heavy metal ions present in the specimen The phosphate buffer neutralizes the NaOH and dilutes the homogenate to lessen the viscosity and specific gravity prior to centrifugation Mycobacteria have a low specific gravity and may remain buoyant during centrifugation A relative centrifugal force of 3 000 xg not 3 000 rpm the centrifuge must be calibrated for 15 minutes is adequate to sediment mycobacteria The rate at which mycobacteria sediment is critically dependent on time of centrifugation and relative centrifugal force applied to the specimen A longer centrifugation time can offset a lower relative centrifugal force but increased centrifugation time increases the temperature of the specimen which leads to additional killing of mycobacteria hence a refrigerated centrifuge is highly recommended Procedure Materials Tuberculocidal disinfectant 7096 ethanol Waste receptacle splash proof for liquids Discard bucket with biohazard bag insert containing appropriate disinfectant e Sterile cylinder and break resistant glass bottle to prepare NaOH NALC Na citrate Clean cylinder flask and beaker to prepare phosphate buffer 50 ml conical polypropylene tubes with plastic screw cap 60 ml Nalgene tubes with plastic screw cap autoclavable
181. sor and record any action taken below Auramine O Acid Alcohol Potassium Batch or Lot Decolorizer Permanganate Expiration Batch or Lot Batch or Lot Date Expiration Date Expiration Date Positive Positive Negative Negative Control control slide control slide control slide Slides date of date of date of date of preparation expiration preparation expiration Results Neg Confirmation Day Required Scanty 1 2 3 Positive control Negative control QC Pass Yes No Technician Initials re m ees lala ww aA o R CN ER O e 31 MTB strain tested positive control Strain tested negative control Reviewed by Date Signature Comments Confirmation required by another technician or prepare another smear stain and read Date format dd mmm yyyy 105 Mycobacteriology Laboratory Manual 106 Month cess icem cec Year Note If QC is out of range inform the supervisor and record any action taken below Fuss Acid Alcohol Potassium Batch or Lot Decolorizer Permanganate Batch or Lot Batch or Lot Expiration Date Expiration Date Positive Positive Neg
182. spection confirms proper color texture and homogeneity of medium Corrective Actions a If all tubes are contaminated notify supervisor immediately discard entire batch and prepare new media b If one tube is contaminated repeat exercise with at least 10 additional tubes c If gt 1 tube is contaminated upon repeat testing notify supervisor immediately and discard entire batch d Investigate and resolve all problems and then prepare new media Documentation Record results on the Reagent Media QC form Appendix E If contamination is seen prepare an Appendix K form to document the corrective action Performance QC il 2 Frequency each new batch of prepared medium Controls 1 396 of LJ tubes from a batch tested with 10 2 10 and 10 dilutions of M tuberculosis H37Rv or H37Ra in 7H9 broth e g testing 296 of a batch of 100 tubes of media would include 6 tubes in total 2 tubes inoculated with each of 3 working dilutions See procedure below Acceptable Results Growth on all tubes is consistent with M tuberculosis and within 20 of the laboratory s own established reference ranges for each dilution Corrective Actions If tubes show no growth notify supervisor immediately discard entire batch and prepare new media Ifgrowth still not in acceptable range after repeat testing notify supervisor immediately and discard entire batch Investigate and resolve problems then prepare new media d
183. stigation into the root cause of the problem Oftentimes there is a bigger underlying problem that causes a QC failure or deviation to occur The issue should be analyzed and the root cause written in this section Ex TTD QC exercise was not assigned to a particular laboratorian 30JAN1 1 Corrective action to fix the current problem The first action taken to resolve the immediate issue is written here along with the expected date of resolution Ex TTD QC exercise performed immediately on 30JAN1 1 Preventative action required to eliminate root cause of problem This action often differs from the action taken to fix the immediate problem mentioned above and should address the root cause of the problem Ex A schedule was prepared to ensure each MGIT user performs the TID exercise OTFEBT1 Person s responsible for monitoring the effectiveness of action This section should explain how the effectiveness of the previously mentioned action is evaluated Ex Ms Pim was assigned to check the schedule in March and verify the appropriate laboratorian signed by their name after performing the TTD QC exercise for both February and March 21MAR11 All persons involvedin the incident the person identifying the issue the QA delegate and the lab supervisor should review the form and sign the document attesting that s he has been notified of the issue and agree to the actions taken 99 Mycobacteriology Laboratory Man
184. t Second Edition Geneva Switzerland 2008 www who int diagnostics laboratory 8 World Health Organization Policy Guidance on Drug Susceptibility Testing DST of Second line Anti tuberculosis Drugs WHO HTM TB 2008 392 Geneva Switzerland 2008 APPENDIX A SPECIMEN TRANSFER FORM Attach a study label to this page This form should accompany any specimen generated by a patient enrolled in the Sponsor study to the laboratory Use a separate Appendix A form for each sample submitted Once the form is completed it should remain in the lab files Dispatch Section This section should be completed by Principal Investigator or delegate Site name Site Screening ID Subject ID PT initials 3 characters Visit amp circle 2 3 A 5 15 16 17 18 27 28 29 30 31b 32 6789 1 19 20 21 22 23 32b 33 34 ET 0 11 12 13 14 24 25 26 UNS Date time collected or received if patient collected 24h clock Specimen Number circle one only circle N A if Visit 2 or 3 O1 02 ON A Sputum induced Yes O No Specimen collected by circle one patient site staff Specimen volume Time at room temp Record hr min if immediately Attach study label here refrigerated or shipped Date Time of dispatch to laboratory 24h clock Mode of transport circle in
185. t and results recorded on the lab worksheet and Appendix B Refer to Section 13 Rapid Identification of M tuberculosis Complex for details On specific visits growth which has been confirmed as MTB complex will be used for drug susceptibility testing if the corresponding MGIT culture is not usable See Section 2 Sample Specimen Timetable and Section 12 Drug Susceptibility Testing MGIT System 11 3 Working up Growth MGIT Subculture on Media ill To check for AFB and purity select 2 to 3 colonies of representative growth using a loop or applicator stick and perform a ZN stain Section 8 Acid fast Bacilli Microscopy AFB Preparation and Staining and Section 9 Acid fast Bacilli Microscopy AFB Examination If growth on solid medium is contaminated or insufficient to archive prepare another tube using growth from original LJ slant Save the culture in the isolate storage bank See Section 11 5 below 11 4 Recording Results of Primary Solid Culture 1 Record weekly growth results on the laboratory worksheet 2 At weeks 1 through 7 if there is no growth record neg If at the 8th read date the culture is negative record no growth on the laboratory worksheet 3 If there is growth at any reading interval re incubate the tube and read again the following week f the same approximate count is seen reading can be stopped and this
186. t Drugs 1 Vortex original MGIT tube diluted MGIT culture or diluted inoculum from LJ as applicable to mix well let suspension settle for 5 10 minutes Aseptically pipette 0 5 ml of the organism suspension from the supernatant into all drug containing tubes STR INH RIF EMB PZA any 2nd line drug tubes using a micropipettor and sterile aerosol resistant tips a separate tip must be used for each tube Take care not to disturb the sediment Tightly recap the tubes Mix tubes by gently inverting 3 4 times 12 7 Entering and Removing AST Sets in the MGIT 960 Instrument for SIRE and PZA Place the tubes in the appropriate carrier set ensuring they are in the correct sequence from left to right For SIRE use the 5 tube AST carrier GC STR INH RIF EMB or the 8 tube carrier if applicable For PZA use the 2 tube AST carrier GC PZA 59 Mycobacteriology Laboratory Manual 60 Mycobacteriology Laboratory Manual Open desired drawer in MGIT instrument and follow instructions below Function Directions Entering new AST sets 1 Press the tube entry soft key 2 Scan AST carrier s barcode label The carrier barcode indicates an AST set how many tubes are in the set and the set s sequence number For PZA testing select PZA as the drug in the two tube carrier definition as it is a longer protocol 3 Scan the accession barcode if available if not present press the accession barc
187. teps 1 6 for each tube When finished close the drawer or press exit to continue with the next task NOTES The MGIT instrument will record the date each tube was entered Do not turn tubes after placing them in the station Do not remove tubes unless they are positive or out of protocol negatives negative at 42 days Do not reassign tubes to a new station 10 3 Incubation of MGIT Tubes Since the MGIT system automatically and continuously incubates and monitors tubes once they are placed in a station there is no need to remove the tubes from the instrument Cultures remain in their stations until signaled positive or if no growth is detected after 42 days incubation are signaled negative If an instrument positive tube is determined to be smear negative for either mycobacteria or contaminants the tube may be re entered into the instrument but within 5 hours of removal If the tube is returned to the instrument via the lt tube entry gt operation described above positivity routines are reset the start of incubation date is retained and monitoring of the tube resumes If the tube is not returned within the 5 hour re entry window the associated data is removed from the instrument s database and the tube will be monitored as a newly entered tube 10 4 Dealing with Negative Tubes Negative cultures exist as ongoing negatives while they are in the routine 42 day incubati
188. tests b If results still unacceptable notify supervisor immediately and prepare new reagents and or new controls as applicable to resolve issue c When QC results are acceptable repeat patient tests and report results Documentation Record results for new staining reagents on the Reagent Media QC Form Appendix E For each batch of smears record results on the appropriate Daily Staining QC Form Appendix C If QC results are not acceptable prepare an Appendix K form to document the corrective action Preparation of Smears Prepare a batch of positive and negative control smears in advance heat fix and store unstained in a closed container in a dry area until used It is preferable to use a seeded sputum sediment to prepare these controls but if not available a suspension of M tuberculosis H37Rv or H37Ra for the positive control and Escherichia coli or another non acid fast bacillus for the negative control should be used The coli suspension may be prepared from the stock culture used in Section 16 2 1 2 3 QC Protocol for Blood Agar Medium 16 3 2 Sputum Digestion Decontamination Reagents For each new batch of the NALC NaOH and Na citrate reagents record preparation details Quality control for these reagents is performed as part of Quality Monitoring of Sputum Processing below Additional QC for the phosphate buffer is performed as follows il Frequency Each new batch of phosphate buffer
189. th appears turbid A ZN smear is prepared to determine the presence absence of acid fast bacilli in the tube A blood agar plate BAP is inoculated with the tube broth to determine the presence absence of contaminants Materials Discard bucket with biohazard bag insert containing tuberculocidal disinfectant Sterile transfer pipettes with graduations marking volume individually wrapped Blood Agar Plates TSA base Sterile loop or disposable applicator stick Ziehl Neelsen stain carbol fuchsin 396 acid alcohol methylene blue Permanent marker Microscope slide with frosted end new and clean Pencil for labeling slide Wax pencil for encircling smears on slide Lowenstein Jensen LJ slant Work with positive MGIT tubes must be done inside the biosafety cabinet using full PPE 1 If multiple positive tubes are being worked up smears from two specimens can be examined on one slide as long as they can be spaced sufficiently so as not to cause interfering results Blood agar plates can be divided into 4 quadrants so that four specimens can be subcultured onto one plate Label glass slide and blood agar plate accordingly with lab accession numbers screening and or subject ID numbers visit numbers sputum specimen number and date Take care not to confuse the inoculations and labeling 2 Vortex the MGIT tube well unscrew tube cap and sample an aliquot of broth using a sterile disposable pipette Remove about 200 ul of br
190. thermore WHO and other experts have published recommended critical concentrations for testing many second line drugs for the MGIT system Principle Susceptibility testing in the MGIT 960 system is based on the same principles as isolation from sputum detection of growth DST is performed using an AST antibiotic susceptibility testing set which consists of a Growth Control tube and one tube for each drug as well as a bar coded tube carrier that holds the set A known concentration of drug is added to a MGIT tube along with the specimen and growth is compared with a drug free control of the same specimen If the drug is active against the mycobacterial isolate isolate susceptible growth will be inhibited and fluorescence will be suppressed in the drug containing tube meanwhile the drug free control will grow and show increasing fluorescence If the isolate is resistant growth and its corresponding increase in fluorescence will be evident in both the drug containing and the drug free tube 51 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual The MGIT 960 system monitors these growth patterns and can automatically interprets results as susceptible or resistant An isolate is defined as resistant if 196 or more of the test population grows in the presence of the critical concentration of the drug BACTEC MGIT 960 AST overview When the AST feature is enabled in the MGIT 960 instrument the Positive
191. thin the defined range for the specific piece of equipment environment Corrective actions If the temperature reading is not acceptable a Adjust temperature control and monitor until correct range is achieved if temperature has not returned to normal within the next working day notify supervisor immediately to decide on further corrective action b If equipment is not functioning notify supervisor immediately so service can be instituted i Relocate specimens reagents etc as applicable to a functioning piece of equipment environment with proper temperature range Documentation a Record temperatures on Appendix D Equipment Temperature Record Form b Document minor adjustments and corrective actions on Appendix D c Document all major unacceptable results e g equipment failure corrective actions and resolutions on an Appendix K form 16 4 2 Equipment Cleaning Maintenance Required 16 4 2 1 Equipment cleaning Keeping laboratory equipment clean and performing recommended routine maintenance are essential for accurate performance of laboratory tests and maintaining the longevity of equipment Suggested equipment cleaning activities are given in the table below however the manufacturers specific recommendations and procedures should be followed Maintain documentation for the performance of routine cleaning in a lab worksheet or logbook 91
192. tube carrier for the AST set you are testing The number of tubes in the set is encoded in the carrier s barcode Using a carrier of the incorrect size will cause the system to interpret the absence of a tube as an error which invalidates the results for the entire AST set 12 2 Preparation of Drug Stocks for Susceptibility Testing 12 2 1 SIRE and PZA Drug Stock Preparation Drug stocks and preparation of MGIT tubes must be carried out inside the biosafety cabinet using full PPE Reconstitute the drugs with the appropriate volume of diluent Volumes vary with different drugs Failure to use the appropriate volume will invalidate these tests Refer to Table 12 1 Drug Concentrations for DST in MGIT below 58 Mycobacteriology Laboratory Manual 54 Mycobacteriology Laboratory Manual Task Instructions Prepare drugs in MGIT 960 1 Reconslitule each Streptomycin lyophilized drug vial with 4 ml of sterile SIRE Kit distilled deionized water to make a stock solution of 83pg ml 2 Reconstitute each Isoniazid lyophilized drug vial with 4 ml of sterile distilled deionized water to make a stock solution of 8 3yg ml 3 Reconstitute each Rifampicin lyophilized drug vial with 4 ml of sterile distilled deionized water to make a stock solution of 83pg ml 4 Reconstitute each Ethambutol lyophilized drug vial with 4 ml of sterile distilled deionized water to make a stock solution of 41 5yg ml Prepare drugs MGIT 960 1 R
193. ture section on Appendix B Specify the ID test used to rule out GenoType Mycobacterium CM or Genolype MTBDRplus 4 Record any comments in the MGIT or LJ culture section on Appendix B 71 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 14 LONG TERM STORAGE OF ISOLATES Purpose This section provides detailed instructions on the proper preparation and freezing of MTB isolates Principle MTB isolates from all positive cultures using the LJ subculture from a MGIT tube positive for MTB or a subculture from the original LJ culture if MGIT is unavailable will be frozen in 7H9 broth plus glycerol to preserve them for any repeat or additional microbiology tests that may need to be performed either in house or at a central laboratory It is recommended that at least four aliquots from baseline visits and two aliquots from subsequent visits be frozen for each MTB isolate Procedure Materials Positive LJ slants from Section 11 Solid Culture Lowenstein Jensen LJ Media e 2 ml sterile cryotubes with screw top externally threaded Water bath 7H9 media with glycerol ADC or OADC enrichment broth Sterile transfer pipettes with graduations marking volume individually wrapped Sterile loop disposable applicator stick or cotton swab Permanent marker Cryobox and rack Study specific labels Forms Appendix B Study Source Document Worksheet Append
194. ty and volume are sufficient for processing 5 Estimate the volume of each specimen by comparing with a 50 ml tube that has been calibrated with known volumes of water This volume must be checked against the volume recorded on Appendix A and adjusted if necessary by drawing a single line through the written volume dating and initialing Record the new volume in the appropriate location on the form 6 Evaluate the quality of each sputum specimen and record on Appendix B Those with noticeably high salivary content will be noted but processed a watery specimen is an indication of high saliva content 7 Compare the date time of collection on Appendix A with the current date time Sputum specimens must be processed within 72 hours of collection 6 1 2 Sputum Specimen 2 site collected sputum these instructions also apply to site collected Sputum Specimen 1 ill Site collected sputum specimens will arrive in a 50 ml tube Upon arrival at the laboratory verify specimen labels with the corresponding Laboratory Specimen Requisition Form and Appendix A Check for any leaks or cracks in the tube If a leak is seen decontaminate the container in an autoclave and discard Contact the site immediately and request that another sputum specimen be collected Record this occurrence in the laboratory Comments section on Appendix A and in the Lab Specimen Logbook Registry The individual receiving the specimens records their name sig
195. ual 100 Mycobacteriology Laboratory Manual REFERENCES 1 Becton Dickinson and Company BACTEC MGIT 960 System User s Manual BD Document Number MA 01 17 Sparks Maryland 2004 06 2 Becton Dickinson and Company BACTEC MGIT 960 AST Instructions BD Document Number MA 0126 Sparks Maryland 2007 01 3 Centers for Disease Control and Prevention National Institutes of Health Biosafety in Microbiological and Biomedical Laboratories HHS Publication No CDC 21 1112 US Government Printing Office Washington 2009 4 Clinical and Laboratory Standards Institute Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters Approved Guideline Third Edition CLS Document M23 A3 Wayne PA 2008 5 Clinical and Laboratory Standards Institute Susceptibility Testing of Mycobacteria Nocardiae and Other Aerobic Actinomycetes Approved Standard Second Edition NCCLS Document M24 A2 Wayne PA 2011 6 Clinical and Laboratory Standards Institute Quality Control for Commercially Prepared Microbiological Culture Media Approved Standard Third Edition NCCLS Document M22 A3 Wayne PA 2004 7 Clinical and Laboratory Standards Institute Laboratory Detection and Identification of Mycobacteria Proposed Guideline CLS Document M48 P Wayne PA 2007 8 Clinical and Laboratory Standards Institute Application of a Quality Management System Model for Laboratory Services Approved
196. udy specimen label and on the Laboratory Specimen Requisition Appendix A and Appendix B forms The study specific labels provided by the Sponsor will be used to label tubes for all subsequent downstream processing of this specimen microscope slides LJ tubes MGIT tubes BAP plates cryotubes etc and will be affixed to the Appendix B form These labels must be completed with the following information lab accession number screening ID number and or subject ID number visit number sputum specimen number 1 or 2 unless specimen is from V2 or V3 site number and applicable date e g the collection date is written on the Appendix B label the date of freezer storage on the cryotube label etc Original Appendix A and Appendix B forms will be stored at the laboratory Send completed copies of Appendix A signed by the laboratory technician and Appendix B signed by the lab supervisor to the study coordinator to be stored with the trial documents 17 Mycobacteriology Laboratory Manual Mycobacteriology Laboratory Manual 8 In addition send a copy of Appendix B to the study coordinator whenever new information is added These routine transmittals allow the study coordinator to update the study database with new microbiology lab information The supervisor or technician must initial and date in the margin next to the new information being sent so updated information is readily evident NOTES All specimens for smea
197. uffer avoiding any solid medium and or contaminants present 4 Vortex the cryovial for 30 seconds to create a uniform suspension 5 For TBc ID be sure the suspension turbidity is adjusted to approximately 0 5 McFarland a Insufficient density in the suspension can lead to false negative results b Subculture to a new LJ slant to obtain adequate growth and repeat testing as necessary NOTES 1 Positive MGIT tubes can be stored at 2 37 and tested in the TBc ID for up to 10 days after positivity testing can be performed for two additional months when tubes are stored at 20 to 8 2 Positive cultures in liquid media and colonies on solid media can be tested with Capilia up to one year when stored at 20 or at 2 to 8 C 13 3 Inoculation of ID Test Device The rapid ID test device should be stored at 2 30 C preferably refrigerated at 2 8 C Direct sunlight excessive humidity and high temperatures should be avoided Foil pouches containing devices should not be opened until test is to be performed Avoid touching the specimen well on the device with your hands 1 If devices are refrigerated bring to room temperature in the foil pouch prior to testing 2 Place the device on a flat surface inside the BSC Remove the rapid ID device from its foil pouch immediately before testing 3 Label one device for each specimen to be tested with a study label containing the identifying information described in Section 6
198. ut log10 organisms Inoculation and Incubation 1 Supplement MGIT medium with Growth Supplement and PANTA as specified in Section 10 Liquid Culture Mycobacteria Growth Indicator Tube MGIT 2 Inoculate one MGIT tube with 0 5 ml of the 1 500 dilution Tube 3 3 Enterthe inoculated tube in the MGIT 960 instrument Take the tube out when indicated positive by the instrument Retrieve data for time to detection 16 4 5 Contamination Rate Assessment Required A monthly assessment of the rates of contamination in MGIT and LJ cultures demonstrates trends of increasing or decreasing contamination and should be monitored 1 Frequency Once a month 2 Results Contamination of liquid and solid cultures occurs a When specimens are inadequately decontaminated because specimens are heavily mucoid or have been improperly stored during transport encouraging bacterial overgrowth b Some highly resistant bacterial species are unaffected by decontamination 3 Corrective Actions Reasonable rates of contamination are unavoidable a If MGIT contamination rate is gt 10 notify the Sponsor immediately for guidance and troubleshooting instructions b If LJ contamination rate is gt 6 notify the Sponsor immediately for guidance and troubleshooting instructions 4 Documentation Report findings on Monthly Data Monitors Form Appendix I Procedure 1 Count the total number of MGIT cultures reported in the month and the
199. ve been removed the instrument will beep 3 times the drawer indicator light extinguishes the barcode scanner turns off and the ok icon appears on the display screen Report Print Unloaded 1 Press print report soft key and select lt unloaded positive tubes gt soft key Positive Tubes 2 When report finishes printing compare unloaded tub discrepancies If tubes report press the lt ok gt soft key When you press the ok soft key the information contained in the report is removed from the database if the tube is not re entered within 5 hours es with report Resolve any NOTES The MGIT instrument will record the date the tube signaled positive and the number of days hours taken to reach positivity TTD Place printouts of results of all positive tubes in the study binder Stain all instrument positive tubes for AFB and subculture to a blood agar plate BAP upon removal from the instrument see procedures in Section 10 6 below n the unlikely event of a broken tube in the instrument close the drawer and turn off the instrument Vacate the room and follow the laboratory s SOP for actions following a spill 10 6 Sampling Tubes for Further Analysis Tubes that signal positive in the MGIT instrument require further analysis to determine the type of growth The tubes should be observed visually MTB growth appears granular and not very turbid while contaminating bacterial grow
200. ve on for 1 hour before turning off If available turn on UV light inside BSC for at least 1 hour 7 3 Specimen Digestion Decontamination il Follow the login procedure for specimen registration described in Section 6 Receipt and Login of Sputum Specimens in the Laboratory Record all specimens processed in a batch using the Lab Processing Worksheet Workbook Also record the technician technologist processing each batch Label slides and tubes using the study specific labels provided with information described in Section 6 2 Login of Sputum Specimens Allow refrigerated specimens and reagents to come to room temperature before testing The ideal maximum volume of a sputum specimen is 10ml If the volume is greater than 10ml vortex the specimen and remove the excess volume with a pipette to bring the total volume to 10 ml prior to processing Using a transfer pipette add to the sputum tube a volume of NaOH NALC Na citrate solution prepared above that is equal to the specimen volume 1 1 volume to the centrifuge tube Use a new sterile pipette for each specimen The final NaOH concentration at this stage is 1 5 Close tube tightly and vortex the suspension until liquefied 15 30 seconds Invert the tube several times so the tube walls and cap are exposed to the NaOH NALC Na citrate solution Start the timer for 15 minutes when the digestion decontamination solution is added to the first specimen Place tube in rack on shaker
201. vity The assay units may differ widely from the actual weight of the powder and often may differ between drug production lots Thus the lab must standardize the antimicrobial solutions based on potency of the individual lot of each drug powder Formula for potency of drug Potency assay purity x active fraction x 1 water content In some cases the potency may be expressed as a percentage The following example shows how to calculate the potency in units of ug mg w w Assay purity 99 896 Measured water content 12 1 Active fraction 10096 Calculation Potency assay purity x active fraction x 1 water content Potency 998 x 1 0 x 1 0 121 877 ug mg or 87 7 The antimicrobial powder must be weighed on an analytical balance that has been calibrated with standard weights If possible weigh more than 100 mg of powder To calculate the appropriate amount of solvent needed to obtain this concentration the potency of the drug powder must be considered is advisable to weigh out a larger amount of the drug than required for the specific concentration and then calculate the volume of solvent needed to obtain the required concentration using the formula below Solvent for drugs Follow the manufacturer s guidelines on the COA for recommendation of solvent If a solvent other than water is recommended only use sufficient solvent to solubilize the antimicrobial powder and then dilute to the f
202. wenty four hours prior to use streak one drop of thawed well mixed E coli or S aureus stock suspension onto a blood agar plate or other appropriate medium and incubate overnight at 35 37 C Inoculation and Incubation 1 Prepare a suspension of colonies from a solid bacteriologic medium in sterile distilled water to McFarland No 0 5 standard Alternatively thaw the stock suspension and dilute to the same density Make 100 fold dilution 10 by adding a 10 ul suspension to 1 ml sterile distilled water Mix well Make another 100 fold dilution using 10 ul of the 107 dilution in 1 ml distilled water Using this 107 dilution inoculate the plate with 10 ul and streak for isolation Incubate at 35 37 for 48 hours and check for growth with typical colony size and morphology 16 2 1 3 Commercially Prepared Media Commercial media are thoroughly tested by the manufacturer and require either less QC testing or are categorized as generally exempt from user quality control according to the US Clinical and Laboratory Standards Institute CLSI Recommendations for New Shipments of Media Upon receipt of each new media shipment check the following 1 Manufacturer s QC records are provided these records must be retained with the lab s QC records 2 Expiration dates notify the vendor of recurring short expiry dates 3 Tubes or plates are not damaged or cracked Mycobacteriology Laboratory Manual 84
203. x on Appendix B In addition note in the MGIT Comments section that test was repeated It is preferable to test pure cultures without contamination although observations show that slight contamination with bacteria does not interfere with the test For MGIT cultures decontaminate the culture and repeat testing if necessary For LJ cultures attempt to subculture a few colonies well separated from the contaminants and repeat testing if necessary However grossly contaminated cultures may cause interference interpret with caution Staphylococcus aureus is known to produce protein A which may interfere and or cause false positive results in all lateral flow assays A negative test does not always rule out MTB as mutations are known to arise in the MPT MPB 64 gene If MTBc is highly suspected and identification results persist in being negative refer to Flowchart 2A for additional testing algorithms using a molecular assay HAIN GenoType Mycobacterium CM or MTBDRplus If the test is invalid Investigate causes for the invalid result and try to resolve e g decontaminating a heavily contaminated culture Repeat the test e f test remains invalid refer to Flowchart 2A for additional testing algorithms using a molecular assay HAIN GenoType Mycobacterium CM or Genolype MTBDRplus Record observations for invalid or uninterpretable results e g a contaminated culture 13 5 Reporting of Id
204. xpiry Date Phosphate Name Prepared p d Prepared puffer nly Use Initials Reagent Spore ilie Monthly Supervisor Review date initials New Lot Batch Reagents Brand Received Expiry Quantity Date Component Manufacturer Lot Number Date Date Received Opened NALC Sodium hydroxide Sodium citrate Disodium phosphate Monopotassium phosphate Date format dd mmm yyyy Mycobacteriology Laboratory Manual MGIT Results Date N nd 7 Supplement Other Result ie Number Se mos 975 Strains Tech Initials 9 Monthly 1MTB strain Other Supervisor esed strains Review if tested date initials New Lot Batch Media Component Brand Received Expiry Quantity Date Put Manufacturer Lot Number Date Date Received in Use BD 7 ml tubes BD MGIT 7 ml tubes BD MGIT 7 ml tubes Date format dd mmm yyyy 123 Mycobacteriology Laboratory Manual 124 Appendix E New Reagents Media QC Form MGIT Supplement Supplement MGIT Media Mar Results MTB Erud RE GC pass Lot Number Lot Number Tech Initials 6 10 days Strains Tech Initials Yes No Monthly strain Other strains tested if tested date initials New Lot Batch Reagents
205. y are described in Section 16 Quality Assurance 41 Mycobacteriology Laboratory Manual 42 Mycobacteriology Laboratory Manual 10 13 Quality Control Quality Monitoring of MGIT Instrument Perform daily functional and temperature checks of the MGIT instrument and record on the MGIT Maintenance Log Each month run the MGIT QC Report a report generated by the MGIT 960 instrument which lists the status of all the detectors in the instrument along with the date and time of their last verification The report also lists all manually blocked stations Print out the report and maintain in the laboratory files 10 14 Study Data Reporting All liquid culture data date of inoculation date of MGIT result culture result original time to detection in days and hours from the MGIT printout final time to detection in days and hours the final TTD would only differ from the original if reincubation was required and Appendix M completed or if a negative culture was incubated longer than 42 days 0 hours ZN and ID results final date of MGIT culture completion and any re decontamination are reported on Appendix B Culture results and TTD are reported according to the guidelines listed in section 10 11 above NOTES 1 Date of MGIT result on Appendix B should be the date the actual culture result was determined This will be the date on the MGIT printout from the original incubation of the culture or if the culture was re
206. ypical growth characteristics and ZN staining properties o MOTT Report as No MTB complex growth but positive for other mycobacteria 2 If growth appears to be or resembles MTB and the MPT MTB 64 antigen test is indeterminate due to presence o contaminants presumptively ID the culture by looking for typical growth characteristics and ZN staining properties of MTB complex and report as Positive for MTB and contaminated 49 Mycobacteriology Laboratory Manual 50 Mycobacteriology Laboratory Manual NOTE In case of either 1 or 2 above leave the ID test method blank and write a comment in the LJ Culture Comments section such as Rapid ID test result indeterminate due to contamination presumptive identification made for culture 11 5 Logging in U Cultures for Isolate Storage Bank Ensure growth is consistent with MTB prior to storing for short term storage If this tube is removed to conduct additional testing a new subculture must be prepared to ensure there is always a short term storage tube 1 Allisolates grown on the LJ subculture from the MGIT culture are stored in numerical order according to screening ID number and or subject ID number sputum specimen number lab accession number and visit number These isolates should be stored at room temperature preferred or in the refrigerator for at least one year from the date of preparation 2 Seal the cap of the U tubes with parafilm Be sure that tubes are c
207. yyy Final date of MGIT culture completion dd mmm yyyy Tech initials Comments AFB Acid Fast Bacilli BAP Blood Agar Plate ZN Ziehl Neelsen Storage of Specimens Was specimen banked for short term Was specimen banked for storage medium Yes No N A long term storage at 70 C Yes N A Supervisor signature My signature confirms that all data have been reviewed for accuracy and that all pre filled information on this form is accurate for this specimen Screening Subject iG 1 ID ID 4 Visit Site Sputum Specimen 10 20 N AQ Onlytick N A for V2 or LJ Culture of Sputum Date of inoculation Date of U result dd mmm yyyy dd dd mmm yyyy Result tick box E Negative TB growth growth TB growth growth No TB Contami Positive Unknown 1 9 colonies 10 100 more than innumerable growth nated for MTB colonies 100 colonies or confluent but positive complex and growih for other contaminated mycobacteria Confirmatory ZN Stain Results Tick one box O Negative for AFB Positive for AFB ON A Identification of AFB jo for Positive ID Test 4 Other Tech in

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