1 Global Laboratory initiative advancing TB diagnosis First Edition, April 2014A publication of the Global Laboratory Initiativea Working Group of the Stop TB PartnershipMycobacteriology Laboratory 111/04/2014 16:39 Mycobacteriology Laboratory ManualiThis 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).
2 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 fi rst 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 current version of the :Kelly W. Stinson, Clinical Manager - Microbiology, Otsuka Pharmaceutical Development & Commercialization, Inc.
3 (OPDC)Kathleen Eisenach, University of Arkansas for Medical SciencesSusan Kayes, OPDCM akoto Matsumoto, Otsuka Pharmaceutical Co, Ltd. - Japan (OPCJ)Salman Siddiqi, Consulting MycobacteriologistSuguru Nakashima, OPCJH iroyuki Hashizume, OPCJJ uliano Timm, OPDCAnne Morrissey, OPDCM arra Mendoza, Otsuka Manila Research Center (OMRC)Princy Mathai, OPDCA cknowledgement: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 Directors/Managers:Jun Yue, Shanghai Pulmonary Hospital Clinical Laboratory , Shanghai, ChinaZhao Yanlin, Tuberculosis Reference Laboratory of China, Beijing, China Mona Abdelmonis Khalifa, El Seka El Baidaa-Abbassia Chest Hospital, Cairo, EgyptTiina Kummik, Tartu University Clinics United Laboratories, Dep of Mycobacteriology , Tartu, EstoniaKlavdia Levina, North Estonian Medical Centre,Diagnostica clinics, Laboratory , Mycobacteriology Section, Tallinn, EstoniaAkio Aono, Japan Anti-Tuberculosis Association Fukujuji Hospital, Tokyo, JapanMotohisa Tomita, Kinki-chuo Chest Medical Center, National Hospital Organization, Osaka, JapanSungweon Ryoo, Molecular Mycobacteriology Unit, Korean Institute of Tuberculosis, Seoul, Korea Chang Ki Kim, Molecular Mycobacteriology Unit.
4 Korean Institute of Tuberculosis, Seoul, Korea Nackmoon Sung, Clinical Research Center (CRC), National Masan TB Hospital , Masan, KoreaGirts Skenders, State Agency Infectology Center of Latvia , Clinic of Tuberculosis and Lung Diseases, Riga, LatviaJurgita Kazlauskaite, Public Institution Republican Siauliai Hospital Subsidiary Hospital of Tuberculosis and Lung Diseases, Siauliai, LithuaniaSandra Svambarytes, Public Institution Republican Siauliai Hospital Subsidiary Hospital of Tuberculosis and Lung Diseases, Siauliai, LithuaniaFront page photo credit: Edwin Cortero i11/04/2014 17:3711/04/2014 17:37 Mycobacteriology Laboratory ManualiiEdita Pimkina, Infectious Diseases and Tuberculosis Hospital Affi liate of Public institution Vilnius University Hospital, Vilnius, LithuaniaElena Romancenco, Public Medical Sanitary Institution (PMSI) Institute of Phtysiopneumology Chiril Draganiuc , Blufstein Laboratorio Clinico, Chisinau, Moldova Karina Vidarte, Blufstein Laboratorio Clinico, Lima, PeruGrace Egos, Tropical Disease Foundation, Inc, Manila, Philippines (in memoriam)
5 Thelma Tupasi, Tropical Disease Foundation, Inc, Manila, PhilippinesHenry Evasco, Tropical Disease Foundation, Inc, Manila, PhilippinesMara Gibson, Clinical Laboratory Services, Johannesburg, South AfricaSaloshini Natalie Ramsamy, Clinical Laboratory Services, Johannesburg, South AfricaAnn Acker, Women s Health Laboratory , San Antonio, Texas, United States of AmericaMicrobiology Consultants:Qinghua Zou, Beijing, China; Junqi Zhang, Shanghai, China; Cheng Wang, Beijing, China; Somaia Abdel Latif Eissa, Cairo, Egypt; Erki Sala, Kohtla-J rve, Estonia; Karmen Laurson, Tallinn, 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; Jolanta Miciuleviciene, Vilnius, Lithuania; Valentina Vorojbit, Chisinau, Moldova; Ivan Sabogal, Lima, Peru; Esperanza Cabrera, Manila, Philippines; Delia Ontengco, Manila, Philippines; Kavindhran Velen, Pretoria, South AfricaThe 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 fi nal version of this Manual .
6 Karin Weyer, Christopher Gilpin, Jean Iragena, 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 Iademarco and Richard ii11/04/2014 17:3711/04/2014 17:37 Mycobacteriology Laboratory ManualiiiTABLE OF CONTENTSLIST OF ABBREVIATIONS AND ACRONYMS V1 INTRODUCTION 12 SAMPLE SPECIMEN TIMETABLE 63 SPECIMEN FLOWCHART 74 Laboratory BIOSAFETY AND INFECTION CONTROL 85 SPECIMEN COLLECTION, STORAGE, AND TRANSPORT 116 RECEIPT AND LOGIN OF SPUTUM SPECIMENS IN THE Laboratory 157 PROCESSING SPUTUM FOR SMEAR MICROSCOPY AND QUALITATIVE CULTURE 198 ACID-FAST BACILLI MICROSCOPY (AFB) PREPARATION AND STAINING 259 ACID-FAST BACILLI MICROSCOPY (AFB) EXAMINATION 2910 LIQUID CULTURE MYCOBACTERIA GROWTH INDICATOR TUBE (MGIT) 32 Flow Chart 1: General Algorithm MGIT 960 Cultures 43 Flow Chart 2: Contaminated MGIT Cultures 44 Flow Chart 2A: Suspected MOTT Cultures 45 Flow Chart 3.
7 MGIT Early Positive Cultures (ZN/BAP negative) 4611 SOLID CULTURE: LOWENSTEIN JENSEN (LJ) MEDIA 4712 DRUG SUSCEPTIBILITY TESTING: MGIT SYSTEM 51 Flow Chart 4: Invalid x200 Errors from MGIT DST 65 Flow Chart 5: Invalid x400 Errors from MGIT DST 6613 RAPID IDENTIFICATION OF M. TUBERCULOSIS COMPLEX USING AN IMMUNOCHROMATOGRAPHIC ASSAY 6714 LONG-TERM STORAGE OF MTB ISOLATES 7215 SHIPPING OF FROZEN ISOLATES 7516 QUALITY ASSURANCE 77 REFERENCES iii11/04/2014 17:3711/04/2014 17:37 Mycobacteriology Laboratory ManualivAPPENDIX A: SPECIMEN TRANSFER FORM 101 APPENDIX B: STUDY SOURCE DOCUMENT WORKSHEET 102 APPENDIX C: DAILY QC STAINING FORMS 104 APPENDIX D: EQUIPMENT TEMPERATURE RECORD FORMS 108 APPENDIX E: NEW REAGENTS/MEDIA QC FORMS 115 APPENDIX F: DST QC/INVENTORY FORMS 131 APPENDIX G: MTB IDENTIFICATION QC FORM - ICA SLIDE TEST MPT64/MTB 64 135 APPENDIX H: WEEKLY MGIT/LJ CULTURE QC FORM 136 APPENDIX I: MONTHLY DATA MONITORING FORMS 137 APPENDIX J: STORAGE LOG FORMS 142 APPENDIX K.
8 CONTINUOUS QUALITY IMPROVEMENT FORM 144 APPENDIX L: MGIT TTD WORKSHEET 145 APPENDIX M: EARLY MGIT POSITIVES/ EARLY CONTAMINATED CULTURES - TRACKING WORKSHEET 146 APPENDIX N: Laboratory VISITOR LOG iv11/04/2014 17:3711/04/2014 17:37 Mycobacteriology Laboratory ManualvLIST OF ABBREVIATIONS AND ACRONYMSAFB Acid fast bacilliAST Antimycobacterial susceptibility testing; see DSTBAP Blood agar plateBD Becton DickinsonBSC Biological Safety CabinetBSL Biosafety levelCOA Certifi cate of analysisCDC US Centers for Disease Control and PreventionCFR US Code of Federal RegulationsCFU Colony forming unitCLSI Clinical Laboratory Standards InstituteCRA Clinical Research AssociateCRC Clinical Research CoordinatorDST Drug susceptibility testing.
9 See ASTEMB EthambutolFIND Foundation for Innovative New DiagnosticsGC Growth controlGU Growth unitICA Immunochromatographic assayINH IsoniazidIUATLD International Union Against Tuberculosis and Lung DiseaseLJ L wenstein-Jensen MDR-TB Multidrug resistant tuberculosisMTB Mycobacterium tuberculosis complexMGIT Mycobacteria Growth Indicator TubeMOTT Mycobacteria other than TBNALC N-acetyl-L-cysteineNaOH Sodium HydroxideNTF Note to FileNTM Nontuberculous mycobacteriaPANTA Antibiotic supplement for MGIT v11/04/2014 17:3711/04/2014 17:37 Mycobacteriology Laboratory ManualviPCR Polymerase chain reactionPPE Personal protective equipmentPZA PyrazinamideOADC Oleic acid, albumin, dextrose, and catalaseQA Quality AssuranceQC Quality ControlQI Quality ImprovementQM Quality ManagementRIF RifampicinSIRE Streptomycin, isoniazid, rifampicin, ethambutolSOP Standard Operating ProcedureSTR StreptomycinTB TuberculosisTIP Time in protocol (from the MGIT printout)TNTC Too numerous to countTSA Trypticase Soy AgarTTD Time to detectionUV UltravioletWHO World Health OrganizationZN Ziehl-Neelsen vi11/04/2014 17:3711/04/2014 17.
10 37 Mycobacteriology Laboratory Manual11 INTRODUCTIONThe 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 effi cacy 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 for clinical trials for the treatment of multidrug-resistant tuberculosis (MDR-TB).