GLI Mycobacteriology Laboratory Manual

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

Cover_mycobacteriology_lab.indd 1 11/04/2014 16:39 ggli_mycobacteriology_lab_manual_quadri.indd i l i _ m y c o b a c t e r i o and LungDiseases,Siauliai,Lithuania Sandra Svambarytes,PublicInstitutionRepublicanSiauliaiHospitalSubsidiary HospitalofTuberculosis and LungDiseases,Siauliai,Lithuania Jurgita Kazlauskaite,PublicInstitutionRepublicanSiauliaiHospitalSubsidiary HospitalofTuberculosis Riga, Latvia Girts Skenders,StateAgency“InfectologyCenterofLatvia”,ClinicTuberculosis andLungDiseases, Nackmoon Sung,ClinicalResearch Center(CRC),NationalMasanTBHospital,Masan,Korea Chang KiKim,MolecularmycobacteriologyUnit,Korean InstituteofTuberculosis, Seoul,Korea MolecularmycobacteriologyUnit,Korean InstituteofTuberculosis,Sungweon Ryoo, Seoul,Korea Motohisa Tomita, Kinki-chuo ChestMedicalCenter, NationalHospitalOrganization,Osaka,Japan Akio Aono,JapanAnti-Tuberculosis AssociationFukujujiHospital,Tokyo, Japan Section, Tallinn, Estonia Klavdia Levina,NorthEstonianMedicalCentre,Diagnostica clinics,Laboratory, Mycobacteriology Kummik,TartuTiina UniversityClinicsUnitedLaboratories,DepofMycobacteriology, Tartu, Estonia Mona AbdelmonisKhalifa,ElSekaBaidaa-AbbassiaChestHospital,Cairo, Egypt Zhao Yanlin, Tuberculosis Reference LaboratoryofChina,Beijing,China Jun Yue, ShanghaiPulmonaryHospitalClinicalLaboratory, Shanghai,China Lab Directors/Managers: laboratory andthein-countrymicrobiology consultants. work, dedication,andinputofthelaboratorydirectors/managers andstaff from eachtrial The Otsukaglobalmicrobiology program couldnothavebeenimplementedwithoutthehard Acknowledgement: Princy Mathai,OPDC Marra Mendoza,OtsukaManilaResearch Center(OMRC) Anne Morrissey, OPDC OPDC Juliano Timm, Hiroyuki Hashizume,OPCJ Suguru Nakashima,OPCJ Salman Siddiqi,ConsultingMycobacteriologist Makoto Matsumoto,OtsukaPharmaceuticalCo,Ltd.-Japan(OPCJ) Susan Kayes,OPDC Kathleen Eisenach,UniversityofArkansasforMedicalSciences Commercialization, Inc.(OPDC) Kelly W. Stinson,ClinicalManager-Microbiology, OtsukaPharmaceuticalDevelopment& Editor: prompted severalrevisions incorporatedinthiscurrent versionofthedocument. Otsuka trialsnetwork,aswellreview ofdatageneratedfrom theclinicaltrialsthemselves, ﬁ TBlaboratories,draftedthe technologist withclinicalexperienceworkinginseveralinternational TBresearch,associate (CRA)withpriorexperienceininternational andaseniorlaboratory mycobacteriology laboratory-basedresearch settings,aseniorclinicalresearch ininternational A specializedOtsukateam,includingamicrobiology expertwithextensiveexperiencein (sputum culture conversioninliquidandsolidmedia). focus ontestingthathasthegreatest impactonmicrobiology endpointsforMDR-TBclinicaltrials standardized procedures related tosputumcollection,handling,analyses,andreporting, witha rst versionofthismanual.Inputfrom multiplelabdirectors/managers andconsultantsinthe l o g y Front pagephotocredit:EdwinCorteroTuyay. tuberculosis (MDR-TB)clinicaltrialsforthedevelopmentofDeltyba Pharmaceutical DevelopmentandCommercialization (OPDC)-sponsored multidrug-resistant tuberculosisa networkofinternational laboratorieshandlingsputumspecimensforOtsuka This documentwasdevelopedtoensure highqualityresults andcomparabilityofdatafrom _ l a b _ m a n u a l _ q u a d r i . i n d d

i ® (delamanid).Itconsistsof 111/04/2014 17:37 1 / 0 4 / 2 0 1 i 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd ii l i _ m y ii c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Richard Lumb. Cleeff, LuciaBarrera, DanielaCirillo,HaraldHoffmann, SvenHoffner, MichaelIademarco and Alexander, ArmandVan Deun,AmyPiatek,SabineRüsch-Gerdes, MariaAliceTelles, Maartenvan Zallocco, FuadMirzayev, Wayne Van Gemert,Tom Shinnick,RickO’Brien,RuminaHasan,Heather endorsing theﬁ from theWHO/GLITBSupranationalReference LaboratoryNetworkare thankedforreviewing and The GlobalLaboratoryInitiative(GLI)Secretariat andCore Group membersaswellrepresentatives Manila, Philippines;KavindhranVelen, Pretoria, SouthAfrica Moldova; IvanSabogal,Lima,Peru;EsperanzaCabrera, Manila,Philippines;DeliaOntengco, Lithuania;Valentina Lithuania;JolantaMiciuleviciene,Vilnius, Kiveryte, Vilnius, Vorojbit, Chisinau, Ho Roh,Daejeon,Korea; ArtaBalode,Riga,Latvia;GintarasMakstutis,Siauliai,Lithuania;Silvija Maris Kauk,Tallinn, Estonia;Toru Mori,Tokyo, Japan;HyukminLee,Kyunggi-do,Korea; Kyoung Abdel LatifEissa,Cairo, Egypt;ErkiSala,Kohtla-Järve,Estonia;KarmenLaurson,Tallinn, Estonia; Qinghua Zou,Beijing,China;JunqiZhang,Shanghai,ChengWang, Beijing,China;Somaia Microbiology Consultants: Ann Acker, Women’s HealthLaboratory, SanAntonio,Texas, UnitedStatesofAmerica Saloshini NatalieRamsamy, ClinicalLaboratoryServices,Johannesburg,SouthAfrica Mara Gibson,ClinicalLaboratoryServices,Johannesburg,SouthAfrica Henry Evasco,Tropical DiseaseFoundation,Inc,Manila,Philippines Thelma Tupasi, Tropical DiseaseFoundation,Inc,Manila,Philippines Grace Egos,Tropical DiseaseFoundation,Inc,Manila,Philippines(inmemoriam) BlufsteinLaboratorioClinico,Lima,Peru Karina Vidarte, “Chiril Draganiuc”,BlufsteinLaboratorioClinico,Chisinau,Moldova Elena Romancenco,PublicMedicalSanitaryInstitution(PMSI)InstituteofPhtysiopneumology Lithuania University Hospital,Vilnius, Edita Pimkina,InfectiousDiseasesandTuberculosis HospitalAfﬁ _ m a n u a l _ q u a d r i . i n d nal versionofthismanual.KarinWeyer, ChristopherGilpin,JeanIragena,Diego d

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd iii l i _ m y c o b a c t e r i o l o g y 6QAIYASRNE 77 75 72 REFERENCES 16 QUALITY ASSURANCE SHIPPINGOFFROZENISOLATES 15 LONG-TERMSTORAGEOFMTBISOLATES 14 lwCat5 nai 40Err rmMI S 66 65 51 45 47 44 13 5:Invalidx400ErrorsfromMGITDST FlowChart 4:Invalidx200ErrorsfromMGITDST FlowChart 46 43 DRUGSUSCEPTIBILITYTESTING:MGITSYSTEM 12 SOLIDCULTURE: LOWENSTEINJENSEN(LJ)MEDIA 11 3:MGIT“EarlyPositive”Cultures(ZN/BAPnegative) FlowChart 2A:SuspectedMOTTCultures FlowChart 29 2:ContaminatedMGITCultures FlowChart 1:GeneralAlgorithmMGIT960Cultures FlowChart 32 1 7 6 LIQUIDCULTURE 25 –MYCOBACTERIAGROWTHINDICATOR TUBE(MGIT) 11 10 ACID-FAST BACILLIMICROSCOPY(AFB)EXAMINATION 8 9 15 ACID-FAST BACILLI MICROSCOPY(AFB)PREPARATION ANDSTAINING 8 19 V PROCESSINGSPUTUMFORSMEARMICROSCOPYANDQUALITATIVE CULTURE 7 RECEIPTANDLOGINOFSPUTUMSPECIMENSINTHELABORATORY 6 SPECIMENCOLLECTION,STORAGE,ANDTRANSPORT 5 LABORATORY BIOSAFETYANDINFECTIONCONTROL 4 3 SPECIMEN SAMPLESPECIMENTIMETABLE FLOWCHART 2 1 INTRODUCTION LIST OFABBREVIATIONS ANDACRONYMS TABLE OFCONTENTS _ l a b _ m SN NIMNCRMTGAHCASY 67 USING ANIMMUNOCHROMATOGRAPHIC ASSAY RAPID IDENTIFICATION OF a n u a l _ q u a d r i . i n d d

i i i M. TUBERCULOSIS COMPLEX 100 111/04/2014 17:37 1 / 0 4 / 2 0 1 iii 4

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd iv l i _ m y iv c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b PEDXN LBRTR IIO O 147 145 142 144 137 APPENDIX N: LABORATORY VISITORLOG 136 131 APPENDIX M: MGITTTDWORKSHEET APPENDIX L: CONTINUOUSQUALITYIMPROVEMENT FORM APPENDIX K: 115 104 STORAGELOGFORMS APPENDIX J: MONTHLY DATA MONITORING FORMS APPENDIX I: 108 101 WEEKLY MGIT/LJCULTURE QCFORM APPENDIX H: 135 MTBIDENTIFICATION QCFORM-ICASLIDETESTMPT64/MTB64 102 APPENDIX G: DSTQC/INVENTORY FORMS APPENDIX F: NEWREAGENTS/MEDIAQCFORMS APPENDIX E: EQUIPMENTTEMPERATURE RECORDFORMS APPENDIX D: DAILY QCSTAINING FORMS APPENDIX C: STUDYSOURCEDOCUMENTWORKSHEET APPENDIX B: SPECIMENTRANSFERFORM APPENDIX A: _ m a n u a l _ q u a d r i . i n d RCIGWRSET 146 TRACKING WORKSHEET EARLY MGITPOSITIVES/EARLY CONTAMINATED CULTURES - d

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd v l i _ m y c o b a c t e r i o l o g y PANTA NTM NTF NaOH NALC MOTT MGIT MTB MDR-TB LJ IUATLD INH ICA GU GC FIND EMB DST CRC CRA CLSI CFU CFR CDC COA BSL BSC BD BAP AST AFB LIST OFABBREVIATIONS ANDACRONYMS _ l a Löwenstein-Jensen b Growth control Becton Dickinson Growth unit Immunochromatographic assay _ Biosafety level Isoniazid USCodeofFederalRegulations NotetoFile BiologicalSafetyCabinet ClinicalResearch Coordinator Colonyformingunit Acidfast bacilli Drugsusceptibilitytesting;seeAST Antimycobacterialsusceptibilitytesting;seeDST Bloodagarplate ClinicalResearch Associate USCentersforDiseaseControl andPrevention ClinicalLaboratoryStandards Institute m Ethambutol Certiﬁ Nontuberculous mycobacteria FoundationforInnovativeNewDiagnostics MycobacteriaGrowth IndicatorTube N-acetyl-L-cysteine a Sodium Hydroxide MycobacteriaotherthanTB n AntibioticsupplementforMGITtubes International UnionAgainstTuberculosis International andLungDisease u Multidrugresistant tuberculosis a l _ Mycobacterium tuberculosiscomplex q u a d r i . cateofanalysis i n d d

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd vi l i _ m y vi c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b ZN WHO UV TTD TSA TNTC TIP TB STR SOP SIRE RIF QM QI QC QA OADC PZA PPE PCR _ m Quality Improvement Tuberculosis Ziehl-Neelsen stain a Ultraviolet Rifampicin Quality Control Time inprotocol (from Time theMGITprintout) Quality Assurance Personalprotective equipment n Quality Management Streptomycin Polymerasechainreaction Time todetection Time Trypticase SoyAgar Pyrazinamide Standard OperatingProcedure Streptomycin, isoniazid,rifampicin,ethambutol u Too numerous tocount World HealthOrganization a Oleicacid,albumin,dextrose, andcatalase l _ q u a d r i . i n d d

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 1 l i _ m y c o b a c t e r i o l o g y provide clarifi A NotetoFile(NTF)isadocumentuseddescribeanerror (i.e.protocol/lab manualdeviation)or NotestoFiles 1.1.2 All originalsource documentsmustbemaintained;donotdestroy ordiscard theserecords. Items thatconstitutesource documentsinclude,butare notlimitedto: i.e., theoriginalrecords where results andobservationsare entered. it mayberecorded, isconsidered ‘source data’.Source dataare containedin‘source documents’, All informationrecorded forthepurposesofstudy, regardless offormorthemediaonwhich 1.1.1 Source StudyData:MaintenanceofDocuments Documents 1.1 detailed QA/QCprocedures, aswellcomprehensive appendicestorecord thesedata. (QC) andqualityassurance(QA)are ofutmostimportanceinthelaboratory. Themanualprovides consistent reporting across laboratories.Finally, asclinicaltrialsare highlyregulated, qualitycontrol In addition,qualityandstandardized reporting ishighlightedthroughout themanualtoensure cultures (positiveMGITsignalwithnegativeconfi charts designedtoaddress scenariossuchasmixedcultures, contamination,andearlypositive investigational compound.Thus,recovery efforts forMGITcultures are highlightedinseveralfl of contaminants,toprovide anaccuratepicture ofthemicrobiological performanceofthe The underlyinggoalofthelaboratorymanualistomaximizerecovery ofMTB,eveninthepresence required forthetest,alongwithapplicablequalitycontrol procedures. Disease Control andPrevention (CDC).Eachprocedure liststhematerials,equipment,andforms biosafety recommendations from theWorld HealthOrganization(WHO)andtheUSCentersfor and laboratoryprocedures from theNationalInstitutesofHealthTBResearch Unit,aswell Institute (CLSI),theFoundationforInnovativeNewDiagnostics(FIND) The laboratorymanualreferences bestpracticesfrom theClinicalandLaboratoryStandards tuberculosis (MDR-TB). randomized, placebo-controlled, PhaseIIforclinicaltrialsthetreatment ofmultidrug-resistant that standardizes keylaboratoryprocedures wasdevelopedandimplementedforamulti-country, and comparabilityofdatafrom allparticipatinglaboratories,acomprehensive laboratorymanual culture conversion(SCC)of trials fornewtuberculosis drugsbaseeffi laboratory facilitiesworldwide,usingavarietyofmethods,equipment,andcapacity. Clinical The diagnosis,treatment, andmonitoringoftuberculosis are conductedinawiderangeof 1 INTRODUCTION _ l a b _ m a n • QA/QCrecords • StudySource Document,AppendixB • SpecimenTransfer Form,Appendix A • • u a and DSTreports Data generatedfrom automatedinstruments;e.g.,MGITunloadedpositive,negative, (AFB smearlog,DSTworksheets,QCrecords, etc.) Manually-collected datasuchaslabrequisitions, labregisters, andlaboratoryworksheets l _ q u cation for aresult. TheSponsor(an individual,institution,companyororganization a d r i . i n d d

1 Mycobacterium tuberculosis cacy on microbiologic endpoints,such assputum rmatorytests). (MTB).To ensure highqualityresults MGIT ProcedureManual, ow 111/04/2014 17:37 1 / 0 4 / 2 0 1 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 2 l i _ m y 2 c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b NTFs mustbefi template withthefollowingthree sections: that takestheresponsibility toinitiate,manageorfi For thoselaboratoriesusingcomputerizedsystemsthatare confi they mustbestored inamannerthatfacilitateseasyretrieval andreview, whileprotecting the Since laboratorydocumentsare subjecttoreview byCRAs,auditors,andregulatory inspectors, and labeling,greatly assistsinthesuccessful managementofthestudy. The timelyfi DocumentFilingandAccess 1.1.4 and/or theSponsor. and aNTF. Ifthere isanyuncertaintyaboutwhichformtocomplete,discusswiththelabdirector example ofhowtocompletethisform.Insomecases,asituationmayrequire bothanAppendixK any qualityimprovements madebythelaboratory. SeeSection16.5:QualityImprovement foran An AppendixKformisrequired whenaQCtestfailstogiveproper results. Itisalsousedtodocument AppendixKForms 1.1.3 capture andretain source documentselectronically, thefollowingprocedure mustbefollowed: tabs, listedbelow. Ifadditionalbindersare required, pleasenotifyyourCRA. The Microbiology LabWorksheets binderprovided bytheSponsormustcontainatleastseven _ m a n dentialityofallpatients(studyandnon-study). u a l _ • Resolutionoftheissue(s) • Stepstakentomitigatetheissue(s) • Descriptionoftheissue(s) • • • • q u written ininkorbymeansofapre-printed label: The followinginformationmustbecaptured onthelastpageofprintouteither Each pageoftheprintoutmustbeinitialedanddatedbylabdirector ordesignee. subject IDnumber, dateofprintout,andclearpagenumbering. and visitinterval,inthelabbinder. Paperprintoutsmustidentifythescreening and/or The labmustensure thatalldataforeachsubjectare printedoutandfi printed andstored appropriately. maintained). Ifqualitycontrol records are stored electronically, thesedatamustalsobe (e.g., onlyauthorizedindividualshaveaccesstothesystemandanaudittrailis (including location/documentationofeachdatapoint)andhowthesystemiscontrolled The labdirector ordesigneemustverify(inwriting)thedatathatsystemcaptures documented bytheappropriate laboratorystaff. data entryperson,andsitemonitorofthemodifi ( initial anddatethechange.Theprevious printoutmustremain inthelaboratorybinder appropriate page/sectionmustbeprintedagainandthelabdirector ordesigneemust Each timemodifi − Astatementthatreads: ‘Iconfi − Dateandsignature ofthelabdirector ordesignee − Total numberofpagesin theprintout data captured forthissubjectasoftoday’. a do notdiscard d ling ofessentialdocumentsinthelaboratory, usinganorganizedsystemofindexing r i . i n ledinthestudylabbinder. d d

2 ). Thelabdirector ordesigneemustalsoinformthesitecoordinator, cations are madetodatathathaspreviously beenprinted,the rm thatthecontents ofthisprintoutfullyreﬂ nance aclinicaltrial)musthavestandard cations. This notiﬁ This cations. not 21CFRPart11compliantto cation should be should cation led, bypatient ect the ect 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 3 l i _ m y c o b a c t e r i o l o g y QC Forms Appendix B-StudySourceDocumentWorksheets Appendix A-SpecimenTransfer Forms VisitorLog Appendix N-Laboratory _ l a b _ m a n • • • • • • • • • • • • • • u a Examples ofdocumentsthatmaybefi the binder, particularlyattheconclusion ofthetrial. information related tostudyspecimensshouldbefi be placedunderthe“QCForms”tabofbinder, oracopyoftheformscontaining that system.However, eitheranote-to-fi If thelabhasaseparatefi Appendix Bform. MGIT printoutsandASTReportscanalsobefi accurate. the specimenhavebeencompleted,tosignifythatdatahasreviewed andis Supervisor mustsignanddatetheformindesignatedspaceswhenalltestsfor the marginnexttonewinformationthatisbeingsentsite. material fortheCRC;technicianorsupervisormustsigntheirinitialsanddatein Remove theformfrom thebinderonlywhennecessarytomakeacopywithupdated manner. Update informationfrom labsource registers andworksheetsontotheforminatimely volume ofspecimens. Insert ‘tabs’toseparatepatientsbySubjectIDs,especiallywhenthelabreceives alarge sequentially byvisitinterval. File theoriginalforminlaboratorybinder(s)consecutivelybySubjectID,and according tosubjectIDandvisitinterval. Alternatively, fi receives alargevolumeofspecimens. Insert additional‘tabs’toseparatepatientsbySubjectIDs,especiallywhenthelab (CRC) forinclusioninthesubject’s binder. Upon completionofeachform,copyandsendittotheClinicalResearch Coordinator tab bySubjectIDandvisitnumber. File theoriginalforminlaboratorybinder(s)under“SpecimenTransfer Form” site staff. include,butareVisitors notlimitedto:auditors,CRAs,Sponsorrepresentatives, and countersign foreachvisitor. N) withthedateandpurposeofvisit.Thelaboratorydirector ordelegatemust Each timesomeonevisitsthelabtodiscussatrial,s/hemustsignvisitorlog(Appendix l _ q u • Monthlydatamonitoringforms/worksheets • Profi • Equipmentmaintenancerecords • MediaQCforms a d r i . i n d ciencytestingresults d

3 le theAppendixAforminfront oftheappropriate AppendixBform ling systemforthesedocuments,itisacceptabletomaintain ledinthebinderinclude,butare notlimitedto: le explainingthelocationoftheseformsshould led inthissection,behindthecorresponding led underthe“QCForms”tab of 111/04/2014 17:37 1 / 0 4 / 2 0 1 3 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 4 l i _ m y 4 c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Trial Correspondence Shipping Records Equipment Temperature Logs following oversightactivities: the strictadherence tothislaboratory manual.Ataminimum,thispersonisresponsible forthe A supervisormustbeassignedtooverseethetriallaboratorystaff andprocedures toensure oftheStudy Supervision 1.3 is agreed uponbetweenthelaboratoryandSponsor. Records willbestored inthelaboratory’s facilitiesunlesspermissiontotransferandstore elsewhere their disposalhasbeengivenbytheSponsor. retained andbemadeavailableforreview underappropriate circumstances, untilpermissionfor Laboratory studyrecords, includingallsource documentsasdescribedinSection1.1,mustbe all timesshallremain theproperty oftheSponsor. treatment ofhumansubjectsandtheintegritytrialdata)onbehalfSponsor, butat individual responsible forconductingtheclinicaltrialandwhoassumesaccountability The retention ofalloriginalstudydatashallbetheresponsibility ofthePrincipalInvestigator(the StudyData:RetentionofDocuments 1.2 maintained atalltimes. such asfrom fi must alsominimizetheriskofenvironmental damageandthethreat ofaccidentaldestruction, as alockedroom orfi Records mustbekeptinasecure locationthatmaintainstheirintegrityandconfi 1.1.5 Document Storage _ m a n u a l _ • • • • • • Semi-annualreview andfollow-upofstudy-related profi • • Routineandtimelyreview ofAppendixBtoverifycompletionandaccuracyalltests • q u this section. All trial-related correspondence, includingemailsandfaxes,shouldbemaintainedin in thissection,includingcopiesofrequisition forms andairwaybills. All records fortheshipmentofspecimenstocentrallaboratorymustbemaintained Temperature Logs”tabofthebinder, particularlyattheconclusionoftrial. forms usedduringthetimeperiodofstudyshouldbefi placed underthe“EquipmentTemperature Logs”tabofthebinder, oracopyofthe system. However, eitheranote-to-fi If thelabhasaseparatefi as outlinedinthismanual Adherence toanddocumentationofequipmentcleaningmaintenanceschedules corrective actionsandimprovement activities Investigation ofunacceptableoroutrangeQCresults, andproper documentationof including signingofallQCforms/worksheetsandconfi Monthly review ofqualitycontrol, qualitymonitoring,andimprovement activities, read, understood,andwillfollowthelaboratorymanual Ensuring relevant staff are trainedonthemanualguidelinesandprocedures, andhave a d r i . i re orfl n d d

4 le cabinetwithaccessrestricted toauthorizedpersonnel.Storageconditions ood.Conﬁ dentialityofbothstudypatientsandnon-studymustbe ling systemfortheselogs,itisacceptabletomaintainthat le explainingthelocationoftheseformsshouldbe rmationoftestresults ciencytestingresults led underthe“Equipment dentiality,such 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 5 l i _ m y c o b a c t e r i o l o g y delegation mustbecaptured inthestudydocumentation. Supervisory roles maybedelegatedtoqualiﬁ _ l a b _ m a n • • • Maintenanceofpatientconﬁ • • u a tion, answerqueries,andresolve issues Coordination withstudy-related staff, includingroutine meetingstoreview documenta- to CRAs,auditors,regulatory inspectors,etc. Organization ofstudydocumentation,andprovision ofessentialdocumentsasapplicable study Adherence tobiosafetyandinfectioncontrol principlesandpracticesthroughout the Management ofinventoryforallstudy-related consumables l _ q u a d r i . i n d d

5 dentiality ed personnelasnecessary. Documentationofthis 111/04/2014 17:37 1 / 0 4 / 2 0 1 5 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 6 l i _ m y 6 c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b glycerol at–70to–80°Cforsixmonthsaftertheconclusionofstudy, unlessotherwiseinstructedbythesponsor. storage performed bysubculturingtoLJslantsforstorageatroom temperature orat4°Cuntiltheendofstudy. d required forinjectable(s) andfl c Susceptibilitytestingforfi b Defi to produce sputumduringthevisit,twoadditionalattemptstocollectspecimenmustbemadeovernext48 hours. using inductionifthepatientisunabletospontaneouslyexpectorateasampleduringsitecollection.If patients -thisspecimenwillbecollectedbysitestaff). Thesecondspecimenwillbecollectedatthesitebystudystaff andpreferably meal(exceptforhospitalized collected bythepatientathome,earlyinmorning, priortomorning only onesputumsamplewillbecollectedbythesitestaff). Whentwospecimensare collected,thefi a Table 2-1 SAMPLESPECIMENTIMETABLE 2 _ Short/long term storage term Short/long susceptibility testing 1st/2nd-line drug (isolate identifi cation) Confi ofMTB rmation MGIT culture LJ culture Sputum AFBsmear Current diagnosisofMDR-TB Day/Week Visit Number Two m Short-term storage a n nitiveidentifi u sputumsampleswillbetestedateachscheduledvisitexceptfor a ofMTBisolatesrecovered from positivecultures (MGITand/orLJ)willbestored induplicate7H9broth w/ l _ a q u

Treatment Period Sample scheduleofMicrobiological AssessmentsforAllTreatment Groups -Intensive a d a r i . i n cationofMTBwillbeperformedfrom bothMGITandLJmediawhengrowth isdetectedfrom both. d d a c

b 6 forMTBisolatesrecovered from apositiveMGIT(orLJculture ifMGITnotavailable)willbe d rst-line anti-TBdrugsandkeysecond-lineutilizedateachsite(second-lineDSTonly uoroquinolone(s)) willbeperformedwithMGITsystem. 12345678910111213141516171819202122 Visits 2and3 Visits (Day-1andDay1;onthesedays, rst specimenwillbe Long-term 111/04/2014 17:37 1 , / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 7 l i _ m y c o b a c t e r i specifi o l points o all procedures inthefl Section 4:LaboratoryBiosafetyandInfectionControl andSection16:QualityAssuranceapplyto time g At y 3 SPECIMEN FLOWCHART _ Examination Microscopy Preparation l ed a AFB Fluorescent Stain b _ m (section 8) (section 5) MGIT specimen Decontaminate a Collection n Sputum u a l _ q u Drug SusceptibilityTesting Defi a (section 9) ZN positiveforMTB and growth onBAP d r i nitiveidentifi . (section 10.7) i and n (section 13) d d owchart. (section 12)

of MTB

7 MGIT cation Liquid Culture –MGIT Sample from positive Receipt andloginof ZN positiveforMTB; Sputum Processing Sputum Specimens no growth onBAP (section 10.7) specifi NaOH/NALC (section 10) points (section 7) (section 6) MGIT tube time At ed retention forshort-termstorage Isolates forlong-termstorage Confi instructions for and growth on rmatoryZNSmearand Subculture onL J section 10.7) at–70to–80°C BAP (Review (section 11.3) ZN negative (section 11.5) work-up in (section 14) L J SolidCulture identifi

(section 11.2) and Deﬁ (section 11) (section 13) ZN positive contaminated MTB cationof culture is negative If MGIT nitive or 111/04/2014 17:37 1 / 0 4 / 2 0 1 7 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 8 l i _ m y 8 c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Materials/Equipment for all TB Laboratory Procedures Materials/Equipment forallTBLaboratory LABORATORY BIOSAFETYANDINFECTIONCONTROL 4 _ m and thequalityofwork.Insummary, thesestandards require: conducting clinicaltrials,require alaboratorydesignthatensures thesafetyofpersonnel Clinical LaboratoryPractice(GCLP)standards, asetofregulations thatguidelaboratories endorsed byWHO,USCDC,andNationalInstituteofHealth(NIH).Inaddition,Good all laboratorieshandlingspecimensfrom suspectedorknownTBcases.Thesestandards are Regardless ofthelevelriskforspread ofTBinfection,standards are recommended for Principle containment andpractices. of aerosol production. Thus,allaforementioned procedures necessitatespecialbiosafety testing procedures generatehighconcentrationsoforganismsthatposeanincreased risk handled withappropriate precautions. Furthermore, MTBcultures anddrugsusceptibility specimens from suspectedorknownTBcasesmustbeconsidered potentiallyinfectiousand media. Althoughclinicalspecimensfrom TBcasescontainalowinfectivedoseofMTB,all smears andmaybeaerosolized duringtheprocessing ofspecimensandinoculationculture cerebrospinal fl aerosols inthelaboratory. Tubercle bacillimaybepresent insputum,gastriclavagefl for laboratorypersonnel,aswellahazard tootherswhomaybeexposedinfectious Transmission oftuberculosis, includingdrug-resistant TB,isarecognized occupationalrisk Purpose a n u a l _ • Spore testforautoclave • Autoclavetape • Autoclave • Centrifugewithbiosafetycanisters/lids • Waste receptacles • Tuberculocidal disinfectant • Biohazard bags • Disposablegloves • Labgowns • N95,FFP2,orequivalentrespirators • ClassIIBiosafetyCabinet(BSC) q u a • • Waste managementprocedures • Useofpersonalprotective apparel/equipment appropriate forthetask • Engineeringcontrols, suchasacontrolled ventilationsystem • d r electrical safety General labsafetyprocedures –includingphysical,biohazard, fi management plans Administrative controls, includinggoodlabpractices,SOPs,andaccident i . i n d d

uid, urine,andinavarietyoftissues.MTBbacillimaysurviveheat-ﬁ

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 9 l i _ m y c o b a c t e r i o l o g y . RecommendedBiosafetyPractices 4.2 RecommendedFacilitiesandEquipment 4.1 _ l a b _ m a n • • • • • • • • • • • • • Accesstothelabmustberestricted, preferably through ananteroom. • Thelabmustbecontained,i.e.,physicallyseparatedfrom otherlabs. u a BSC andautoclaved. that containsdisinfectantsolutionwhichcanbesealedbefore beingremoved from the Place allwastescontainingMTBinaleak-proof containerorautoclavableplasticbag killing MTB),before andaftereveryprocedure. Disinfect theBSCandallworksurfaceswithatuberculocidal disinfectant(capableof the airfl Keep theamountofequipmentinsideBSCtoaminimumsoasnotinterfere with is completed.Theseprecautions willminimizeinterruptionofairfl arm movements;oncebeginningwork,donotmovehandsoutofthehooduntilwork Inside theBSC,keeparmsparalleltoworksurface,incenter, andminimize but theyare noteasilyaerosolized ifdriedonaslidewarmerfortwohours. before handlingoutsidetheBSC.Heat-fi Heat-fi vortexing, mixing,pipetting,pouring,andinoculationofmedia.Forexample: Take cautionwhenperforming aerosol-generating procedures suchascentrifugation, Microcentrifuges shouldbeoperatedinsideBSCusingtubeswithcapshavingO-rings. containment lids). Clinical centrifugesmustbeequippedwithbiosafetycanisters(bucketsaerosol- BSCs mustbecertifi and positivecultures. work area; UVlampoptional)mustbeusedforallmanipulationsofclinicalspecimens Class IIbiosafetycabinets(BSC;vertical,laminar-fl within theventilationsystem. this environment canbeachievedthrough high-effi Air from thecontainmentlabshouldnotbere-circulated tootherareas inthebuilding– maintained atalltimes. lab, supportedbyavisualmonitoringdeviceshowingthatproper directional airfl Controlled ventilationshouldbeinstalledwhichmaintainsadirectional airfl – – – – – l _ manufacturer’s recommendations forthespecifi in contactwiththecontaminatedmaterialforatleast15minutes. Checkthe (not “hard”) water, to2-5%.Bothtypesof disinfectants are onlyeffective ifleft Similarly, phenol-based disinfectantsmustbediluteddaily, preferably withdeionized stay atorabove0.5%chlorine–undilutedcommercial bleachisusually4-5%. based disinfectants.Diluted(working)bleachsolutionsmustbeprepared dailyand The mostcommontuberculocidal disinfectantsare bleach(hypochlorite)andphenol- Eject micropipette tipsdowninsidediscard bucket Use pipettesthatare easytocontrol Open centrifugecanistersonlyinsidetheBSC Delay openingcapsuntilaerosols havesettled q u a d r x slidesonawarmerintheBSC,heatingthemat65-75°Cforleasttwohours i . owpattern. i n d d

9 ed atleastannuallybypersonneltrainedinthecertifi xed smearsmaycontainviabletubercle bacilli, ow which blowsHEPA-fi cdisinfectanttobeused. ciency particulateair(HEPA) fi owinsidetheBSC. cation process. ltered airover owintothe ltration ow is ow 111/04/2014 17:37 1 / 0 4 / 2 0 1 9 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 10 l i _ m 10 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b will enterthelung. nuclei. Arespirator canserveasanadditionalbarriertoreduce thelikelihoodthattubercle bacilli normal aerosol containmentcapabilityiscompromised, thereby permittingescapeofdroplet NOTE: . PersonnelProtection-Training andMonitoring 4.4 RecommendedPersonalProtectiveEquipment 4.3 _ m a n u Laboratoryinfectionsare nearlyalwayscausedbypoorlymonitored BSCsoraBSCinwhich a l _ • • • • • • • • • • • • q u guidelines forpreventing TBinfection. Staff shouldbeparticipatinginaTBscreening program, perthelaboratory’s national Staff shouldreceive frequent re-training andbemonitored toensure compliance. with personneltrainingrecords. (especially theBSC)forproper operation.Documentationofthistrainingmustbekept procedures, useofpersonalprotective equipment,andhowtomonitorallequipment All personnelworkinginthecontainmentlabmusthaveproper trainingonbiosafety Respirator protection ismore thanjustwearingamask!Attentionmustbegivento: equivalent isacceptable. Health (NIOSH)N-95rating,European CommitteeforStandardization ‘FFP2’rating,or lab. Anyrespirator conformingtotheNationalInstituteforOccupationalSafetyand Respiratory protective devicesare highlyrecommended whileworkinginthecontainment All outerprotective clothingmustberemoved whenleavingthecontainmentlaboratory. Hair covers(caps)andshoeare recommended. andmustbelongenoughtooverlapthesleevesofgown. Gloves mustbeworn long sleeveswithsnug(knit)cuffs. ontop.Thescrubsuitsshouldbechangeddaily.worn Theprotective gownmusthave a solid-front orwrap-around gown.Ifscrubsuitsareprotective worn, gownsshouldbe Staff workinginthecontainmentlabmustwearprotective laboratoryclothingsuchas BSC. Theprocedure shouldinclude: Write aprocedure fortheappropriate handlingofaspill,bothinsideandoutsidethe transported oncartsinprotected racksorbaskets. improperly stackingracksorbaskets.Alltubes,plates,andothercontainersshouldbe Avoid practicesthatcanresult inspills,e.g.,hand-carryingtubes,vials,andbottles,or sterility isachieved. The autoclaveshouldbemonitored withaspore testatleastmonthlytoensure that – – – − − a d Training personnelontheuse,ﬁ Conducting ﬁ Selecting theappropriate respirator fortheindividual room, andallowtheBSCtorunforatleast30minutesbefore enteringtheroom. to theautoclave.Ifaliquidculture isspilled,allpersonnelshouldexitthecontainment be appliedtothespillforatleast20minutes,afterwhichitemscantransported Inside theBSC:BSCshouldcontinuetorunandappropriate disinfectantshould enter immediately(wearingarespirator), without waitingfortheaerosols tosettle. laboratory, allstaff shouldleavetheroom, andthepersondisinfectingspillcan wear arespirator whendisinfectingthearea. Ifthelabisabiosafetylevel3(BSL3) 30 minutestoallowtheaerosols tosettle.Thepersoncleaningthespillshould Outside theBSC(butwithinlab):allstaff shouldleavetheroom foratleast r i . i n d d

1 0 t-testing tchecking,andstorageoftherespirator 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 11 l i _ m y c o b a c t e r i o l o This specimenwillbecollectedbythesitestaff inasterile 50mlcentrifugetube. Sputum Specimen#2 laboratory. case, thespecimenwillbesentbysiteto 50 mlcentrifugetubewillbeused.Ineither If thespecimeniscollectedbysite,asterile • • • • container hasthefollowingparts: leakage andeliminatestheneedtotransfersputumfrom acontainerto50mltube.The patient willuseaspecialsputumcollectioncontainerthatminimizescontaminationand and anydosing),willbelabeled“ meal specimen, consideredspecimen(collectedpriortothemorning tobethe‘morning’ the patient.Thereafter, twosputumspecimenswillbecollectedateachtimepoint.Theﬁ Onvisits2and3,onlyonesputumspecimenwillbecollectedfrom Specimen Timetable. This specimenwillbecollectedbythepatientathome, Sputum Specimen#1 a ‘spot’specimen,willbelabeled“ g y Materials PROCEDURE SPECIMENCOLLECTION,STORAGE,ANDTRANSPORT 5 Sealed label Outer containerandlinedcap Built-in funnel 50 mlcentrifugetubeandcap _ l a Sputum specimenswillbecollectedaccording tothevisitschedulesin Principle staff. of thespecimen. specimens foranalysisinthemycobacteriologylaboratoryandmaintainingcorrect identity results. Adherence toprocedural detailswillresult incollectingadequateandqualitysputum Proper collectionandtransportofsputumspecimensisrequired toensure qualitylaboratory Purpose b _ m a n • • • • • • • • u Patientsputumcollectioninstructionsare documented elsewhere. a Cold packs Cooler Refrigerator withdetachedthermometer Disposable gloves Study Labels Permanent marker Sponsor-provided SputumCollectionKit Sterile, disposable,single-use,screw-capped, conicalcentrifugetubes(50ml) l _ q u a d r i . i n d d

The procedurebelowdetailssputumcollectionatthesiteby

1 1 Sputum Specimen#1 Sputum Specimen#2 ”. Thesecondspecimen,considered ”. unless s/heishospitalized Section 2 : Sample . The rst 111/04/2014 17:37 1 / 0 4 / 2 0 11 1 4

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 12 l i _ m 12 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Forms 8. 7. 6. 5. Ifable,instructthepatienttostand. 4. 3. Discussthefollowingcollectionprocedures withthepatient: 2. 1. 5.1 Sputum Collection _ m a n circling ‘Yes’ or‘No’. mark thatthesputumhasbeeninducedundersectionlabeled“Sputum Induced?”by Form mustalsoaccompanytheappropriately-labeled inducedsputumtothelab.Clearly the site.TheLaboratorySpecimenRequisitionFormandAppendixA: SpecimenTransfer this procedure. Sputuminductionwillbeperformedaccording tothestandard procedure at to inducesputumproduction byaerosol inhalation.Contactthestudyphysiciantoconduct If apatientisunabletospontaneouslyexpectoratesputumsample,anattempt willbemade and holdthebreath momentarily. Repeatingthisseveraltimesmay induce coughing. If thepatientcannotcoughspontaneously, instructthepatienttotakeseveraldeepbreaths demonstrator glassandtube/containertoshowthepatientprocedure ifthiswillhelp. Instruct thepatienttoproduce sputum,afterrinsingmouthasdescribedabove.Usea Instruct thepatienttorinsetwice. Give thepatientaglassofwater(bottledorboiled)torinsemouthfree offoodparticles. collected. Placeonestudylabelontube/containerinwhichthesputumwillbe 3)andvisitnumberforwhichthespecimenisbeing 2orVisit number (#1or#2,N/AifVisit with thescreening and/orsubjectIDnumber, dateandtimeofcollection,sputumspecimen Positively identifythepatient.Prepare twostudylabels(three forpatient-collectedsamples) avoid leakage.50mlpolypropylene centrifuge tubesare preferred. Collect specimensinappropriate sterile,disposablecontainers.Screw capsmustfi u a l _ • • • • • • • • • • q u and equivalent) Appendix D-EquipmentTemperature Record Form(orusesite-specifi Appendix A-SpecimenTransfer Form laboratory requests. Laboratory SpecimenRequisitionForm–thisisasite-specifi lid. Avoid spillsorsoilingtheoutsideofcontainer. Close thetube/containertightlywithscrew-on lidwithouttouchingtheinsideof releasing thespecimen. Release sputumintothelabeledtube/containerbyholdingittolowerlipandgently Cough deeplyandvigorously atthesametimebreath iscomingout. Hold breath foramoment. Take adeepbreath. fi Instruct thepatientnottotouchinsideofcollectioncontainerorlidwiththeir Emphasize thenature ofthedesired specimen. − − Informthepatientthatnasalsecretions andsalivaare notsputum. a ngersorotherobjects. d white-yellow, andsometimesblood-tinged.Itisfrom thelowerairwaysandlung. Explain thatthedesired specimenisproduced byadeepcoughandisthick,mucoid, r i . i n d d

1 2 c formusedforroutine c form,ifavailable ttightlyto 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 13 l i _ m y c o b a c t e r i o l o g y 1. Transport ofSputumSpecimens 5.3 (Appendix D). 2-8°C. Record thetemperature dailyontheappropriate EquipmentTemperature Record Form temperature readings detachedthermometer. usinganinternal Theacceptablerangeis NOTE: 2. 1. StorageofSputumSpecimens 5.2 Thelaboratorysectionwillbecompletedbystaff whentheyreceive thespecimen. 10. Afterthespecimeniscollected,completefollowing: 9. _ l a b _ in thesamecityasmycobacteriologylaboratory. Specimens shouldbedelivered tothelaboratorysamedayascollected,ifstudysiteis be heldincoolerswithicepacks. the growth ofcontaminantsinthespecimen.Ifarefrigerator isnotavailable,specimenscan Refrigerate specimensat2-8°Cuntilready fortransporttothelaboratory. Refrigerationreduces forms. ID onthetubematchesscreening and/orsubjectIDontherequisition andAppendixA Check thatthetubeistightlycapped,properly labeled,andthatthescreening and/orsubject m a Refrigeratorsinwhichsputumspecimensare stored mustbemonitored withdaily n • • • • • u a early thefollowingmorning. may bestored inthesite’s refrigeratorbutmustbedelivered overnight, tothelaboratory Sputum specimenscollectedattheveryendofdaywhentransport is notavailable processing thesameday. on thesameday, for processing thenextday, for orearlythefollowingmorning, shouldeitherarrivetothelaboratory Sputum specimenscollectedinthelateafternoon sothattheycanbeprocessed thesameday.or earlyafternoon should reachSputum specimenscollectedinthemorning laboratory in themorning Appendix A–mustcontainthefollowing: Laboratory SpecimenRequisitionForm–allrelevant ﬁ – – – – – – – – – – – – – – – l _ q complete thissectionifacourierisused) transport/courier information(tobecompletedbydriver, orsitepersonnelcan name andsignature ofpersoncompletingtheform name ofpersoncollecting(orreceiving ifpatient-collectedsample)thespecimen mode oftransport date/time ofdispatchtolaboratory study label time sputumwasatroom temperature total volume who thespecimenwascollectedby(eitherpatientorsitestaff) whether sputumwasinducedornot specimen number(#1,#2,orN/AforV2V3) date andtimeofcollection visit number screening IDnumberand/orsubjectnumber, subjectinitials site nameandnumber u a d r i . i n d d

1 3 eldsmustbecompleted. 111/04/2014 17:37 1 / 0 4 / 2 0 13 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 14 l i _ m 14 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 6. 5. 4. 3. 2. _ m a n provided, insteadofthedrivernameandsignature. ﬁ the “Transport Section”ofAppendixA.Ifacourierserviceisbeingused,thissectioncanbe The nameandsignature ofthetransportdriver, dateandtimeofpick-upare recorded in transport container. enclosed, thenplaceenvelopeinasealed,leak-proof bagandplacetheinside to theoutsideoftransportcontainer. Ifthecourierservicerequests thattheformsbe Place theLaboratorySpecimenRequisitionandAppendixAformsinanenvelopeattach packs. be packagedinaseparatesealedplasticbagbefore placinginatransportcoolerwithice/cold tubes inaracktokeepuprightduringtransport.Forlongerdistances,eachspecimenmust If bagsare notavailable,checktoensure thatcapsare tightlyclosedandplacecontainersor packs. Fortransportonsiteorwithinthecity, eachspecimenshouldbeinasealedplasticbag. When thisveriﬁ for eachtransportboxthat: Before transportingfrom theclinic,adesignatedstaff memberofthestudyteammustverify made onSaturdays. NotifylabifaSaturday deliveryisplanned. be delivered onaweekday, unlessthelaboratoryisopenonSaturdays anddeliverycanbe refrigerator andtransportedonice.Arrangeapick-uptimewhichwillallowthespecimensto but nolaterthan48hoursfrom timeofcollection.Thesespecimensmustbestored inthe If specimensare transportedlongerdistances,theymustbedelivered assoonpossible, lled inbythesitestudyteammember, andthenameofcouriertrackingnumber u a l _ • • • • q u the transportservice. who collectedthespecimenorpersonresponsible forhandingoff thespecimensto The “DispatchSection”ofAppendixAmustbecompletedandsignedbytheindividual information foreachpatient. The LaboratorySpecimenRequisitionandAppendixAformscontainalltherequested that ontheLaboratorySpecimenRequisitionandAppendixAforms. The screening IDnumberorsubjectonthecontainer/tubecorresponds to specimen). of LaboratorySpecimenRequisitionandAppendixAforms(oneeachformper The totalnumberofsputumtubes/containersintheboxcorresponds tothenumber a d r i . i n d d

1 4 cation iscomplete,putthespecimens inthetransportcontainerwithice 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 15 l i _ m y c o b a c t e r i o l o g y 1. SputumSpecimen#1–patient-collectedsputum 6.1.1 ReceiptofSputumSpecimens 6.1 Forms RECEIPTANDLOGINOFSPUTUMSPECIMENSINTHELABORATORY 6 _ l a b _ Specimen RequisitionFormandAppendixA. arrival atthelaboratory, specimenlabelsmustbeverifi placed onthecapofoutercollectioncontainerandsideplasticlabel.Upon Patient-collected specimenswillarriveinasealed,plasticbiohazard bag.Studylabelswillbe m a n • • • • • • • • u a Appendix A. of receipt, aswelladescriptionofthespecimens,on“LaboratorySection” The individualreceiving thespecimensrecords theirname,signature anddate/time collection containerunlessspecimenreceiptisperformedinabiosafety cabinet. Appendix A. information onthestudylabelagainstLaboratorySpecimenRequisition Formand Open theplasticbiohazard bag,remove thesputumcontainer, andverifythespecimen and/or thecourier. leaks. Ifacontaineriscrackedornotclosedproperly, sitestaff canre-educate patients Specimen Logbook/Registry. It isimportanttonotifythesiteaboutreason forany this occurrence inthelaboratory“Comments”sectiononAppendixAandLab do notopentheplasticbag.Autoclavecontainerandbagdiscard. Record Check theplasticbagforanyleaksorcracksinoutercontainer. Ifaleakisseen, Appendix B-StudySource DocumentWorksheet Appendix A-SpecimenTransfer Form Study Labels patients. laboratory requests. Ifpossible,useaseparatelogbookforstudypatientsandnon-study Laboratory SpecimenLogbook/Registry–thisisasite-specifi laboratory requests Laboratory SpecimenRequisitionForm–thisisasite-specifi l _ q u a d r i . i n d d

1 5 Do notremovethewhitecaporplasticlabelfromsputum ed withthecorresponding Laboratory c formusedforroutine c formusedforroutine 111/04/2014 17:37 1 / 0 4 / 2 0 15 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 16 l i _ m 16 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b specimens mustbeprocessed within Compare thedate/timeofcollectiononAppendixAwithcurrent date/time.Sputum 7. of highsalivacontent). noticeably highsalivarycontentwillbenotedbutprocessed (awateryspecimenisanindication Evaluatethequalityofeachsputumspecimenandrecord onAppendixB.Thosewith 6. volume, dating,andinitialing.Record thenewvolumeinappropriate locationontheform. recorded onAppendixAandadjusted,ifnecessary, bydrawingasinglelinethrough thewritten calibrated withknownvolumesofwater. Thisvolumemustbecheckedagainstthe Estimatethevolumeofeachspecimenbycomparingwitha50mltube thathasbeen 5. volume withthelabdirector anddetermineifthequalityvolumeare suffi Sputumspecimensmustbe2-3mlinvolume.Ifthespecimenislessthan2ml,discuss 4. 3. procedures mustbefollowed: Ifthelaboratoryfi 2. _ m a n container atatimetoprevent mislabeling. Collection containersmustbeopenedinabiosafetycabinet.Onlyopenonespecimen u a l _ • • • • • – – – – • • q u Container, funnel,andwhitecapmustbeautoclavedpriortodisposal. Specimens andplaceonthe50mltube. Immediately prepare astudylabelwiththeinformationin Remove the50mlcentrifugetubeandclosetightlyusingcapprovided. Unscrew thefunnelportionfrom thecontainer. Remove theplasticlabelfrom thecontainerbytearingalongperforations. specimen logbookordatabase(ifavailable). entered inthelaboratory“Comments”sectionofAppendixAform,andlab The factthatthespecimenwasreceived unlabeledandsubsequentlylabeledwillbe be fi site whowillsignastatementtakingresponsibility forthelabeling.Thisstatementmust The specimenwillremain inthelaboratory, andmustbelabeledbysomeonefrom the Notify thesitewhenanunlabeledspecimenisreceived. labeled atthetimeofcollection. The laboratorywillnotprocess unlabeledspecimens,andspecimensmustalwaysbe For unlabeledspecimens: requisition: For aspecimenwithanIDonthetubeandmismatched,incomplete,ormissing – – a d requisition formisreceived. Thelaboratorywillcontactthesite. Specimens withoutrequisitions are unacceptable,andwillnotbeprocessed untila specimen isprocessed. The laboratorywillcontactthesitetoobtainanyneededinformationbefore the r i . ledwiththeAppendixAforminstudylaboratorybinder. i n d d

1 6 nds amislabeledormismatchedspecimenandrequisition, thefollowing (There arenoexceptionstothispolicy.) 72 hours ofcollection. Section 6.2 cient forprocessing. : LoginofSputum 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 17 l i _ m y c o b a c t e r i o l o g y 2. 7. OriginalAppendixAandBformswillbestored atthelaboratory. 6. 5. 4. 3. 2. 1. LoginofSputumSpecimens 6.2 1. site-collected SputumSpecimen#1) SputumSpecimen#2-site-collectedsputum(theseinstructionsalsoapplyto 6.1.2 _ l a b _ (signed bythelabsupervisor)to study coordinator tobestored withthetrialdocuments. Send on theAppendixBlabel,dateoffreezerstoragecryotube label,etc.). is fromV2orV3)sitenumber, andapplicabledate(e.g.,thecollectioniswritten subject IDnumber, visitnumber, sputumspecimennumber(#1or#2,unless with thefollowinginformation:labaccessionnumber, screeningIDnumberand/or cryotubes, etc.-andwillbeafﬁ downstream processing ofthisspecimen-microscope slides,LJtubes,MGITBAPplates, The study-speciﬁ Specimen Requisition,AppendixA,andBforms. Place laboratoryaccessionnumberonthestudyspecimenlabelandLaboratory specimen hasbeencollectedtoensure theappropriate work-upofapositiveculture. to thelaboratory’s standard operatingprocedure. Notethestudyvisitnumberforwhich Record patientandspecimeninformationinthelabdatabaseorlogbookaccording the generallabreceiving area orinthemycobacteriologylab. #1 andsputumspecimen#2musthaveseparateaccessionnumbers).Thismaytakeplacein Assign auniquelaboratoryaccessionnumbertoeachspecimen(notethatsputum 2-8°C ifnotprocessed immediately. the mycobacteriologylabifreceived inaseparatereceiving area. Specimensmustbestored at Specimens, LaboratorySpecimenRequisitionForms,andAppendixAformsare transferred to Follow steps2,4,5,6,and7above. Appendix A. verify specimenlabelswiththecorresponding LaboratorySpecimenRequisitionFormand Site-collected sputumspecimenswillarriveina50mltube.Uponarrivalatthelaboratory, m a n information inthe“Comments”sectiononAppendixAform. to instructthepatienttryagainwithanewpre-labeled specimencontainer. Record this provide anotherspecimenwithin48hoursofthescheduledvisit.Callsiteimmediately contaminated withfoodparticlesshouldonlybeprocessed ifthepatientisnotaccessibleto collection. Thesespecimensare acceptablefortesting.Specimensthatare visibly Note: • • u a completed Appendix A. of receipt, aswelladescriptionofthespecimen,on“LaboratorySection” The individualreceiving thespecimensrecords theirname,signature, anddate/time section onAppendixAandintheLabSpecimenLogbook/Registry. sputum specimenbecollected.Record thisoccurrence inthelaboratory“Comments” in anautoclaveanddiscard. Contactthesiteimmediatelyandrequest thatanother Check foranyleaksorcracksinthetube.Ifaleakisseen,decontaminatecontainer l _ q u a Inducedsputummayappearwateryandresemble salivaduetothemethodof d r i . i n d d c labels(provided bytheSponsor)willbeusedtolabeltubesforallsubsequent

copiesofAppendixA(signedbythelaboratorytechnician)and B

1 ae Labaccess#: SubjID: Date: Site#: V#: Specimen#: Screen ID: 7 xed totheAppendixBform. These labelsmustbecompleted 111/04/2014 17:37 1 / 0 4 / 2 0 17 1 4

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1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 18 l i _ m 18 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Record thefollowingonAppendixB: StudyDataReporting 6.3 • • NOTES: 8. _ m Specimens mustberefrigerated ifnotprocessed withinonehour. laboratory, especiallyifonthefollowingday(s)labisclosed. All specimensforsmearandculture are expectedtobeprocessed onthedayofreceipt inthe a the marginnexttonewinformationbeingsentsoupdatedisreadily evident. with newmicrobiology labinformation.Thesupervisorortechnicianmustinitialanddatein added. Theseroutine transmittalsallowthestudycoordinator toupdatethestudydatabase In addition,sendacopyofAppendixBtothestudycoordinator whenevernewinformationis n u a l _ • • • • • • • • q u any comments name ofpersoncollectingthespecimen induced ornot,patient-collectedsite-collected) date andtimespecimenwasreceived, specimenvolume,andtypeof(i.e. sputum specimennumber(#1,#2,orN/A) visit number screening IDnumberand/orsubjectnumber, patientinitials site number lab accessionnumber a d r i . i n d d

1 8 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 19 l i _ m y c o b a c t e r i o l o g y Materials Procedure PROCESSINGSPUTUMFORSMEARMICROSCOPYANDQUALITATIVE CULTURE 7 _ l a buoyant duringcentrifugation.Arelative centrifugalforce of3,000 gravity priortocentrifugation.Mycobacteriahavealowspecifi buffer neutralizestheNaOHanddiluteshomogenatetolessenviscosityspecifi effect ontheNALCbychelatingheavymetalionspresent inthespecimen.Thephosphate loses activityrapidlyinsolution,soitismadefresh daily. Sodiumcitrateexertsastabilizing decontaminating agent,NaOH,tobeusedatlowerfi N-acetyl-L-cysteine (NALC),amucolyticagent,isusedforrapiddigestion,whichenablesthe Principle processed inthismannerforpreparation ofAFBsmearsandliquid(MGIT)/solid(LJ)cultures. as possiblewhileharmingfewmycobacteriapossible.Allsputumspecimenswillbe specimen. Areasonable compromise istodestroy asmuchofthecontaminatingbacteria will selectivelydestroy onlycontaminatingfl there are severaltechniquesavailable,noneofthemare ideal,meaningnoneofthem mycobacteria andliquefactionofmucousorganicdebrisinthespecimen.Although Processing sputumspecimenshastwoobjectives:decontaminationofbacteriaotherthan Purpose centrifuge ishighlyrecommended). of thespecimen,whichleadstoadditionalkillingmycobacteria(hence,arefrigerated lower relative centrifugalforce, butincreased centrifugationtimeincreases thetemperature relative centrifugalforce appliedtothespecimen.Alongercentrifugationtimecanoffset a rate atwhichmycobacteriasedimentiscriticallydependentontimeofcentrifugationand centrifuge mustbecalibrated)for15minutesisadequatetosedimentmycobacteria.The b _ m a n • • • • • • • • • • • • u a Phosphate buffer pH6.8(orcomponentsifmadein-house) 2.9% Nacitrate 6% NaOH NALC powder 60 mlNalgenetubeswithplasticscrew cap,autoclavable 50 mlconicalpolypropylene tubeswithplasticscrew cap Clean cylinder, fl Sterile cylinderandbreak-resistant glassbottletoprepare NaOH/NALC-Nacitrate Discard bucketwithbiohazard baginsert,containingappropriate disinfectant Waste receptacle, splash-proof forliquids 70% ethanol Tuberculocidal disinfectant l _ q u a d r i . i n d d

1 9 ask,andbeakertoprepare phosphatebuffer ora andachievecompleteliquefactionofthe nal concentration(insputum).NALC c gravityandmayremain xg ( not 3,000rpm–the c 111/04/2014 17:37 1 / 0 4 / 2 0 19 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 20 l i _ m 20 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Forms . To prepare 1Ldigestant-decontaminant solution: 1. 6% SodiumHydroxide,2.9%Citrate Advance PreparationofSolutions: PreparationofNaOH/NALC-NaCitrateSolutionand PhosphateBuffer 7.1 _ m a n u a l _ • • • • • • • • • • • • • • • • • • • • • • • • • • • q u Appendix B-StudySource DocumentWorksheet equivalent) Appendix D-EquipmentTemperature Record (orusesite-specifi laboratory tests. Specimen Processing Worksheet/Workbook –thisisasite-specifi laboratory requests. Laboratory SpecimenRequisitionForm–thisisasite-specifi Paper towels LJ tubes MGIT tubes Microscope slides,frosted oneend,newandclean Shaker Permanent marker Pencil forlabelingslides Study labels Slide warmer(hotplate) pH meterorappropriately sensitivepHpaper Magnetic stirringbarandstirrer Sterile, transferpipetteswithgraduationsmarkingvolume(individuallywrapped) Pipette aid(DrummondScientifi 2 mldisposable,serological pipette,sterile,individuallywrapped Timer Refrigerated centrifugewithcovered bucketinserts for50mlconicaltubes Clean rackfor50mlcentrifugetubes Vortex mixer Weigh boats Precision balance Analytical balance 2.9% Nacitrate:dissolve14.5gcitratedihydratein500mldistilledwater. 6% NaOH:dissolve30gNaOHin500mldistilledwater. a d r i . i n d d

2 0 c,PortablePipet-AidXPorsimilar) c formusedforroutine c formusedforroutine cform,ifavailableand 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 21 l i _ m y c o b a c t e r i o l o g . To prepare NALC-NaOHworking solution: 1. NaOH/NALC-Na citrate Daily PreparationofSolutions: 2. y . To prepare 1.5Lofphosphatebuffer: 1. Phosphate BufferSolution 2. _ l a b 50 mlinto60sterileNalgenetubes,asdescribedabove. volumes inasterilecontainerandcheckpHusingaseptictechnique.Thenproceed todispense be madeinlargevolumes,sterilized,andstored separately. Whenready touse,mixequal Alternatively, stocksolutionsofdisodiumphosphateandmonopotassium phosphatecan later use.Forexample: Alternatively, thesolutionsmaybemixed,sterilized,andstored inscrew-cap containersfor _ m a (KH • • • • • • • • n • • • • • • • • • u NALC (seetablebelow),tothefl solution. Forexample,ifatotalof200mlNaOH-NaCitratesolutionisneeded, add1g Using thetable,addappropriate amountof0.5%NALCtotheNaOH-NaCitrate contains 3%NaOH. in asterilefl If solutionsare stored separately, mixequalvolumesoftheNaOHandNacitrate solutions Just before use,determinehowmuchreagent willbeneededfortheday’s work. the digested-decontaminatedspecimenandresuspend thepellet. Cap thetubesandsterilizeinautoclave.Thesesmalleraliquotswillbeusedtowash and technicianinitials. Label containerswiththebuffer name,dateprepared, expirydate,batchlotnumber, - 200mlsterilecontainerscanbeused). storage (ideally, 50mlare dispensedinto60mlsterileNalgenetubes;alternatively, 100 Using asmallerfl pH; addmonopotassiumphosphatetolowerpH. Check pH,whichshouldbe6.8.Adjustifnecessary. Adddisodiumphosphatetoraise Stir withmagneticstirringbaronstirrer. a Combine 7.1gdisodiumphosphate(Na Store reagents refrigerated untilneeded. and technicianinitials. Label containerswiththebuffer name,dateprepared, expirydate,batchlotnumber, Sterilize byautoclavingat121°Cfor15minutes. Mix wellandaliquotinto500mlbottles. In a4Lfl Store reagents refrigerated untilneeded. and technicianinitials. Label containerswiththebuffer name,dateprepared, expirydate,batchlotnumber, Sterilize eachreagent byautoclavingat121°Cfor15minutes. l _ q u 2 a PO d r i 4 . i ), and1,500mldistilledwaterina2or4Lfl n askcombine1Lof6%NaOHand2.9%sodiumcitrate. d d

ask usingasterilecylinder, e.g.,100mlofeach.Theresulting solution 2 1 ask orbeaker, dispensebuffer intosmallervolumecontainersfor ask.Mixgently–donotshakevigorously. 2 HPO 4 ), 6.8gmonopotassiumphosphate ask. 111/04/2014 17:37 1 / 0 4 / 2 0 21 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 22 l i _ m 22 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 2. 11. 10. 9. 8. 7. Placespecimentubesatleastonespaceapartinracktoaidpreventing contamination. 6. 5. 4. 3. 2. 1. All processingmustbedoneinabiologicalsafetycabinet(BSC)usingfullPPE. PreparationofBSCandSpecialMicrobiologyPractices 7.2 Table 7.1 _ m a Volume ofdigestant for contaminatingthelargerstockbottle. aliquot fordigesting-decontaminatingspecimens. Pour asmallervolumeofthisworkingsolutioninto50mlconicaltubeanduse leave onfor1hourbeforeoff. turning Ifavailable, 70% ethanol,letstand3minutesandwipedry. off IfBSCisturned intheevening,besure to allBSCsurfaceswith completion ofwork,placepapertowelsindiscardUpon bucket.Wipe soaked towelandchangeglovespriortoprocessing additionalspecimens. if anysuspicionofdroplets from aspecimenisseenongloves,wipegloveswithdisinfectant- paper towelsoakedintuberculocidal disinfectantpriortoremoval from theBSC.Inaddition, are free from anydroplets/potential contaminants. Ifnecessary, wipetherackortubewitha Ensure thattubes,bottles,racks,pipetteaid,etc.are removed from thesafetycabinet cross-contamination andmisplacingthecapsonwrong tubes. Remove onlyonecapatatimefrom thetubes(50mlspecimentube,culture tubes)toavoid contact withtheedgeofspecimencontainer. To reduce theriskofcross-contamination, ensure thatreagent containersdonotcomein avoid spillage/confusionofsamples. Work methodicallywiththetubesononesideanddiscard bucketsclosetothespecimens, 20 minutesbetweenbatches,priortorepeatingsteps1-4. turn onUVlights(ifavailable)for20minutes,andallowairtocirculateintheBSC When processingmultiplebatchesonthesameday, cleanthe BSCwithdisinfectant, Do notworkwithmorespecimensthancanbeplacedinthecentrifugeatonetime. for disposalofcontaminatedmaterials. Place adiscard bucketwithabiohazard bagcontainingtuberculocidal disinfectantinsideBSC the BSC. Place papertowelssoakedintuberculocidal disinfectantovertheentire worksurfaceinside concentration andplaceinsideBSCfordisposalofliquids. Prepare asplash-proof wastereceptacle withtuberculocidal disinfectantattheappropriate 3 minutes priortodryingwithapapertowel. Before processing theday’s specimens,cleanBSCsurfaceswith70%ethanoland letstand n u a needed (ml) l _ • • q u 0050505.00 500 500 1000 needed. Discard anyunusedNaOH/NALCsolutionattheendofday. If more orlessofthissolutionwillbeusedinoneday, prepare theappropriate volume brought toroom temperature priortousingonsubsequentspecimens. mucolytic activityonstandingatroom temperature. However, ensure thesolutionis Store NaOH/NALCsolutionat2-8°Cbetweenprocessing batches,asNALCloses Preparation ofNaOH/NALC-Nacitratedigestantsolution 0 5 5 2.50 1.25 1.00 0.50 250 125 100 50 250 125 100 50 500 250 200 100 a 02 50.25 25 25 50 d r i . i n d d

2 2 %NO 2.9%Nacitrate 6% NaOH turn onUVlightinsideBSC turn This techniquereducesthepotential Amount NALCtoadd for atleast (grams) 1 hour. 111/04/2014 17:37 1 / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 23 l i _ m y c o b a c t e r i o l o g y 14. Transfer tubesina50mltuberacktothecentrifuge. 13. 12. 11. 10. Repeatsteps6-8forsubsequentspecimensat 30secor1minuteintervals. 9. 8. 7. 6. 5. Allowrefrigerated specimensandreagents tocomeroom temperature before testing. 4. 3. 2. 1. 7.3 Specimen Digestion-Decontamination _ l a b _ (lids) are properly placedoneachofthebuckets. Place tubesincentrifugebuckets,ensuringtheyare equallybalancedandthebiosafetycovers be killedoff ifexposedtoNaOHlongerthan20minutes. 20 minutesofaddingthedigestion-decontaminationsolution maximum of20minutes. above), sothateachspecimenisONLY exposedtodigestion-decontaminationsolutionfora Continue toaddphosphatebuffer toallspecimensat30secor1minuteintervals(as inversion orvortex.Discard unusedbuffer. 50 mlmarkonthecentrifugetubeusingpre-dispensed, 50mlaliquotsofbuffer. Mixby When timehaselapsed,remove tubefrom shaker. Addphosphatebuffer (pH6.8)tothe of NALCpowderandvortex15-30sec. Make sure thespecimeniscompletelyliquefi fi Start thetimerfor15minuteswhendigestion-decontaminationsolutionisaddedto several timessothetubewallsandcapare exposedtothe NaOH/NALC-Nacitratesolution. Close tubetightlyandvortexthesuspensionuntilliquefi is 1.5% Use anewsterilepipetteforeachspecimen( (prepared above)thatisequaltothespecimenvolume(1:1volume)centrifugetube. Using atransferpipette,addtothesputumtubevolumeofNaOH/NALC-Nacitratesolution to 10mlpriorprocessing. vortex thespecimenandremove theexcessvolumewithapipettetobringtotal The idealmaximumvolumeofasputumspecimenis10ml.Ifthegreater than10ml, Section 6.2 Label slidesandtubes,usingthestudy-specifi Also, record thetechnician/technologistprocessing eachbatch. Record allspecimensprocessed inabatchusingtheLabProcessing Worksheet/Workbook. Login ofSputumSpecimensintheLaboratory. Follow theloginprocedure forspecimenregistration describedin m rstspecimen.Placetubeinrackonshakerplatformtoimprove homogenization: a n • • u a times duringtheincubationperiod. If ashakerisunavailable,vortexthetubesgently(tomimicshakingaction)2-3more sample isexposedtothedigestion-decontaminationsolution. Adjust speedofrotator sothatitmixesthespecimenwellwhileensuringentire – – – l _ q Once theidealspeedisidentifi NALC, makingitineffective forliquefying sputum. Donotagitatethetubetoovigorously; depending upontheinstrument. a gentle,buteven,mixingofthesampleoccurs,usuallybetween60and80rpm Start withanrpmof60,increasing speedinsmallincrements, e.g.,5rpm,until ). u a d r i : LoginofSputumSpecimens. . i n d d

2 3 It isveryimportantthatthephosphatebuffer isaddedwithin ed,useforprocessing ofallsputum samples. ed. Ifstillmucoidafter10minutes,add~50mg c labelsprovided, withinformationdescribedin The ﬁ nal NaOHconcentrationatthisstage extensiveaerationcausesoxidationof ed (15-30seconds).Invertthetube , sincemycobacteriacan Section 6 : Receiptand 111/04/2014 17:37 1 / 0 4 / 2 0 23 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 24 l i _ m 24 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b on AppendixB. Record thedateandtimeofsputumprocessing, aswellthecharacter(quality)ofsputum, StudyDataReporting 7.5 for smearmicroscopy andculture (MGITandLJmedia).See must beincludedneartheendofbatchandhandledaspatientspecimensbeingprocessed control sample(toverifyaccuracyofmethods)weeklywithabatchspecimens.Thesesamples Includeonenegativecontrol sample(tocontrol forcross-contamination), andonepositive 2. such aslotnumbers,expirydates,pHofbuffer, etc.See Record preparation information foreachnewbatchofdigestion/decontaminationreagents, 1. QualityControlandDataMonitors Internal 7.4 15. 21. 20. 19. 18. 17. 16. _ m a n Centrifuge at3,000 is confi It isrecommended tostore anyremaining sedimentat2-8°C,leastuntilthesputumsmear When fi the samepipette. culture mediaandprepare smearforAFBstainimmediatelyaftersuspendingthepellet,using the smearforAFBmicroscopy Solid Culture: LowensteinJensen(LJ)Media.Thesamepipettecanthenbeusedtoprepare Culture –MycobacteriaGrowth IndicatorTube (MGIT),andculture onLJmedia Use thesuspendedpelletforculture intheMGITBACTEC960TBSystem a pre-measured 2.5mlvolumeofwater. Useanewpipetteforeachspecimen. expelling buffer. Ifnecessary, compare thevolumeintubetoareference 50mltubewith fi Add sterilephosphatebuffer (pH6.8)(from individual50mlaliquots)tothepelleta disinfectant, takingcare nottodisturbthesedimentatbottomoftube. Carefully intoasplash-proof decantthesupernatant containercontainingatuberculocidal the BSCpriortoopeningbiosafetycovers. Once centrifugationiscomplete,remove thecentrifugebucketswithoutdelayandcarryto centrifugation periodshouldbeginonly nal volumeof u a l _ q u a rmedasacceptable.Autoclavesputumsedimentbefore discarding. d nished,disposeofthepipetteintobiohazard discard bucket. r i . i n d d

2 4 2.5 ml xg , andresuspend thesedimentusingapipette--gentlyaspiratingand (notethisis Section 8 not : AFBMicroscopy Preparation andStaining.Inoculate after 3,000rpm)for15minat4-12°C.Theminute 3000 xg Section speedhasbeenachieved. Section 16.3formore details. 16.4.3.1formore details. Section 10 Section 11 : Liquid 111/04/2014 17:37 1 : / 0 4 / 2 0 1 4

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 25 l i _ m y c o b a c t e r i o l o g y Materials Procedure ACID-FAST BACILLIMICROSCOPY(AFB)PREPARATION ANDSTAINING 8 _ l a stains blue.TheZNstainconfi methylene bluecounterstain.Acid-fastorganismsstainred, whilethebackground ofdebris brightly. TheZiehl-Neelsenmethodusesacarbolfuchsinstain,acidalcoholdecolorizer, and power, thusmore fi stain ismore sensitivethanZiehl-Neelsenbecausethesmearcanbeexaminedunderalower rhodamine stain,organismsfl non-specifi auramineOstain,organismsfl as forZiehl-Neelsenstaining.With For thefl Principle Section of thesputumsmearmustconformtoreading andreporting procedures describedin fl If analternative (MGIT, LJ). reported. TheZiehl-Neelsenstainisusedtoconfi Neelsen staincanbeusedtoconfi are stainedwithfl ( All sputumsmearsare prepared from decontaminatedandconcentratedspecimens AFB observedinstainedsmears. stain andbecounted.Asemi-quantitativegradingsystemisusedtoreport thenumberof of clinicalspecimensandcultures. Bothlivinganddead(viablenon-viable)bacilliwill The purposeofAFBmicroscopy istodetectacid-fastbacilli(AFB)bymicroscopic examination Purpose b Section _ m a n • • • • • • • • • • • u a Sterile loopordisposableapplicatorstick Sterile, transferpipetteswithgraduationsmarkingvolume(individuallywrapped) (orspiritlamp) Bunsen burner Hot plateorslidewarmer Study labels Pencil forlabelingslides Microscope slides,frosted atoneend,newandclean Paper towelsoakedinappropriate disinfectant Discard bucketwithbiohazard baginsert,containingappropriate disinfectant Waste receptacles (includingsplashproof receptacle forliquids) Tuberculocidal disinfectant l _ 9:Acid-fastBacilliMicroscopy (AFB)Examination. uorochrome stain,theprincipleofdecolorizerandcounterstainissame 7:Processing SputumforSmearMicroscopy andQualitativeCulture). Thesesmears q u c debrisstainspaleyellow, auramine/ andthebackground isalmostblack.With a d r i . i n d d uorescent stainsuchasacridineorangeisused,microscopic examination

uorescent stains,eitherauramineOorauramine/rhodamine.TheZiehl-

2 elds canberead inthesameamountoftime,andbacillistandout 5 uoresce yellow-red inanalmostblackbackground. Fluorochrome rmstheacid-fastproperty ofmycobacteria. rm ﬂ uorescent smearresults, buttheseresults willnotbe rm thepresence ofAFBinpositivecultures uoresce brightyellow, 111/04/2014 17:37 1 / 0 4 / 2 0 25 1 4

Mycobacteriology Laboratory Manual

1 7 : 3 7 ggli_mycobacteriology_lab_manual_quadri.indd 26 l i _ m 26 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g Forms y _ l a b . Work inaBSCasdescribedabove. 2. 1. PreparationofSmearsfromPositiveCultures 8.2 Ideally, removesmears fromBSCafterheat-ﬁ 5. 4. 3. 2. 1. The slidesmustremaininthebiologicalsafetycabinetuntiltheyhavedried. PreparationofSmearsfromProcessedSputum 8.1 _ m a • • • • • • • • • • n ID number, sputumspecimen number (ifapplicable),anddate. Label frosted endofslideinpencilwithlaboratoryaccessionnumber, screening and/orsubject a time.Disposeofthetransferpipetteintobiohazard discard bucket. close proximity (oftenbestatbackofcabinet).Remembertoopenonlyonespecimentube Work systematicallythrough thesampleswithslidesononesideanddiscard bucketin light. least 2hours(longertimeispreferable), toheat-ﬁ Place theslidesonahotplateorslidewarmerattemperature between65°Cto75°Cforat 1 x 2 cm.Air-dry thesmear. the digested-decontaminatedspecimenontoslide,spreading overanarea approximately Use atransferpipettetoplace~100μl(2drops) ofwell-mixedresuspended pelletfrom Processing SputumforSmearMicroscopy andQualitativeCulture) tomixthoroughly. Working inabiologicalsafetycabinet,vortexthedecontaminatedsediment(see specimen isfrom V2orV3),anddate. number and/orsubjectIDnumber, visitnumber, sputumspecimennumber(#1or#2,unless Label thefrosted endoftheslideinpencilwithlaboratoryaccessionnumber, screening ID u Appendix C-DailyAFBStainingQCForm Appendix E-Reagent/MediaQCForm Wash bottle Distilled water Vortex mixer Timer Forceps Slide dryingrack Staining rack Staining sink alcohol decolorizer) a l _ • • • • q u Acridine orangestain(acridinesolutioninbarbitonebuffer, 2NH permanganate) Auramine/Rhodamine (auramine/rhodamine,0.5-1%acidalcohol,0.5%potassium Auramine stain(auramineO,0.5-1%acidalcohol,0.5%potassiumpermanganate) Ziehl-Neelsen stain(carbolfucshin,3%acidalcohol,methyleneblue) a d r i . i n d d

2 6 or xationforstaining. x thesamples.DonotexposeslidestoUV 2 SO 4 Section , acid or 7: 111/04/2014 17:37 1

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1 7 : 3 7 ggli_mycobacteriology_lab_manual _2coul.indd 27 l i _ m y c o b a c t e r i 8. Allowslidestoairdryinthesliderack.DONOTBLOT! 7. Wash off theacidalcoholwithdistilledwater. 6. Floodtheslidewith2%acidalcoholandletstandfornomore than3min. 5. Wash off thestainwithdistilledwaterandtiltslidetodrain. 4. Floodslideswithacridineorangeworkingsolutionandletstandfor10min. 3. Wash off the2NH2SO4withdistilledwater. 2. o l o g y 1. AcridineOrangeProcedure 8.3.2 8. Allowslidestoairdryinthesliderack.DONOT BLOT! 7. Wash off thestainwithdistilled water. 6. 5. Wash off theacidalcoholwith distilled water. 4. Floodtheslidewith0.5%acidalcoholandlet standfor2min. 3. Rinsethestainawaywithdistilledwater andtiltslidetodrain.Water mustbechlorinefree. 2. 1. insert. in whichcasethemanufacturer’s instructionsshouldbefollowedexactlyaswritteninthepackage NOTE: AuramineorAuramine/RhodamineProcedure 8.3.1 ProcedureforFluorescentStaining 8.3 Ideally, removesmearsfromBSCafterheat-fi 6. 5. Air-dry smear. 4. 3. unable toread rightaway, placeslidesincovered box. Protect smearsfrom lightandexamineimmediatelyusingthefl for 10min. _ l a b _ Place slidesonstainingracksotheyare atleast1cmapart,fl unable toread rightaway, placeslidesincovered box. Protect smearsfrom lightandexamineimmediatelyusingthefl permanganate toactover2min,oritmightquenchthefl Flood slideswith0.5%potassiumpermanganatefor1-2min.Donotallow auramine/rhodamine stainandletstandfor20min. Place slidesonstainingracksotheyare atleast1cmapart,andfl least 2hours(longertimeispreferable), toheat-fi Place theslidesonahotplateorslidewarmerattemperature between65°Cto75°Cforat into thebiohazard discard bucket. to thewaterandgentlymixmakeasmooth,thinsuspension.Disposeofapplicatorstick a transferpipette.Usingsterileloopordisposableapplicatorstick,2to3colonies If examiningcoloniesonsolidmedium,dispense~100μlofdistilledwateraglassslidewith biohazard discard bucket. spreading ittocoveranarea approximately 1x2cm.Disposeoftransferpipetteintothe an aliquotofbroth usingadisposablepipette.Place~100μl(2drops) ofbroth ontotheslide, If examiningapositiveMGITculture: vortextubewell,unscrew MGITtubecapandsample m a Theprocedures belowshouldbestrictlyfollowed,unlessacommercial stainingkitisused, n u a l

_ 2 c o u l . i n d d

2 7 xationforstaining. xthesamples. uorescence ofacid-fastbacilli. ood with2NH uorescent microscope. If uorescent microscope. If ood withauramineor 2 SO 4 , andletstand 111/04/2014 17:40 1 / 0 4 / 2 0 27 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 28 l i _ m 28 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b • • • NOTES: Allowslidestoairdryinthesliderack.DONOTBLOT! 10. theslidestodrain. Tilt 9. Wash off themethylenebluewithdistilledwater. 8. Floodtheslideswithmethyleneblueandletstandfor1-2minute. 7. Wash off theacid-alcoholwithdistilledwaterandtiltslidestodrain. 6. Letstandfor2-3min(more acid-alcoholshouldbeusedifthesmearisheavilystained). 5. Floodslideswith3%acid-alcohol. 4. Wash off thestainwithdistilledwater. 3. 2. Placeslidesonstainingracksotheyare atleast1cmapart,andfl 1. ProcedureforZNStaining 8.4 . Labeltheseslideswithstudylabelandsampledetailsafterstainingsmearisdry. 2. Store allfl 1. StorageofSlides 8.6 See Section16.3.1:AcidFastStainsformore details. 2. 1. QualityControl Internal 8.5 _ m covered andexaminewithin24hoursofstaining. Fluorochrome stainedsmearslosefl from oneslidetoanother. Slides mustnottoucheachotherwhenplacedonstainingracktoprevent transferofmaterial handling slides. Heat fi a n not letthestainboilordry. Addadditionalstainifnecessary. also beused.Applyonlyenoughadditionalheattokeeptheslidesteamingfor5minutes.Do Heat theslidetosteamingwithfl (Appendix E). when newlotsofstainsare received. Record theseresults ontheReagent/MediaQCForm Positive andnegativecontrol slidesmustbeincludedwitheverybatchofpatientslides,and Form (AppendixC). Record lotnumbers,expirydates,etc.forthestainingreagents ontheDailyAFBStainingQC u a l

_ xing (slidewarmerorﬂ 2 c o u l . uorescent-stained sputumsmearsinaslideboxuntiltheendofstudy. i n d d

2 8 ame) doesnotalwayskillmycobacteria;exercise care when uorescence withtimeandexposure tolight. ame from aBunsenburner. Anelectricheatingblockmay oodwithcarbolfuchsin. Keep smears 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 29 l i _ m y c o b a c t e r i o l o g y Forms Materials Procedure ACID-FAST BACILLIMICROSCOPY(AFB)EXAMINATION 9 _ l a A positivesmearisapproximately 10 including may bepartiallyacidfast,somicroscopy cannotbeusedtodetermineindividualspecies, All mycobacteriaare acidfast,andothergenera,suchas The Ziehl-Neelsenstainsacid-fastorganismsred andthe background debrisstainsblue. fl fl yellow, Auramine/Rhodaminestain,organisms andthebackground isalmostblack.With AuramineOstain,organismsfl With Principle according totheWHO/IUATLD system. used scoringsystemsare publishedbyWHO/IUATLD andCDC-USA.Reportingmustbe results canbecompared andusedinamannerthatisrelevant topatientcare. Thecommonly The results ofexaminationstainedsmearsare reported inastandardized waysothat Both viableandnon-viablebacilliwillstainbecounted. The purposeofAFBmicroscopy istodetectacid-fastbacilliinclinicalspecimensandcultures. Purpose b uoresce red-orange inablackbackground. uoresce yellow-red AcridineOrange,organisms inanalmostblackbackground. With _ m a n • • • • • • • • • • • • u a Appendix B-StudySource DocumentWorksheet Appendix C-DailyAFBStainingQCForm of smearresults. Laboratory worksheet/workbook–thisisasite-specifi laboratory requests. Laboratory SpecimenRequisitionForm–thisisasite-specifi Dark room (forexaminationunderfl Lens cleaningsolution Lens paper Slide storagebox Slide holder Immersion oilforZiehl-Neelsenstain Fluorescent microscope forfl Light (brightfi l

_ 2 M. tuberculosis c o u l . i n d d

2 9 eld)microscope forZiehl-Neelsenstain . 4 uorescent stain bacillipermlorgreater. uoresce brightyellow, non-speciﬁ uorescence) Nocardia c formused forinitialrecording and c formusedforroutine c debrisstainspale Corynebacterium

111/04/2014 17:40 1 / 0 4 / 2 0 29 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 30 l i _ m 30 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Table 9.1 2. 1. Save allsputumsmearsinslideboxes. Examine fl ExaminationofFluorescentStain 9.1 new lotsofreagents. See A positiveandnegativecontrol smearmustbeincludedineachbatchofpatienttestsandwith Quality Control Internal 9.4 and transferred toAppendixB. concentrated sputumspecimensandpositivecultures are reported onthelaboratoryworksheet If QCsmearresults are acceptable,patientsmearresults canbereported. Smearresults of SmearResults Reporting 9.3 2. 1. ExaminationofZiehl-NeelsenStain 9.2 ** Confi *The numberofAFBindicateshowinfectiousthepatientis.Itisimportanttorecord exactlywhatyousee. 5. 4. 3. _ 5 F noefied>6 F noefied3+ 1+ 2+ Scanty >60AFBinonefi Confi eld required** rmation NoAFBobserved 7-60AFBinonefi eld 1-6AFBinonefi eld 3-24AFBinonelength > 250AFBinonefi eld 1-2AFBinonelength 25-250 AFBinonefi eld NoAFBinonelength 3-24 AFBinonefi eld 5-49 AFBinonelength 1-4 AFBinonelength No AFBinonelength m a Using 20Xmagnifi eyepiece foratotalof200Xmagnifi afl With blue. culture (MGIT).TheAFBappearbrightred againstthebackground materialcounterstained may beaggregated sidebyandendtoformcords, especiallywhengrown inliquid from veryshortrods tolongfi AFB willhavesimilarmorphologyasfl AFB-positive smearisread. tip ofthedropper whendispensingoil.Alwayswipeoilfrom theoilimmersionlensaftereach (10X eyepieceforatotalof1000Xmagnifi Using abrightfi record onthelabworksheetappropriate result according tothetable. the smearpositivity, thefewerfi Count bacilliinthenumberoffi large clumps. and stainingintensity, andmayoccursingly, insmallgroups containingafewbacilli,oras Mycobacteria appearasrod-shaped, coccoid,orfi morphology athighermagnifi Occasionally usethe40Xobjectivetoseemore detailedbacterialmorphology. Confi report anegativeresult. n u a htyuse(0x Whatyousee(400x) What yousee(200x) rmationrequired by anothertechnicianorprepare anothersmear, stainandread. l

_ 2 uorescent-stainedslideswithin24hoursofstaining. c Grading ScaleforFluorescent Stain o u l uorescent microscope, scantheentire smearwiththe20Xobjective(with10X . i n d d

3 eld microscope, Ziehl-Neelsensmearsare examinedwiththe100Xoilobjective 0 cation, one2cmlengthisequivalentto30fi Section 16 laments. Oftentheyare bent,containheavilystainedbeads,and cationavoidsafalse-positivereport duetofl elds counted).Average thecountfornumberoffi elds appropriate forthedegree ofpositivity(e.g.,thehigher : QualityAssuranceforfurtherdetails. cation). uorescence-stained bacilli.Theyare variableinshape, cation). Take care nottotouchtheslidewith lamentous bacilli.Theymaybevaryinshape elds, whichissuffi What toreport uorescing debris. * elds and cient to cient rming 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 31 l i _ m y c o b a c t e r i o l o g y . DetectingSourceofFalseAFBSmearResults 9.6 seen). ZN Stain:Smearresults willberecorded as“positive”foracid-fastbacilli,or“negative”(noAFB Fluorescent Stain:Refertothestudyreporting schemeinthetable positive cultures onAppendixB. Record thetypeofsmearandresults ofbothconcentratedsputumspecimensand StudyDataReporting 9.5 _ odto as CorrectiveAction Cause Condition l a negative positive b False- False- _ m a n u a l

_ Incomplete slide reading Search smear in a uniform mannerandread Searchsmearinauniform Settemperatureat65-75°Candmonitoronadaily/ Incomplete slidereading temperature Incorrect slidewarmer Examineﬂ uorescent stainedsmearswithin24hours. Nonstaining orpoorlystainingAFB. smear toothin. Requestanotherspecimen. Useonlyfreshstains,withoutprecipitatesor Smear areaistoolarge,makingthe duringstaining. washed off are notclean,causingmaterialtobe Smears thataretoothick,orslides lens orthroughcontaminatedoilbottles. AFB transferredinoilontheimmersion Precipitated stains. Food particles. a negativesmear. AFB transferredfromapositivesmearto material fromprevioussmears. Old, usedmicroscopeslidesretain 2 c o u l . i n d d

3 1 suggested numberoffi elds. weekly basis to avoidingdilutingstainingreagents. excessrinsewaterbetweensteps staining, drainoff flrinse waterwillaffect uorescence stain.During reagents indarkbottles;highchlorinecontent under heatingduringsmearfi xation; storestaining Protect smearfromUVlight,directsunlight,over/ Apply smeartoa1x2cmarea. smears. Proper digestionofspecimen.Avoid makingthick whenever thereisevidenceofcontamination. each AFB-positivesmearisread.Changeoilbottles Always wipeoilfromtheimmersionlensafter contaminating organisms. each other;donotusestainingjars. Use astainingrackandkeepslidesfromtouching Use onlynewslides. Section 9.1above. 111/04/2014 17:40 1 / 0 4 / 2 0 31 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 32 l i _ m 32 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 0 LIQUIDCULTURE –MYCOBACTERIAGROWTHINDICATOR TUBE(MGIT) 10 _ m Sputum specimensare processed ( computer algorithms. The instrumentdetectsthisfl indicated byfl by theadditionofacocktailantibiotics.Growth ofbacteria,includingmycobacteria,is Mycobacteria multiplyinanutrient-richmedium,whilecontaminatingbacteriaare inhibited Principle of thepresence ofviableMTBinthesputum. culture result, alongwithconfi tubes tosignalpositive(timedetection,TTD)intheBACTECMGIT960system.The quantitative assessmentofthebacterialloadbydeterminingtimetakenforculture a liquidculture media(MGIT)andtodetectpositivesamplesrapidly. To makeasemi- To amplifythenumberof Purpose MGIT mustbefamiliarwiththismanual. and 4,whichshouldbestored withineasyaccessoftheMGITsystem.Theoperator Additional detailsare foundintheBACTECMGIT960System’s User’s Manual,Chapters3 perform routine instrumentmaintenance. and stationstatus,toenterremove tubes,setuptheinstrument,printreports, and LCD displayandkeypad specimen identifi Barcode scanner a positive(+),oneindicatesnegative(–),andstationerror (!). Drawer statusindicators the 20stationcolumnsandcalibratortube. stations. Theassemblymovesfrom lefttorightandback,takingtestreadings foreachof The detectorassembly Stations Tube rack of whichholdsupto320tubes.Eachdrawercontainsanapparatusconsistingof: The tubesare arrangedinthree continuouslyincubateddrawers,labeledA,B,andC,each The BACTECMGIT960instrumentiscapableofmonitoringatotal960,7 ml MGITtubes. BACTEC MGIT960–Instrumentoverview fl concentrated sputumspecimensmetabolizeoxygenintheculture medium,allowingthe the emissionsfrom thecompoundandlittlefl presence ofoxygendissolvedinthebroth. Initially, thelargeamountofoxygenquenches contain afl with OADC(Growth Supplement)andacocktailofantibiotics(PANTA). TheMGITtubes and QualitativeCulture), andinoculatedinto7mlMGITtubes,whichare supplemented a uorescence tobedetected.Bloodsamplesare notsuitablefortheMGITsystem. n u a l

_ 2 c o –individualwellsintherackintowhichtubesare inserted. u l . uorescent compoundembeddedinthebaseoftube,whichissensitiveto –rackinthedrawerthatholdsMGITtubes i n d d uorescence, whichincreases proportionally asoxygendecreases inthetube.

3 cation. The scanner turns onautomatically. cation.Thescannerturns 2 –locatedatthefront oftheinstrument;itisusedtoscantubelabelsfor –sitsbelowtherackandhas16detectors,oneforeachrow of –presents alltheinformationneededtomonitorinstrument –three lampslocatedonthefront ofeachdrawer;oneindicates Mycobacterium tuberculosis rmatory ZN,BAP, andrapidIDtests,are theprimaryindicators uorescence inthemediumusingaUVlightandcomplex Section 7:Processing SputumforSmearMicroscopy uorescence isdetected.Bacteriapresent inthe (MTB)organismsinasampleusing 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 33 l i _ m y c o b a c t e r i o l o g is nodirect correlation ofbiomassandGUatthetimeinstrumentpositivity. the tubecansignalpositivewhenCFUistoolowtoyieldaZNsmear. Thus,there y Materials Procedure _ l a fl The GUisnotanindicationofbiomasswithinaculture tube.AMGITtubethathasbeen blood agar(seethe“SamplingTubes forFurtherAnalysis”sectionbelow). contaminated. Contaminationwillbeconfi records a curve generatedbythe Represented graphically, thisgrowth curvewouldbeverysteep,compared tothegradual of incubation If theMGITsignalstubeaspositiveandrecords aGUof staining andcontaminationchecks–see“SamplingTubes forFurtherAnalysis”sectionbelow. units. Thisvalueisidentifi algorithmswhentheGUreachesautomatically viainternal orexceedsthecutoff valueof75 The MGIT960takesareading everyhouronthehour. An‘InstrumentPositive’tubeissignaled fl The Growth Unitisanalgorithmicmeasure ofsensorfl Defi confi When positivetubesare identifi on theLCDscreen. indicator lightonthefront ofthedrawer(andanoptionalaudiblealarm)andare displayed drawers iscompletedevery60minutes.Positivecultures are immediatelyfl activating theirfl The instrumentcontinuouslyreads allthetubes.Arow ofLEDsbelowthetubesilluminates, Summary ofhowtheMGITinstrumentworks b agged aspositiveusuallycontainsabiomassofapproximately 10 uorescence voltagesignalproduced byopticalintegrationofaMGITtubeinthe960 instrument. _ m a nition ofaGrowthUnit(GU) rmationofresults, andforisolationdetectionoftheorganism. n • • • • • • • • • • • • u a Permanent marker Study labels Discard bucketwithbiohazard baginsert,containingappropriate disinfectant Tuberculocidal disinfectant p1000 pipette(orequivalent)withsterileaerosol resistant tips Pipette aid Sterile serological pipettes(10or20ml) Sterile, transferpipetteswithgraduationsmarkingvolume(individuallywrapped) 15 mltuberacks MGIT Growth Supplement MGIT PANTA MGIT tubes,7ml l

_ 2 T c o inthegrowth column.Explosivegrowth usuallymeansthattheMGITtubeis u l . , growth hasoccurred veryrapidlyandexplodedpastthe75unitcut-off. i n d d uorescent sensors.Photodetectorstakethereadings. Atestcycleofall

3 3 ed asa True Positive ed, theyshouldberemoved from theinstrumentfor True Positive . Ifexplosivegrowth hasoccurred, thesoftware rmed byanegativeZNsmearandgrowth on , andisconﬁ uorescence derivedfrom theraw rmed byfurthertestssuchas ZN 5 to10 0 orhigher 6 CFU/ml.However, within 5hours agged byan 111/04/2014 17:40 1 / 0 4 / 2 0 33 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 34 l i _ m 34 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Forms 1. for use.InoculationofMGITtubesmustbedoneinsidethebiosafetycabinet withfullPPE. Prior touse,examineallMGITtubesforevidenceofcontaminationordamage, astheseareunsuitable InoculationofMGITTubes 10.1 Record thismaintenanceontheMGITMaintenanceLogdayitwasperformed. 5. 4. Drythoroughly withpapertowelsandreplace intheinstrument. 3. Removethefi 2. 1. The instrumentairfi loading oftubes. All functionchecksmustbeperformedDAILY preferably everymorning, before unloadingor MGIT DailyInitiationRoutine lamp verifi Drawer indicator lamp verifi indicator External verifi Temperature _ m a the dateofpreparation, dateofexpiry, andpreparer initials. 5 daysifstored at2°-8°C.Iftheremaining mixture willbestored, labelitwiththecontents, Reconstitute MGITPANTA with15mlofMGITGrowth Supplement.Thismixture isstablefor and QualitativeCulture), prepare theantibioticsupplement(PANTA) fortheMGITtubes. While thespecimensare beingprocessed ( instrument. Thefaceplatewillsnapintoplace. The cut-outinthefaceplateshouldsurround theon/off switch.Firmlypress intowards the fi Remove thefaceplatebygraspingitalongbottomedgeatfi n cation rmlypullstraightout. ucin Instructions Functions u a l

_ • • • • 2 cation cation c MGIT MaintenanceLog–thisisasite-specifi Appendix B-StudySource DocumentWorksheet equivalent) Appendix D-EquipmentTemperature Record (orusesite-specifi Lab-specifi o u l . i n d d

lterandwashinanantimicrobial disinfectant.

1. OpendrawerA.Pressthe< 6. RecordthisfunctioncheckonMGITMaintenanceLog. 5. 4. Pressthe< 3. Pressthe< 2. Pressthe< 1. 4. 3. 3. 2. 7. RecordthisfunctioncheckonMGITMaintenanceLog. 6. Repeatsteps1-5forDrawersBandC. 5. 4. Pressthe< 2. 1. 3 4 ltermaintenancemustbeperformedatleast cformsforrecording MGITdata Closealldrawers. instrument Alarm indicator.instrument Alarm indicatorlampsonallthreedrawersshouldlight,aswellthe All threeexternal Record allmanualandinstrumentdrawertemperaturesontheMGITMaintenanceLog. each ofthedrawers. Verify thatthedrawertemperatureiscurrentlywithin1.5°Cofmanualreadingfor temperature readings. Press the< the station(s). All thegreenLEDsatallstationsshouldlight.Ifanydoesnot,youblock station(s). Pressthe< All theredLEDsatallstationsshouldlight.Ifanydoesnot,youblock From themainstatusscreen,press< locatedineachdrawer. thermometers Check thetemperaturereadingsoninternal test drawerindicators test indicators maintenance test redLEDs test greenLEDs test redLEDs > softkey. > softkey. > softkey. > softkeyagaintoextinguishthegreenLEDs. Section test greenLEDs > softkey. > softkeyagaintoextinguishtheredLEDs. 7:Processing SputumforSmearMicroscopy cform temperature > softkey. monthly > softkeytoaccessdrawer cform,ifavailableand nger holes.Gentlybut . To cleantheﬁ lter: 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 35 l i _ m y c o b a c t e r i o l o g y removed from theinstrument’s databaseandthetubewillbemonitored asanewlyentered tube. tube resumes. Ifthetubeisnotreturnedwithin5-hourre-entry window, theassociateddatais above), positivityroutines are reset, thestartofincubationdateisretained, andmonitoringofthe of removal. Ifthetubeisreturnedtoinstrument(via< mycobacteria orcontaminants,thetubemaybere-entered intotheinstrument,butwithin5hours signaled “negative”.Ifaninstrumentpositivetubeisdeterminedtobesmearnegativeforeither in theirstationsuntilsignaled“positive”,orifnogrowth isdetectedafter42daysincubation,are are placedinastation,there isnoneedtoremove thetubesfrom theinstrument.Cultures remain Since theMGITsystemautomaticallyandcontinuouslyincubatesmonitorstubesoncethey IncubationofMGITTubes 10.3 • • • • NOTES: instrument willassignthestations. Enter tubesintotheinstrumentassoonpossible.AlwaysscanMGITbarcodeﬁ EnteringTubes intheMachine 10.2 2. 5. Tightly recap theMGITtubeandmixwellbygentlyinvertingseveraltimes. Tightly 5. 4. 3. or “singletube”operation. removed through the< three drawerindicators,illuminatesGREEN.Theindicatorremains lituntilallnegativetubesare 42 days,thenegativetubeindicator, markedwithaminussign(-)andlocatedinthecenterof region ofthemainscreen menu,nexttotheﬁ period. Acountofthenumbernegativecultures appearsforeachdrawerinthesummary Negative cultures existasongoingnegativeswhiletheyare intheroutine 42-dayincubation DealingwithNegativeTubes 10.4 _ Accession l ucin Instructions Functions a Do notreassign tubestoanewstation. Do notremove tubesunlesstheyare positiveorout-of-protocol negatives(negativeat42days). tubesafterplacingtheminthestation. Do notturn The MGITinstrumentwillrecord thedateeachtubewasentered. b _ described in Label thesideofeachMGITtubeusingstudylabelthatcontainsidentifyinginformation tube. Disposeoftransferpipetteinbiohazard bucket. ( Using asterile,graduatedtransferpipette,add0.5mlofwell-mixedsputumsediment contaminate thetubes. using arepeat pipettorifavailable,oramicropipettor withsteriletip,takingcare notto Once dissolved,add0.8mlofthePANTA/growth supplementmixture toeachMGITtube the barcode label)onthelabMGITworksheet. Section m a n u a l

_ 8. Whenﬁ nished, closethedrawerorpress< 7. Repeatsteps1-6foreachtube. 6. Carefullyandcompletelyplacethetubeinappropriateposition. 5. 4. Ifthetube’s labelisdamaged,useasparebarcodelabel. 3. 2. Pressthe< 1. Openthedesireddrawer. 2 7:Processing SputumforSmearMicroscopy andQualitativeCulture) totheMGIT c o illuminate GREEN. sequence number).Inaddition,thestationLEDofassignedforthattubewill The assignedstationisshowninthemainbodyofdisplay(alongwithscanned was scanned. scanner. Rotatethetubeifnecessary. Theinstrument willbeeponcetoindicatethatthetube Place thetubeinalignmentblockfrontofscannerwithbarcodelabelfacing u l . Section i n d d

3 5 remove negativetubes tube entry 6.2:LoginofSputumSpecimens.Record theMGITtubenumber(from > softkey. lled circle icon.Whennogrowth isdetectedafter > operation.Tubes can beremoved via“batch” exit > tocontinuewiththenexttask. tube entry > operationdescribed rst; the rst; 111/04/2014 17:40 1 / 0 4 / 2 0 35 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 36 l i _ m 36 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b plus ( The positivetubeindicatoristheleftmostofthree drawerindicators.Itismarkedwitha DealingwithPositiveTubes 10.5 • NOTES: tube isidentiﬁ Positive Tubes the drawer. Theindicatorremains lituntilallpositivetubesare removed through the< • • barcodes) the individualtube without havingtoscan from theinstrument ﬁ nal negativecultures batch Remove negatives– Negative Tubes Report Print Unloaded the barcodescanner) must bescannedby negative tuberemoved single tube Remove negatives– Report Positive Tubes Print Unloaded Remove positives _ m be processed according to inspectallnegativetubespriortodisposal.AnythatappearhavegrowthVisually must Autoclave allMGITtubespriortodisposal. Printouts ofresults ofallnegativetubes mustbekeptinthestudybinder. instructions, andnotifytheSponsorofthisoccurrence. a n ucin Instructions Functions u (removalofall + a ucin Instructions Functions ) sign,andilluminates l

_ 2 c (each o u l . i ed. n d > operation.Inaddition,anoptionalaudiblealarmsoundswhenanewlypositive d

2. 1. Press< 9. 8. afterremoval. 7. Placethetubeintoarackorcarriertotransport 6. TheGREENandREDlightswillextinguish. 5. 4. Allpositivetubeswillbeindicatedbyﬂ ashing GREENandREDlights. 3. Pressthe< 2. OpenthedrawerwithilluminatedREDpositivelight. 1. Press<

3 6 removed fromthedatabase(iftubeisnotre-enteredwithin5hours). When youpressthesoftkey, is containedinthereport the information pressthe< discrepancies. Iftubesmatchreport, fiWhen report Resolveany nishes printing,compareunloadedtubeswithreport. appears onthedisplayscreen. andthe< off, drawer indicatorlightextinguishes,thebarcodescannerturns When allpositivetubeshavebeenremoved,theinstrumentwillbeep3times, order intherack. Repeat steps4to7removeadditionalpositivetubes,placingtheminsequential scanner. Rotatethetubeifnecessary. tube inthealignmentblockfrontofscannerwithbarcodelabelfacing Remove onetubeatatime.Scanthepositivetube’s barcodelabel byplacingthe 3. 2. Press< 1. OpendrawerwheretheGREENlightison. 4. 6. 5. Whenallnegativesareremoved,pressthe< 7. softkey, isremoved fromthedatabase. containedinthereport theinformation pressthe< discrepancies. Iftubesmatchreport, fi2. Whenreport Resolveany nishes printing,compareunloadedtubeswithreport. 1. Press< 6. 5. 4. 3. 2. 1. Followsteps1-2,asabove. Press < in thefl ashing greenstations Stations withnegativetubeslightupfl ashing GREENlights.Removealltubes tubes cannotbescanned. the < and off, GREEN drawerindicatorlightextinguishes,thebarcodescannerturns When allnegativeshavebeenremoved,theinstrumentbeeps3times, same orderinrack. atfrontofdrawerandremoveinsequentialorderplacing entered tubes.Start in thedrawerafteritisclosed,instrumentwillregisterthesetubesasnewly Close thedrawergentlyorpress< Follow steps6-7above. Continue toremovedesiredtubesandscantheirbarcodelabels. Place thetubeinaracksequentialorderofremoval. was scanned.TheLEDattheemptystationextinguishes. removing thenexttube.Theinstrumentwillbeep,indicatingthatcorrecttube As eachnegativetubeisremoved,thebarcodelabelshouldbescannedpriorto on. scanner turns Stations withnegativetubeslightupfl ashing GREENlightsandthebarcode steps forotherdrawersindicatingnegativetubes. print report silence alarm ok Section remove positivetubes red remove negative-batch remove negativetubes print report > iconappearsonthedisplayscreen. toinformyouthatoneormore positivetubesare present in > softkeyandselect< 10.6below. Inaddition,refer toFlowChart1forfurther > to mute the audible alarm (if alarm isactivated). (ifalarm > tomutetheaudiblealarm > softkeyandselect< > softkey. prior > softkey. > softkey. and off, Thebarcodescannerturns toclosingthedrawer. Ifnegativetubesremain exit unloaded positivetubes > tocontinuewiththenexttask.Repeat unloaded negativetubes ok ok > softkey. ok > softkey. > softkey. > softkey. When youpressthe > softkey. ok Remove > icon 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 37 l i _ m y c o b a c t e r i o l o g y 4. 3. 2. 1. Work withpositiveMGITtubesmustbedoneinsidethebiosafetycabinet,usingfullPPE. Materials tube broth todeterminethepresence/absence ofcontaminants. presence/absence ofacid-fastbacilliinthetube.Abloodagarplate(BAP)isinoculatedwith while contaminatingbacterialgrowth appearsturbid.AZNsmearisprepared todeterminethe growth. Thetubesshouldbeobservedvisually. MTBgrowth appearsgranularandnotveryturbid, Tubes thatsignalpositiveintheMGITinstrumentrequire furtheranalysistodeterminethetypeof SamplingTubes Analysis forFurther 10.6 • • • NOTES: • _ l a removal from theinstrument(seeprocedures in Stain allinstrumentpositivetubesforAFBandsubculture toabloodagarplate(BAP)upon Place printoutsofresults ofallpositivetubesinthestudybinder. hours takentoreach positivity(TTD). The MGITinstrumentwillrecord thedatetubesignaledpositiveandnumberofdays/ instrument. Vacate theroom andfollowthelaboratory’s SOPforactionsfollowingaspill. In theunlikelyeventofabrokenoff tubeintheinstrument–closedrawerandturn b _ fast bacteria(AFB)asdescribedin Microscopy (AFB)Preparation andStaining.ExaminetheZNsmearforpresence ofacid- Heat-ﬁ culture results are retained intheinstrument database. Re-enter theMGITtubeintoinstrumentwithin5hoursofremoval, toensure theoriginal Dispose oftransferpipetteintothebiohazard discard bucket. broth onaslide.Usewaxpencilorslideswithetchedcircles tohelpcontainsmearcontents. plate with2drops, streaking withthepipettetip.Usesametoplace2drops of drops) andinoculatethebloodagar disposable pipette.Removeabout200μlofbroth (~ 4 Vortex theMGITtubewell,unscrew tubecapandsampleanaliquotofbroth usingasterile, Take carenottoconfusetheinoculationsandlabeling. screening and/orsubjectIDnumbers,visitsputumspecimennumber, anddate. onto oneplate.Labelglassslideandbloodagarplateaccordingly withlabaccessionnumbers, Blood agarplatescanbedividedinto4quadrantssothatfourspecimenssubcultured on oneslide(aslongastheycanbespacedsufﬁ If multiplepositivetubesare beingworkedup,smearsfrom twospecimenscanbeexamined m a n • • • • • • • • • • u a Lowenstein Jensen(LJ)slant Wax pencilforencircling smearsonslide Pencil forlabelingslide Microscope slide,withfrosted end,newandclean Permanent marker Ziehl-Neelsen stain(carbolfuchsin,3%acidalcohol,methyleneblue) Sterile loopordisposableapplicatorstick Blood AgarPlates(TSAbase) Sterile, transferpipetteswithgraduationsmarkingvolume(individuallywrapped) Discard bucketwithbiohazard baginsert,containingtuberculocidal disinfectant x theslideforZiehl-Neelsen(ZN)stainingasdescribedin l

_ 2 c o u l . i n d d

3 7 Section 9:Acid-fastBacilliMicroscopy (AFB)Examination. Section cientlysoasnottocauseinterferingresults). 10.6.below). Section 8:Acid-fastBacilli 111/04/2014 17:40 1 / 0 4 / 2 0 37 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 38 l i _ m 38 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a ii. i. b. 2. 1. b 5. Below are thepossibleresults thatcanbeobtainedfrom theZN/BAPtesting. InterpretingMGITResults 10.7 3. 2. 1. Thistubeiscontaminated;itcanbediscarded. b. a.IfthesubsequentZNispositive,follow step Flow Charts1-3foradditionalguidance. further testing/stepsshouldbedocumentedinthelabworksheets/notebook. are thesubsequentstepstotakedetermineafi ii. i. a. _ m a growth ofcontaminants). is notavailable,incubateat37°C.Thelowertemperature ispreferred asitfacilitatesthe Incubate thebloodagarplateinincubatorat35°C(±1°C)for72hours(ifincubation the descriptionandsmearresult onthelaboratory worksheet. If AFBare seen,describethemastypical,atypical,andnotewhethercording isseen.Record culture (see broth toconfi the source ofcontamination.Inaddition,performaMPT/MPB64antigentestfrom theMGIT Perform aZNsmearfrom theBAPgrowth todetermineifrapidlygrowing mycobacteriaare ensures thatlownumbersof Re-incubate theMGITtubeforanother14daysandrepeat theZNsmearandBAP. This n u culture: If theZNfrom theBAPisnegative,useresults from therapidIDtesttoresult theMGIT GenoType a reincubation andrepeat testinginthe“Comments”sectionofAppendixB. MGIT culture for48hoursandretest withtheMPT/MPB64antigentest.Document If theZNfrom theBAPispositiveandIDtestnegativeforMTB,reincubate the on AppendixM:EarlyMGITPositive/EarlyContaminatedCultures -Tracking Worksheet. discarded. Record theresults from initial/subsequenttestingandreincubation ofthisculture If thesubsequentZNisnegative,thistubeconfi l contaminated.” If rapidtestispositiveforMTB,theculture is“Positivefor retesting. If rapidIDtestisnegativeforMTB,followthestepsin3aabovereincubation and

_ and contaminated”. If theIDtestisnowpositiveforMTB,thisculture isresulted as“PositiveforMTBcomplex If theIDtestisstillnegative,performHAINGenoType 2 c tuberculosis or iftheMTBDRplustestisnegativeforMTBcomplex,culture isresulted as“No If theGenoType complex andcontaminated.” If eithertestispositiveforMTBcomplex,theculture isresulted as“PositiveforMTB o u l . i n d Section d rm thepresence ofMTB.Ifnecessary, oneattemptshouldbemadetopurifythe

3 MTBDRplustests,followingthepackageinsertinstructions. 8 complexgrowth butpositiveforothermycobacteria.” NRsl A eutTTD BAPResult ZN Result TTD BAPResult TTD ZN Result BAPResult ZN Result eaieGot <7days Growth Negative Growth Negative 10.10below),ifthecontaminationinterferes withtheantigen test. oiieGot Any Growth Positive ® MycobacteriumCMtestispositiveforanon-tuberculous mycobacteria, M. tuberculosis donotgoundetected. nal result fortheMGITculture. Allresults and 3 below. rmed ascontaminatedandcanbe ® ≥ MycobacteriumCMorHAIN 7days M. tuberculosis Beneath Please referto eachtable and M. 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 39 l i _ m y c o b a c t e r i o l o g 2. 1. y i. e. IftheIDtestisnowpositiveforMTB,thisculture isresulted as“PositiveforMTBcomplex.” i. ii. b. d. c. b. a. 5. a. 4. _ l a b _ Worksheet. fi TTD from MGITprintout,ifapplicable),dateofresult at42days(ifapplicable),and the culture wasreincubated, locationofincubation,totalnumberdaysreincubated (and testing, includingoriginaldateofpositivityandTTD,results from eachZN/BAPtest,whether the originalTTDanddateofpositiveMGITresult. Record theresults from theinitial/subsequent signal), oruntiltheMGITsignalspositiveagain.RepeatZNsmearandBAP, takingnote of MGIT tubeforatleastanother3days(startcountingfrom thedateoforiginalpositive Although rare, thissituationdoesoccurandrequires furtherinvestigation.Re-incubatethe Perform aMPT/MTB64antigentesttoconfi nal results ofthisculture, onAppendixM:EarlyMGITPositive/EarlyContaminatedCultures m GenoType step If thesubsequentZNsmearisstillnegativeandBAPcontaminated,proceed asin a 3 Repeat theZNandBAPtestsafter42dayincubationperiod,proceed asinsteps Appendix B. of theadditionaldays/hoursincubationtoreport afi If thesubsequentZNispositiveandBAPnegative,proceed asinstep If MTBisconfi for theremainder ofthe42-dayprotocol. Appendix B. antigen test.Documentthereincubation andrepeat testinginthe“Comments”sectionof If thetestisnegativeorinvalid,reincubate for48hoursandretest withtheMPT/MPB64 tests, andproceed asspecifi the machinesignalspositiveagainpriortoendofprotocol, repeat theZNandBAP incubator ifmore than5hourshavepassedsinceremoval) forthefull42-dayprotocol. If MGIT tube(eitherinthemachineifwithin5hoursofremoval, orina37°C[±1°C] If thesubsequentZNsmearisstillnegativeandBAPhasnogrowth, re-incubate the and AppendixB. note oftheadditionaldays/hoursincubationtoreport afi If thesubsequentZNispositiveandBAPcontaminated,proceed asinstep n , or u on AppendixM. test. Documentwhetherornotthetubeisturbidandresult oftheidentifi for signsofgrowth (e.g.turbidity),andtesttheMGITbroth withanMPT/MTB64antigen If after42days,theZNisnegativeandBAPhasnogrowth, visuallyinspectthetube If theIDtestisstillnegative,performHAINGenoType a M. tuberculosis or iftheMTBDRplustestisnegativeforMTBcomplex,,culture isresulted as“No If theGenoType complex.” If eithertestispositiveforMTBcomplex,theculture isresulted as“PositiveforMTB l

2 _ 4 2 above,regardless oftheoriginalTTDforthisculture. above. c o u l . i n ® d MTBDRplustests,followingthepackageinsertinstructions. d rmed,record theculture result as“Positivefor

3 9 NRsl A eutTTD BAPResult ZN Result TTD BAPResult ZN Result eaieN rwhAny Nogrowth Negative oiieN rwhAny Nogrowth Positive ® complexgrowth butpositiveforother mycobacteria.” MycobacteriumCMtestispositiveforanon-tuberculous mycobacteria, ed instep 1 , rmthepresence ofMTB. 3 , or 4 above.Ifstillnegative,re-incubate ofﬂ nal TTDonthelabworksheetand ® MycobacteriumCMorHAIN nal TTDonthelabworksheet M. tuberculosis 4 above,takingnote 3 complex”. above,taking cation test cation ine 1 , 111/04/2014 17:40 1 / 0 4 / 2 0 39 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 40 l i _ m 40 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 1. SubcultureandStorageofMGITCultures 10.9 1. DrugSusceptibilityTesting fromMGITCultures 10.8 iii. ii. . Addphosphatebuffer, pH6.8tothe50mlmarkandmixwell. 5. Mixwellandletstandfor15-20minutes,mixinginvertingthetubeperiodically. 4. Addanequalquantityof4%NaOHsolution,forafi 3. Asepticallytransferentire volumeofbroth intoa50mldisposablecentrifugetube. 2. Vortex contaminatedMGITtube;letstandforabout1minute. 1. Decontamination ofaMGITtubeshouldbeperformedinthefollowingsituations: 10.10 Decontamination ofContaminatedMGITCulture 6. 2. 2. _ m a aliquot (~200ul)ofbroth andinoculate1-2LJtube(s);incubateat37°C(±1°C)( Vortex theMGITtubewellandremove tubecap.Usingadisposablepipette,remove asmall timepoints, followingtheguidelinesoutlinedin MTB growth from theMGITtubewillbeusedfordrugsusceptibilitytestingatspecifi smear, and repeat theZNsmearfrom theMGIT. Ifthesameorganismisagainidentifi has nogrowth at72hoursofincubation.Re-incubatetheBAPforanadditional24 In rare cases,non-acidfastbacteriamaybeseenonaZNnegativesmear, whiletheBAP least 3monthsaftertheculture isfi Store allinstrument-positiveMGITtubes,includingcultures growing MTBorMOTT, forat Lowenstein Jensen(LJ)and is usedfortheshort-termandlong-termstorageofisolates( drug susceptibilitytesting,ifthistestisunsuccessfulusingtheMGITbroth. ThisLJsubculture Solid Culture: LowensteinJensen(LJ)Media).Growth from thisLJsubculture canbeusedfor testing, oneattemptshouldbemadetorecover andtheprimaryLJculture isunavailablefor (see Section2:SampleSpecimenTimetable), If aMGITculture isresulted as“PositiveforMTBcomplexandcontaminated”,aDSTisrequired System. report thisculture result. consult thelabsupervisorandSponsor the presence andtypeofnon-acidfastorganismsonthelaboratoryworksheet.Inaddition, n u a l

_ • • • Record theLJsubculture dateonAppendixM. Jensen (LJ)Media)andincubateboththeLJMGITat37°C(±1°C)inanincubator. In addition,subculture theMGITbroth toaLJslant( 2 result, anddocumentthedateofnotiﬁ Notify theSponsor c corresponding primaryLJculture isnotpositiveforMTB. To prepare theshort-termLJstorageandsubsequentlong-termfrozen isolate,ifthe If theantigentestresult isindeterminate,owingtothepresence ofcontaminants. positive forMTB. is from atimepointthatrequires DST, andthecorresponding primaryLJculture isnot When theculture result is“PositiveforMTBandcontaminated”,thespecimen o and veriﬁ u l . i n d d

4 0 ed byasecondtechnician immediately Section nalized,atroom temperature awayfrom direct light. 14:Long-Term StorageofMTBIsolates). forfurtherassistanceonhowtoreport thisculture cationonAppendixM. immediately , andthe96-hourBAPremains nogrowth, note Section M. tuberculosis nalconcentrationof2%NaOH. Section 12:DrugSusceptibilityTesting: MGIT forfurtherassistanceonhowto 11:SolidCulture: Lowenstein . (seeSection10.10below). Section 11:SolidCulture: Section ed onthe 11: 11: ed 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 41 l i _ m y c o b a c t e r i o l o oiiePstv Growth Positive Positive oiiePstv Negative Positive Positive eaieNANANANegativefor OriginalTTDfrom Contaminated N/A N/A N/A Negative N/A Growth Negative Positive g y following tabletoreport theresult oftheMGITculture onAppendixB. Mycobacterial growth mustbereported immediatelyonthelaboratoryworksheet.Use 10.11 ResultsofMGITCultures Reporting 9. Resuspendthesedimentin0.5mlofphosphatebuffer andmixwell. 8. ﬂ Pouroff thesupernatant 7. Centrifugeat3,000 6. 2. 1. 10.12 Quality ControlofMGITMedia this occurrence. process according totheguidelinesinSection10.6andFlowChart1,notifyingSponsorof If MGITculture signalsnegative,buttubeappearsturbidwhenremoved from theinstrument, NOTE: * RefertoSection10.14StudyDataReportingforadditionalinstructionsrecording theTTD. _ oiiePstv No Positive Positive Machine l a Result MGIT b _ according toSection10.1above,andreincubate followinginstructionsin Inoculate 0.5mlintoafresh MGITtubesupplementedwithgrowth supplement/PANTA described in Quality control procedures forMGITmedia,andtheevaluationofTTDreproducibility, are Reagent/Media QCform,AppendixE. Record lotnumber, expirydates,etc.fornewMGIT mediumandsupplement/PANTA onthe 10.3. m a n u a l

_ Result 2 c ZN o u l . Section i n or growth d d growth

4 Result 1 BAP xg 16:QualityAssurance. for15-20minutes. HAIN GenoType by rapidIDtest, MTB complexdetected HAIN GenoType MTB notdetectedby MTBDRplus tests MTBDRplus tests or HAINGenoType Mycobacterium CM, Mycobacterium CMtest HAIN GenoType MOTT detectedby MTBDRplus test,or or HAINGenoType Mycobacterium CM, uid. HAIN GenoType by rapidIDtest, MTB complexdetected DRsl AppendixBResult ID Result ® ® ® ®

® ®

method complex; recordID ID positiveforMTB tuberculosis Positive for method complex; recordID ID positiveforMTB and contaminated, tuberculosis Positive for method complex; recordID negative forMTB mycobacteria; ID positive forother complex growthbut No tuberculosis M. tuberculosis M. M. complex complex, complex M.

applicable) Appendix M,if (and fi nal TTDfrom MGIT printout Original TTDfrom applicable) Appendix M,if (and fi nal TTDfrom MGIT printout Original TTDfrom applicable) Appendix M,if (and fi nal TTDfrom MGIT printout applicable) Appendix M,if (and fi nal TTDfrom MGIT printout Original TTDfrom 0 hours fi nal TTDof42days, MGIT printout,anda Original TTDfrom Time toResult(TTD) Section days/hours* 10.2and 111/04/2014 17:40 1 / 0 4 / 2 0 41 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 42 l i _ m 42 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 8. 7. 6. 1. NOTES 10.11 above. Appendix B.Culture results andTTDare reported according totheguidelineslistedinsection ID results, fi completed, orifanegativeculture wasincubatedlongerthan42days,0hours),ZN,BAP, and (the fi detection indaysandhoursfrom theMGITprintout,fi All liquidculture data(dateofinoculation,dateMGITresult, culture result, originaltime-to- 10.14 Study DataReporting time oftheirlastverifi instrument), whichliststhestatusofalldetectorsininstrument,alongwithdateand Maintenance Log.Eachmonth,runtheMGITQCReport(areport generatedbytheMGIT960 Perform dailyfunctionalandtemperature checksoftheMGITinstrumentandrecord ontheMGIT 10.13 Quality Control/QualityMonitoringofMGITInstrument 5. 4. 3. 2. and maintaininthelaboratoryfi _ m a the Sponsorofanycultures reported as“Unknown.” provide abriefexplanationintheMGIT“Comments”fi tube, MGITinstrumentmalfunction,etc.),report theMGITculture result as“Unknown”and In theunlikelyeventthataculture result cannotbeobtained(e.g.,broken orlostMGIT Appendix B.Thefi “TIP” valuereported ontheMGIT“unloadednegatives”printoutasoriginalTTD If theMGITculture isdeterminedtobe“Negativefor MGIT printout,andthefi If theMGITculture isdeterminedtobe“Contaminated”,record boththeoriginalTTDfrom the culture, documentthisfactintheMGIT“Comments”fi from the to performID,DST, etc.,record theMGITculture result, theTTD,anddateofMGITresult result wasobtainedandfi culture, oriftheculture wasreincubated, thisdaterepresents thedatewhenfi determined. ThiswillbethedateonMGITprintoutfrom theoriginalincubationof “Date ofMGITresult” onAppendixBshouldbethedateactualculture result was If a“Positivefor revised, fi fast bacilli),twoTTDswillbereported: 1)theoriginalTTDfrom theMGITprintout,and2) original ZNwasnegative(culture wasre-incubated andsubsequentZNwaspositiveforacid- If theMGITculture isdeterminedtobe“Positivefor these testsonAppendixB. If ZN,BAP, andIDare notperformedbecauseculture isnegative,mark“N/A”foreachof MGIT sectioniscomplete(i.e.ZN,BAP, andID). “Final dateofMGITculture completion”onAppendixBshouldbethedatealltestingin successfully recover MTBfrom a“Positivefor noted onAppendixBthattheMGITtubewasdecontaminatedandre-cultured. Ifunableto n u nal TTDwouldonlydiffer from theoriginalifreincubation wasrequired andAppendixM a l

_ 2 c o nal dateofMGITculture completion,andanyre-decontamination) are reported on u nalTTDasreported onAppendixM. l . i original n d d

4 2 cation. Thereport alsolistsallmanuallyblockedstations.Printoutthereport M. tuberculosis nalTTDshouldbewrittenas“42days,0hours”onAppendixB. sputumculture onthelabworksheetandAppendixB.Itshouldalsobe nal TTDasrecorded onAppendix M(ifreincubation was performed). nalTTDcalculatedfrom AppendixM. les. complex andcontaminated”culture isre-decontaminated M. tuberculosis nal time-to-detectionindaysandhours eldonAppendixB.Inaddition,notify M. tuberculosis eldonAppendixB. M. tuberculosis complexandcontaminated” Complex”,record the complex”,andthe nal culture 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 43 l i _ m y c o b a c t e r i o l o g y Flow Chart1: _ applicable). from AppendixM,if (and revised TTD from MGITprintout MTB, originalTTD negative, Positivefor ZN positive,BAP Report: required. term storage;DSTif Short andlong- Action: Rapid IDtest–Positive BAP – Negative ZN –Positive& True Positive report asnegativeforMTB. of growth, doZN&BAP. Otherwise, before discarding. Ifanysuspicion Action: (At theendof42-dayprotocol) Negative Signal l a b _ m a n u Observetubevisually a l

_ 2 c GeneralAlgorithmMGIT960Cultures o u l . i n d d

4 3 chart 2A. Refer toFlow Action: Negative Rapid IDtest – BAP – Negative ZN –Positive& MOTT Suspected Inoculated Tube inMGITInstrument chart 2. Refer toFlow Action: (contamination) BAP –Growth ZN –Positive& Contaminated Positive and chart 2. Refer toFlow Action: BAP –Growth ZN –Negative& Contaminated Action: Positive Signal ZN &BAP chart 3. Refer toFlow Action: tube. growth inMGIT & novisual BAP –Negative ZN –Negative, Early Positive 111/04/2014 17:40 1 / 0 4 / 2 0 43 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 44 l i _ m 44 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Flow Chart2: _ m a Flow Chart2A. Action: Referto invalid If MTB-negativeor applicable). Appendix M,if ﬁ MGIT printout(and original TTDfrom and Contaminated, Positive forMTB BAP contaminated, Report: If MTB-positive test from MGIT. Action: mycobacteria Rapid growing BAP growth ZN –Positivefrom n nalTTDfrom u a l

_ 2 c RapidID o ZNpositive, u l . i ContaminatedMGITCultures n d d Contaminated

BAP –Growth Positive and

ZN –Positive

4 4 Flow Chart2A. Action: Referto invalid If MTB-negativeor applicable). Appendix M,if ﬁ MGIT printout(and original TTDfrom and Contaminated, Positive forMTB BAP contaminated, Report: If MTB-positive test from MGIT. Action: from BAPgrowth ZN –Negative nalTTDfrom RapidID ZNpositive, again. check ZNandBAP 14 more days,then Action: in < 7days Positive signal ZN –Positive Re-incubate Contaminated BAP –Growth ZN –Negative value. contaminated, TTD culture BAP contaminated, ZN negative, Action: in Positive signal Appendix M. ﬁ MGIT printout,and original TTDfrom contaminated, culture contaminated, ZN negative,BAP Action: ZN –Negative nal TTDfrom ≥ 7days Report Report 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 45 l i _ m y c o b a c t e r i o l o g → MTBDRplus. → Mycobacterium CMorHAINGenoType y Sponsor representative. MGIT, repeatrapidIDtestandnotify If testremainsinvalid:decontaminate Flow Chart2A: _ Action: ID test If MTB–negativeorinvalidfromrapid MGIT printout. Positive forMTB,originalTTDfrom Report: If MTB–positivefromrapidIDtest retest withRapidIDtest. Action: Rapid IDtest–Negativeorinvalid BAP –Negative ZN –Positive Suspected MOTT(from FlowChart1)

l If MTBpositiveonGenoType negative onGenoType Mycobacterium CMtest,orMTB MGIT printout. Positive forMTB,originalTTDfrom Report: If positiveforMOTTonGenoType MTBDRplus test a Mycobacterium CMorGenoType MGIT printout. mycobacteria, originalTTDfrom No TBgrowth, butpositiveforother Report: b _ m a n u Perform HAINGenoType Reincubate for48hoursand ZN positive,BAPnegative, a ZN positive,BAPnegative, ZN positive,BAPnegative, l

_ 2 c o u SuspectedMOTTCultures l . i n d d

4 ® 5 MTBDRplus ®

® ®

Original MGITculture = positive signal → MTBDRplus. → Mycobacterium CMorHAINGenoType Sponsor representative. MGIT, repeatrapidIDtestandnotify If testremainsinvalid:decontaminate Action: ID test If MTB–negativeorinvalidfromrapid applicable). (and ﬁ original TTDfrom MGITprintout Positive forMTBandContaminated, Report: If MTB–positivefromrapidIDtest retest withRapidIDtest. Action: Rapid IDtest–Negativeorinvalid BAP –Growth ZN –Positive Suspected MOTT(from FlowChart2)

If MTBpositiveonGenoType negative onGenoType Mycobacterium CMtest,orMTB Appendix M,ifapplicable). MGIT printout(andﬁ contaminated, originalTTDfrom contaminated, PositiveforMTBand Report: If positiveforMOTTonGenoType MTBDRplus test Mycobacterium CMorGenoType applicable). (and ﬁ original TTDfrom MGITprintout positive forothermycobacteria, contaminated, NoTBgrowth, but Report: nalTTDfrom AppendixM,if nalTTDfrom AppendixM,if Perform HAINGenoType Reincubate for48hoursand ZN positive,BAPcontaminated, ZN positive,BAP ZN positive,BAP nalTTDfrom ® MTBDRplus ®

® ®

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 46 l i _ m 46 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Flow Chart3: _ Action: If ZN/BAPstillnegative Action: before endof42days: If signaledpositiveagain Action: BAP –Negative ZN –Negative indicated before reporting. further work-upmaybe Notify Sponsorincase and incubate. Subculture MGITbroth toLJ growth. Savetubeat37°C. MGIT. inspectfor Visually protocol. remainder ofthe42day incubate off-line forthe Note: IfZN/BAPstillnegative, 42 dayswillbecompleted. Mark capoftubewithdate protocol. 960 untilendof42-day m a n u Repeat ZN/BAP a l

_ Rapid IDtestfrom Repeat ZN&BAP. Re-incubate inMGIT 2 c o u l . i n MGIT “EarlyPositive”Cultures (ZN/BAPnegative) d 42 days d

4 6 Action: date ofﬁ again, whicheverislater. RepeatZN/BAPtests. Re-incubateculture foratleast3more daysfrom rstpositivesignal,oruntilMGITsignals Chart 1. Action: BAP –Negative ZN –Positive Regardless ofTTDvalue Follow Flow printout, andﬁ culture contaminated,originalTTDfrom MGIT Report: BAP –Growth ZN –Negative Action: BAP –Growth ZN -Positive Action: BAP –Negative ZN -Positive Follow FlowChart2. Follow FlowChart1. ZN negative,BAPcontaminated, Chart 2. Action: BAP –Growth ZN –Positive nalTTDfrom AppendixM. Follow Flow BAP. and repeat ZN& regardless ofTTD, incubate for14days, Flow Chart2:re- Action: BAP –Growth ZN –Negative Follow 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 47 l i _ m y c o b a c t e r i o l o g y Materials Procedure SOLIDCULTURE: LOWENSTEINJENSEN(LJ)MEDIA 11 _ l a media mustbetestedforsterilityandperformancecharacteristicsbefore being used. product toexcessiveheatorsunlight;andmethodlengthofstorage.Alllab-prepared including qualityofeggs;preparing andsterilizingmediumglassware; exposure offi As withallmediapreparation, attentionmustbegiventopurityofchemicalcomponents, components oftheegg. drugs mustbeadjustedtoaccountfortheirlossbyheatingorinteractionwithcertain drug susceptibilitytestsare more diffi 2) ifcontaminationisslight,itnotevidentwhenmycobacterialgrowth isconfl Disadvantages are: 1)whencontaminationoccurs,itofteninvolvestotalsurfaceofmedium, and 3)isolatedcolonieswithcharacteristiccolonymorphologyforMTBcanbeobserved. 1) itiseasyandeconomicaltoprepare, 2)itisassociatedwithlowercontaminationrates, medium isbestforroutine isolation.Theadvantagesofegg-basedmediasuchasLJare: of egg-potatobaseoralbumin-agarmedia.There isnogeneralconsensusonwhich Many different solidmediaare availableforcultivatingmycobacteria.Mostare variations Principle these positiveslantswillbestored inacool,darkplacetoarchive thepositive Slants willalsobeinoculatedfrom eachpositiveMGITtube.Oncegoodgrowth isobtained, specimens ( on LJmedium.Slantswillbeinoculatedwithdecontaminatedandconcentratedsputum The purposeofthisprocedure istoisolateandsemi-quantifygrowth of Purpose isolates. b _ m a n • • • • • • • • • • • • • • u a Incubator Study labels Pencil forlabelingslides Permanent marker Sterile distilledwater Paper towelsoakedindisinfectant Microscope slides,frosted atoneend,newandclean Parafi Ziehl-Neelsen stain(carbolfuchsin,3%acidalcohol,methyleneblue) Sterile loopordisposableapplicatorstick Discard bucketwithbiohazard baginsert,containingappropriate disinfectant Tuberculocidal disinfectant Sterile, transferpipetteswithgraduationsmarkingvolume(individuallypackaged) LJ medium l

_ 2 c o Section lm u l . i n d d

4 7:Processing SputumforSmearMicroscopy andQualitativeCulture). 7 culttoperformusingegg-basedmediabecausesome M. tuberculosis M. tuberculosis uent, and3) nal

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 48 l i _ m 48 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Forms 1. Working SolidCulture upGrowthonPrimary 11.2 1. Inoculation ofslantsmustbedoneinsidethebiosafetycabinet,usingfullPPE. InoculationandIncubationofSolidCultures 11.1 2. 3. Removeanyexcesswaterintheslantusingasteriletransferpipette. 2. 7. 6. 5. 4. _ m a Microscopy (AFB)Preparation andStaining loop orapplicatorstick,makeasmearandperformZNstain( To checkforAFBandpurity, select2to3coloniesofrepresentative growth usingasterile in Label LJtube,usingthestudy-specifi M. tuberculosis Typical growth characteristics,alongwithZiehl-Neelsenstainingproperties, are suggestiveof in thelaboratoryworksheet/workbook.Also,assesstimeforgrowth toappearonmedium. Note thecolonymorphologyandpigmentationonsolidmedium,describe thesefeatures the labworksheetandAppendixB. incubate toallowMTBgrow. IfaZNsmearisperformed,theresults shouldberecorded on Appendix B.Ifcontaminationisonlypresent onasmallsurfacearea oftheslant,continueto or notpractical(e.g.,whentheentire slantdissolves);inthesesituations,mark“N/A”on NOTE: Examination). Inoculate thetubewith200μlofsample(eitherwell-mixed,processed sputum– will containlargenumbersof care tominimizeaerosol generationwhensamplingfrom positiveMGITtubesasthisbroth graduated disposablepipette.Spread inoculumevenlyoverentire surfaceofmedium.Take Section Processing SputumforSmearMicroscopy andQualitativeCulture; orpositiveMGITtube – distinctive. the slantsurface. To observefi bench, aslongthecapsare NOTloosened. Examine andrecord results forthecultures weekly, for8intervals.Cultures canberead onthe incubation. tube canbeincubatedimmediatelyinanuprightpositionwithcaplooseforthefi then tightencapsecurely andincubateinuprightpositionat37°C(±1°C).Alternatively, the Leave tubeinslantedpositionwithcaplooseneduntilinoculumisabsorbed(aboutaweek), of thetubewithapapertowelsoakedintuberculocidal disinfectant. Replace capandensure there are nodroplets off around theoutside therimoftube.Wipe n u a Section l

_ • • • • • 2 c Appendix J-StorageLogfor Appendix E-Reagent/MediaQCForm Appendix B-StudySource DocumentWorksheet laboratory requests Laboratory SpecimenRequisitionForm–thisisasite-speciﬁ Lab-speciﬁ ContaminationofLJmaybeobvious,andaZNsmeareithernot benecessary, o u 10:LiquidCulture –MycobacteriaGrowth IndicatorTube (MGIT))usingasterile l . i n 6.2:LoginofSputumSpecimens. d ne growth, astrong direct lightfrom theanglepoiselampmustbeshoneonto d

4 complex. 8 cformsforreading/recording LJresults M. tuberculosis M. tuberculosis usuallygrows asabuff-colored, drycolony, whichisvery M. tuberculosis c labelsthatcontainidentifyinginformationasdescribed . Section IsolatesonLJMedia 9:Acid-fastBacilliMicroscopy (AFB) Section c formusedforroutine 8:Acid-fastBacilli rst weekof Section 7: 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 49 l i _ m y c o b a c t e r i o l o g y 4. 3. 2. Record weeklygrowth results onthelaboratoryworksheet. 1. SolidCulture RecordingResultsofPrimary 11.4 2. 1. Working upGrowthfromMGITSubcultureonLJMedia 11.3 4. 3. complex andreport as“PositiveforMTBandcontaminated”. contaminants, presumptively IDtheculture bylookingfortypicalgrowth characteristicsandZNstainingproperties ofMTB 2 Ifgrowth appearstobeorresembles MTB,andtheMPT/MTB64antigentestisindeterminatedueto presence of MOTT. Reportas“NoMTBcomplexgrowth, butpositiveforothermycobacteria”. of contaminants,presumptively IDtheculture bylookingfortypicalgrowth characteristicsandZNstainingproperties of 1 Ifgrowth appearstobeorresembles MOTT, andtheMPT/MTB 64antigentestisindeterminateduetothepresence oeNogrowth None te yoatra rwhPositive for other Other mycobacterialgrowth 2+ 1+ to countorconfl Recordactual uent) > 200colonies(toonumerous > 100-200colonies 10-100 colonies 1-9 colonies _ otmntdContaminated Contaminated contamination ZN+ growthinpresenceof l a b _ the laboratoryworksheetandAppendixB. Use thefollowingstandardized reporting schemetoreport growth from thesolidculture on week. If there isgrowth atanyreading interval,re-incubate thetubeandread againthefollowing is negative,record “nogrowth” onthelaboratoryworksheet. At weeks1through 7ifthere isnogrowth, record “neg“.Ifatthe8thread datetheculture Section tube usinggrowth from originalLJslant.Savetheculture intheisolatestoragebank(See If growth onsolidmediumiscontaminatedorinsuffi Preparation andStaining or applicatorstickandperformaZNstain( To checkforAFBandpurity, select2to3coloniesofrepresentative growth usingaloop On specifi (Refer to the MPT/MTB64antigentest,andresults recorded onthelabworksheetandAppendixB At allvisits,growth thatisAFBpositiverequires confi susceptibility testing, Specimen Timetable and Specimen Timetable m a n • • u a following week,continuetoread theculture weeklyuntilgrowth stabilizes. However, ifthecountincreases substantially(e.g., considered fi If thesameapproximate countisseen,reading canbestoppedandthiscount l

Growth _ 11.5. below). 2 c Section c visits,growth whichhasbeenconﬁ o u l . i n d d

13:RapidIdentiﬁ 4 nal. 9 if thecorrespondingMGITcultureisnotusable Section mycobacteria 3+ number contaminated MTB and Positive for Laboratory Laboratory Report Section 12:DrugSusceptibilityTesting: MGITSystem.) cationof 9:Acid-fastBacilliMicroscopy (AFB)Examination). Result N/A N/A pos pos pos pos pos ZN pos M. tuberculosis Section rmed asMTBcomplexwillbeusedfordrug rmation as Result / NegativeforMTBcomplex N/A T TB growth(innumerableorconﬂ uent); MTB TBgrowth(morethan100colonies); MTB TBgrowth(10-100colonies); MTB TBgrowth(1-9colonies);recordID MTB / Contaminated N/A e NoMTBcomplexgrowth,but neg T PositiveforMTBcomplexand MTB ID 8:Acid-fastBacilliMicroscopy (AFB) cient toarchive, prepare another 1+ Complexfordetails). record IDresultandtestmethod positive forothermycobacteria; record IDresultandtestmethod record IDresultandtestmethod record IDresultandtestmethod result andtestmethod test method contaminated; recordIDresultand atweek2, M. tuberculosis Study Report –SolidCulture Study Report 2 . (See 2+ Section atweek3)the complexusing 2:Sample 1 111/04/2014 17:40 1 / 0 4 / 2 0 49 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 50 l i _ m 50 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b NOTE: 5. 4. 3. 2. 1. always ashort-termstoragetube. removed toconductadditionaltesting,anewsubculture mustbeprepared toensure there is Ensure growth isconsistentwithMTBpriortostoringforshort-termstorage.Ifthistube LogginginLJCulturesforIsolateStorageBank 11.5 presumptive identifi Culture “Comments”section,suchas,“RapidIDtestresult indeterminateduetocontamination; In caseofeither#1or#2above,leavetheIDtestmethodblankandwriteacommentinLJ NOTE: of AppendixB.Inaddition,notifytheSponsoranycultures reported as“Unknown.” the solidculture result as“Unknown”andprovide abriefexplanationintheLJ“Comments”fi Intheunlikelyeventthataculture result cannot beobtained(e.g.,broken orlosttube),report 2. IfZNisnotperformedbecauseculture isnegative,mark“N/A”forthistestonAppendixB. 1. NOTES: to confi Sputum” sectiononAppendixB.Inaddition,checktheappropriate boxonpg.1ofAppendixB date ofinoculation,result, ZNresult, andidentifi Use thereporting schemein StudyDataReporting 11.7 Refer to should bedocumentedontheReagent/MediaQCForm(AppendixE)whenmediaisputintouse. Records ofbatchnumbers,datespreparation, expirationdates,andQCresults ofallmedia QualityControlofMedia 11.6 laboratory-prepared andcommercially prepared media. _ m a stored. necessary testinghasbeencompletedandallfrozen stockaliquotshavebeenappropriately After oneyearofstorage,LJsubcultures canbedesignatedfordisposal,provided thatall after theconclusionofstudy).RefertoSection14:Long-Term StorageofMTBIsolates. Use theseLJsubcultures toprepare thefrozen isolatesforlong-termstorage(i.e.6months Media (AppendixJ). Login thesedataforeachspecimenontheStorageLog Specimens. study labelthatincludesidentifyinginformationasdescribedin Seal thecapofLJtubeswithparafi (preferred) orintherefrigerator foratleastoneyearfrom thedateofpreparation. accession number, andvisitnumber. Theseisolates shouldbestored atroom temperature according toscreening IDnumberand/orsubjectnumber, sputumspecimennumber, lab All isolatesgrown ontheLJsubculture (from theMGITculture) are stored innumericalorder n u a Approval todiscard theseLJsubcultures mustbegivenbytheSponsorpriortodisposal. l rmwhethertheisolatewasarchived forshort-termstorage.

_ 2 Section c o u l . i n d d 16:QualityAssurancefordetailsonsterilityandperformancetestingboth

5 cationmadeforculture”. 0 Section 11.4abovetorecord results ofLJculture growth. Record lm. Besure thattubesare clearlylabeledwitha cation ofspeciesinthe“LJCulture of M. tuberculosis Section 6.2:LoginofSputum Isolates onLJ eld 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 51 l i _ m y c o b a c t e r i o l o g y 2 DRUGSUSCEPTIBILITYTESTING:MGITSYSTEM 12 _ l a drug-containing andthedrug-free tube. is resistant, growth anditscorresponding increase infl meanwhile, thedrug-free control willgrow andshowincreasing fl growth willbeinhibitedandfl same specimen.Ifthedrugisactiveagainstmycobacterialisolate(isolate susceptible), MGIT tube,alongwiththespecimen,andgrowth iscompared withadrug-free control ofthe as abar-coded tubecarrierthatholdstheset.Aknownconcentration ofdrugisaddedtoa testing) set,whichconsistsofaGrowth Control tubeandoneforeachdrug,aswell from sputum(detectionofgrowth). DSTisperformedusinganAST(antibioticsusceptibility Susceptibility testingintheMGIT960systemisbasedonsameprinciples asisolation Principle line drugsfortheMGITsystem. other expertshavepublishedrecommended criticalconcentrationsfortestingmanysecond- automatically detectspresence ofgrowth andcompletionoftest.Furthermore, WHOand system tobeoperational(providing somedegree ofstandardization), and3)theinstrument must becarriedoutaccording totherecommended publishedprocedures inorder forthe yields results fasterthanthegrowth-based methodsusingasolidmedium,2)testing The MGITsystemisrecommended fortestingsecond-linedrugsbecause1)thesystem specifi appropriately managethestudypatients.Theseresults willbereported onaseparate,lab- drugs, theinvestigatorand/orlabmanagermayrequest DSTofthesedrugsasnecessaryto While thestudyprotocol doesnotrequire testing/reporting ofothersecond/third-line routine qualitycontrol testingofreference strains. submit theirapproved SOPforthisprocedure, anddemonstratesatisfactoryperformancein using theproportion methodonsolidmedium.Eachlabperformingmediatestingmust If afl only DSTresults required fortestingpositivecultures from patients. drugs iswell-established,andresults are reliable andreproducible. Therefore, theyare the MGIT methodisunreliable foraspecifi kanamycin) usedinlocalstandard of care willbetestedintheMGIT 960system,unlessthe levofl (PZA)) willbetestedintheMGIT960system.Additionally, thefl All fi on thefi throughout thetrial.Astwospecimensare collectedateachtimepoint,DSTwillbeperformed DST willbeperformedonisolatesfrom newlyenrolled patientsandatregular timepoints resistance inthesepatients. is crucialforthemanagementofMDR-TBandpreventing emergenceofadditionaldrug susceptible drugs.Therefore, reliable drugsusceptibilitytesting(DST)oftheseanti-TBdrugs or acyclicpeptide),compoundsfrom otherdrugclasses,aswellanyremaining fi complex regimens canincludeafl rifampicin, andeffective patientmanagementrequires optimizedtreatment regimens. These Multidrug resistant (MDR)TBiscausedbystrainsofMTBresistant toatleastisoniazidand Purpose b _ m a rst-linedrugs(streptomycin, isoniazid,rifampicin,ethambutol(SIRE);andpyrazinamide uoroquinolone orinjectablecannot betestedinMGIT, itisacceptabletoperformDST n oai, moxifl oxacin, u cform(notonAppendixB). a l rstpositiveculture from thesetonly(notboth).

_ 2 c o u l . i n d d

5 oai, oﬂ oxacin, 1 uorescence willbesuppressed inthedrug-containingtube; oxacin) andinjectabledrugs(amikacin,capreomycin, uoroquinolone, injectabledrugs(eitheranaminoglycoside c second-linedruginMGIT. DSTofthesesecondline uorescence willbeevidentinboththe uoroquinolones (gatiﬂ uorescence. Iftheisolate oxacin, rst-line 111/04/2014 17:40 1 / 0 4 / 2 0 51 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 52 l i _ m 52 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Materials Procedure _ m with thismanual. be stored withineasyaccessoftheMGITsystem.Theoperatormustbefamiliar Additional detailsare foundintheBACTECMGIT960System’s ASTmanual,whichshould set’s Growth Control tube. for thesetilluminateorﬂ aswithindividualMGITtubes.However,same pattern withASTtubesets,ALLthestations the presence ofoneormore drawer, inadditiontoindicatingpositiveMGITgrowth tubesinthedrawer, also indicates When theASTfeature isenabledintheMGIT960instrument,Positiveindicatoron BACTEC MGIT960–ASToverview population grows inthepresence ofthecriticalconcentrationdrug. results assusceptibleorresistant. Anisolateisdeﬁ The MGIT960systemmonitorsthesegrowthandcanautomaticallyinterprets patterns a n u a l

_ • • • • • • • • • • • • • • • 2 c Sterile graduatedserological pipettes(5and10ml) p1000 andp200pipettes(orequivalent)sterileaerosol resistant tips Pipet Aid McFarland standards (0.5and1.0) NaOH, fordissolvingGatifl Sterile distilled/deionizedwater AST carriersets MGIT OADCEnrichment(MGITsupplementforsecondlinedrugs,BDcatalog #245116) BD BACTECMGITSIREandPZAsupplement Second-line drugpowders(dependinguponlocalstandard ofcare recommendations): BD PyrazinamideMGITkitreagents BD SIREMGITkitreagents 7ml MGITPZAmediumtubes 7ml MGITtubes Discard bucketwithbiohazard bagandappropriate tuberculocidal disinfectant – – – – – – – o u Ofl Moxifl Levofl Kanamycin (Sigmacatalog#K1876orK4000) Gatifl Capreomycin (Sigmacatalog#C4142) Amikacin (Sigmacatalog#A1774orA2324) l . i n oxacin(Sigmacatalog#O8757) d d oxacin(Sigmacatalog#G7298)

oxacin(Sigmacatalog#28266)

oxacin(Sigmacatalog#32477)

5 2 ash. TheleftmoststationisALWAYS theassignedlocationof the completed oxacinandOﬂ ASTsets.Thestationindicatorsilluminateinthe oxacin,ifapplicable ned asresistant if1%ormore ofthetest 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 53 l i _ m y c o b a c t e r i o l o g y Forms Concentrations forDSTinMGIT, below. drugs. Failuretousetheappropriatevolumewillinvalidatethesetests.Refer toTable 12.1-Drug Reconstitute thedrugswithappropriatevolumeofdiluent.Volumes withdifferent vary full PPE. Drug stocksandpreparationofMGITtubesmustbecarriedoutinsidethe biosafetycabinet,using SIREandPZADrugStockPreparation 12.2.1 PreparationofDrugStocksforSusceptibilityTesting 12.2 AST set. system tointerpret theabsenceofatubeasanerror, whichinvalidatestheresults fortheentire in thesetisencodedcarrier’s barcode. Usinga carrier oftheincorrect sizewill causethe you mustusethecorrect sizetubecarrierfortheASTsetyouare testing.Thenumberoftubes Appendix A)forASTcarriersizesandoptionalconfi MGIT drugswillbetested,checktheBACTEC960User’s Manual(ASTInstructionsand The susceptibilitytestmaybeconfi PreparingASTCarriers 12.1 _ l a b _ m a n • • • • • • • • • • • • • • • • • • • • • u a Appendix B-StudySource Document Appendix F-DSTQCForm(orusesite-specifi Lab-specifi Study labels Permanent marker BD BactecASTCarriers 2 mlsterilecryotubeswithscrew top(polyethyleneorpolypropylene) Syringe fi Sterile syringes(10or20ml) Erlenmeyer fl Graduated cylinder Analytical balanceandweighboats Vortex BBL Middlebrook 7H9broth Capped steriletubescontaining2mmglassbeads,forpreparing inoculumfrom LJ Sterile disposablelooporapplicatorstick McFarland standards 16.5 x128mmsteriledisposabletubes,ortubesofthesamesize(diameter)aslab’s Middlebrook 7H9broth, forpreparing inoculumfrom positiveLJcultures Blood agarplates Sterile saline Sterile graduatedtransferpipettes,individuallywrapped l

_ 2 c o u l . i n lters(0.22μmpore size;e.g.,Millex-GS) d d cformsforrecording DSTresults

5 askormediabottles 3 gured inavarietyofformats.Sincedifferent proﬁ gurations. WhensettinguptheASTtubesets, cform,ifavailableandequivalent) les ofnon- 111/04/2014 17:40 1 / 0 4 / 2 0 53 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 54 l i _ m 54 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b considered. amount ofsolventneededtoobtainthisconcentration,thepotency drugpowdermustbe standard weights.Ifpossible,weighmore than100mgofpowder. To calculatetheappropriate The antimicrobial powdermustbeweighedonananalyticalbalancethathasbeencalibratedwith Calculation: to calculatethepotencyinunitsof In somecasesthepotencymaybeexpressed asapercentage. Thefollowingexampleshowshow Formula forpotencyofdrug drug powder. the labmuststandardize theantimicrobial solutionsbasedonpotency oftheindividualloteach from theactualweightofpowderandoftenmaydiffer betweendrugproduction lots.Thus, All antimicrobial agentsare assayedforstandard unitsofactivity. Theassayunitsmaydiffer widely Weighing drugs determined from theformulabelow. clearly determinedfrom theCOA.IfpotencyinformationisnotincludedonCOA,itmaybe the Sigmawebsite.Contactsupplierormanufacturer ifanyvalueismissingorcannotbe COA isnotincludedwiththeantibioticshipment,itcanbedownloadedbylotnumberfrom Check the“Certiﬁ it mustcometoroom temperature before itisopenedtoavoidcondensationofwater. The powdersare stored asrecommended bythemanufacturer. Whenremoved from thefreezer, potency (usuallyexpressed inmicrograms [μg]ofdrugpermgpowder),anditsexpirationdate. pharmaceutical company. Acceptabledrugpowdersare labeledwiththegenericname,itsassay All second-linedrugswillbeobtainedinchemicallypure formfrom Sigmaortheappropriate Source ofdrugs SecondLineDrugStockPreparation 12.2.2 not store orrefreeze. the dateoforiginalexpiry, whichevercomesﬁ aliquot anyleftoverdrugsolutionsandfreeze at-70to-80ºC(±10°C)up6monthsor Store lyophilizeddrugsat2-8ºCuponreceipt andreconstitute priortouse.Oncereconstituted, SIRE Kit Prepare drugsinMGIT960 PZA Kit Prepare drugsinMGIT960 _ m a n u a l

_ • • • 2 c Active fraction=100% Measured watercontent=12.1% Assay purity=99.8% o akInstructions Task u l . i n d d

Potency =(assaypurity)x(activefraction)(1–watercontent) Potency =(assaypurity)x(activefraction)(1–watercontent)

cate ofAnalysis”(COA)provided bySigmaforeachdrug/lotnumber. Ifthe Potency =(998)x(1.0)(1–0.121) 5 4 3. 2. 1. 1. 4. to makeastocksolutionof8000µg/ml. Reconstitute each distilled/deionized watertomakeastocksolutionof8.3µg/ml. Reconstitute each distilled/deionized watertomakeastocksolutionof83µg/ml. Reconstitute each Reconstitute PZAdrugvialwith distilled/deionized watertomakeastocksolutionof415µg/ml. Reconstitute each distilled/deionized watertomakeastocksolutionof83µg/ml. μg/mg (w/w) rst. Oncethawed,discard anyleftoverdruganddo R I S E soniazid lyophilizeddrugvialwith thambutol lyophilizeddrugvialwith treptomycin lyophilizeddrugvialwith ifampicin lyophilizeddrugvialwith : 877 μg/mgor87.7% 2.5 ml ofsteriledistilled/deionizedwater 4 ml 4 ml 4 ml 4 ml ofsterile ofsterile ofsterile ofsterile 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 55 l i _ m y c o b a c t e r i o l o weight is180.0mg,thevolumeofsolventneededasfollows: a potencyof750μg/mg,170to200mgpowdershouldbeaccuratelyweighed.Iftheactual Example: . Discard excessdrugandneverre-freeze. 3. 2. Thawindividualcryovialstoroom temperature. 1. Before inoculatingMGITtubes: Preparing workingsolutions whichever comesfi up to12monthsat-70-80°C(±10°C),orthedateoforiginaldrugpowderexpiry, Dispense smallvolumesofsterilestocksolutionsintocryovials,carefully seal,andstore for otherwise recommended bythemanufacturer. concentration (seetablebelow),andsterilizedbymembranefi All stocksolutionsmustbemadeatleast1,000μg/ml,orten-foldhigherthanthedrug’s working g y Formula forvolumeofsolvent 4. 3. Prepare 0.1N(0.1M)NaOH.Forexample,dissolve4gNaOHin1litersteriledistilledwater. 2. Addweigheddrugtoasteriletube,e.g.,50mlFalcontube. 1. if therecommended solventforOfl powder, andthendilutetothefi other thanwaterisrecommended, onlyusesuffi Follow themanufacturer’s guidelinesontheCOAforrecommendation ofsolvent.Ifasolvent Solvent fordrugs formula below. and thencalculatethevolumeofsolventneededtoobtainrequired concentration,usingthe It isadvisabletoweighoutalargeramountofthedrugthanrequired (forthespecifi _ l a tubes are listedinthetablebelow. and usewithoutdelay. Test concentrationsforeachdrugandthevolumesaddedtoMGIT Dilute asappropriate insteriledistilledwatertoachievethecorrect workingconcentration b _ water. Finish dilutingdruguptotheappropriate volume(calculatedbelow)withsteriledistilled drug dissolvescompletelyandthesolutionisclear. Add 0.1NNaOHsolutiondrop-wise, shakinggentlyaftereachaddition.AddNaOHjustuntil m a To prepare astocksolutioncontaining10,000μg/mlofkanamycinwithpowder that has n u a l

_ 2 c o u l . i rst. n d d

5 5 Volume Volume = nal stockconcentrationwithsteriledistilledwater. Forexample, oxacinis0.1NNaOH: 180.0 mgx750μg/mg = (Actual weight)x(Potency) 10,000 μg/ml (Desired concentration) cient solventtosolubilizethe antimicrobial =13.5ml ltration (0.22μmpore size),unless c concentration) 111/04/2014 17:40 1 / 0 4 / 2 0 55 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 56 l i _ m 56 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a eo xcn16 100 166 Levofl oxacin Kanamycin b Table 12.1 . Asepticallyadd0.8mLofBACTECMGITPZAsupplement toeachPZAtube. 3. 2. 1. the pHisdifferentforbothPZAmediumandsupplement. supplied withthePZAkit.DonotuseconventionalMGITtubesor ‘SIREsupplement’,as When preparingPZAtubes,itisimportanttousethemediatubes and‘PZASupplement’ Preparation ofPZA 12.3.2 DonotadddrugstotheMGITGCtube. 6. Itisimportanttoaddthecorrect drugtothecorresponding tube. 5. 4. 3. 2. 1. Preparation ofSIRE 12.3.1 PreparationofTubes forSusceptibility Testing 12.3 testing performed. testing thisdrugmustsubmitawrittenrationaleforthecriticalconcentrationusedtesting,aswellanyvalidation 2 WHOhasnotrecommended acriticalconcentrationforthetestingofthisdruginMGIT960instrument.Laboratories Geneva, WHO,2008(WHO/HTM/TB/2008.392). 1 World HealthOrganization.PolicyGuidanceonDrugSusceptibilityTesting (DST)ofsecondlineanti-tuberculosis drugs. mkcn8 0 1.0 100 1.0 5.0 0.1 1.0 100 100 100 100 100 100 83 8000 415 83 8.3 83 Amikacin MGIT PZA MGIT EMB MGIT RIF MGIT INH MGIT STR aroyi 0 0 2.5 100 100 100 166 20.75 208 Enviomycin Ofl oxacin Moxifl oxacin Gatifl oxacin Capreomycin _ m a PZA. Place thetubesinfollowingsequence2tubeASTsetcarrier, from lefttoright:GC, label tubeswithoneofeachthefollowing:GC(Growth Control) orPZA(pyrazinamide). identifying informationdescribedinSection6.2:LoginofSputumSpecimens. Inaddition, Label two7mLMGITPZAmediatubesforeachtestisolatewithastudy labelthatincludes etc. tube; e.g.,add100μlofthe83μg/mLMGITSTRsolutiontotubelabeled“STR”, Aseptically pipette100μloftheappropriately reconstituted drugintothecorresponding MGIT SIRE tube.Itisimportanttousethesupplementsuppliedwithkit. Aseptically add0.8mLofBACTECMGITSIRESupplement(provided intheSIREkit)toeach STR, INH,RIF, EMB. Place thetubesinfollowingsequence5tubeASTsetcarrier, from lefttoright:GC, RIF (rifampicin),EMB(ethambutol). with oneofeachthefollowing:GC(Growth Control), STR(streptomycin), INH(isoniazid), information describedin Label fi n u a l

_ 2 Drug 2 c 2 ve 7mLMGITtubesforeachtestisolatewithastudylabelthatincludesidentifying DrugConcentrationsforDSTinMGIT 2 o u l . i n d d

5 6 working solution(µg/ml) Concentration ofdrugin Section ––– ––– ––– 6.2:LoginofSputumSpecimens.Inaddition,labeltubes Volume addedtoMGIT tubes fortest(µl) concentration inMGIT tubes (µg/ml) Final/Critical 2.0 1 1 1 1 0.25 1 2.0 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 57 l i _ m y c o b a c t e r i o l o g y 1. UsinganInoculumfrom PositiveMGITCulture 12.4.1 DST ifmorethan5dayshaveelapsedaftersignalingpositive. thus, thefollowinginstructionsmustbeadheredtostrictly. Culturesmustnotbeusedtoset up Bacterial densityoftheinoculumiscriticaltocorrectperformancesusceptibilitytest; outallworkwithpositiveMGITtubesinbiosafetycabinet,usingfullPPE. Carry InoculumforMGITDST 12.4 • NOTES: Preparation ofSecond-LineDrugs 12.3.3 DonotadddrugstotheMGITGCtube. 5. MGIT tube. Asepticallypipette100uLofthe8000μg/mlMGITPZAsolutiontoappropriately labeled 4. 1. • • 4. 3. DSTmustnotbesetuponthesamedayaMGITtubesignalspositive. 2. . DonotadddrugstotheMGITGCtube. 8. Itisimportanttoaddthecorrect drugtothecorresponding tube. 7. 6. 5. 4. Ifaseparatecontrol willbeused,labelaGCtube(Growth Control). 3. 2. _ l a possible “undefi If second-linedrugsare entered intotheMGITinstrumentusingoneofinstrument’s three carrier. If needed,blankMGITtubes(non-inoculated,drug-free) canbeusedtofi carrier, thusrequiring onlyoneGCtube. Alternatively, second-linedrugscanbeaddedbehindasetofSIREusingthe8-tubeAST for useinaseparatecarrierfrom theSIREtubes. b _ Positive MGITcultures musthavepure growth of information describedin Label 7mLMGITtubesforeachtestisolatewithastudylabelthatincludesidentifying 1 mlofMGITbroth in4mlofsterilesaline(1:5dilution). If theculture isusedtosetupDSTbetweenthree andfi inoculate theDSTMGITtubes. If theculture isworkeduponeortwodaysaftersignalingpositive,itcanbeuseddirectly to an Immunochromatographic Assay)inorder tobetestedfordrugsusceptibility. MPT/MTB 64antigentestpositive; second-line drugs. Refer tothetableaboveforappropriate volumesandworkingsolutionconcentrationsofthe Aseptically pipette100μloftheappropriately diluteddrugintothecorresponding MGITtube. to eachMGITtube. Aseptically add0.8mLofBACTECMGITSIRESupplement,orappropriate OADCEnrichment, always intheleft-mostposition. Load tubesinthecarriersetsameorder consistently, ensuringthattheGCtubeis AMK (amikacin),CAP(capreomycin), OFL(ofl In addition,labeltubeswiththeappropriate second-linedrugnameorabbreviation; e.g., Indicator Tube (MGIT) and m a n u a l

_ 2 c o u l . i n ned drug”setconﬁ d d

5 7 Do notuseMGIT960growthsupplementorPZAsupplement. Section Section 6.2:LoginofSputumSpecimens. gurations, aseparatedrug-free GCtubemustbeprepared 13:RapidIdentiﬁ Section oxacin),etc. 10:LiquidCulture –MycobacteriaGrowth M. tuberculosis cation of ve daysaftersignalingpositive,dilute M. tuberculosis (ZNpositive,BAPnegative, ll emptyslotsina ComplexUsing 111/04/2014 17:40 1 / 0 4 / 2 0 57 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 58 l i _ m 58 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b

9. 8. 7. 6. 5. 4. 3. 2. 1. outallworkwithpositiveLJslantsinbiosafetycabinet,usingfullPPE. Carry UsinganInoculumfrom PositiveLJCulture 12.4.2 5. _ m a patient population,the3-5dayculture canbeused NOTE: tubes. mmsteriletube.Thisdilutedinoculumisusedforpreparation oftheMGITDST 16.5 x 128 Dilute 1mloftheadjustedsuspensionin4sterilesaline(1:5dilution) usinganew above a0.5McFarlandStandard. Using 7H9broth, adjustthesuspensiontoa0.5McFarlandstandard. Donotadjustbelowor sterile tube. Transfer fl thesupernatant transferring anyofthesediment)andletsuspensionstandforanother 15min. Transfer fl thesupernatant of thetube. Let thesuspensionstandfor30minwithoutdisturbing.Sedimentshouldsettletobottom exceed a1.0McFarlandstandard inturbidity. Vortex thesuspensionfor2-3mintobreak upthelargerclumps.Thesuspensionshould Middlebrook 7H9broth. no more than14daysold.Donotremove anysolidmedium.Suspendthecoloniesin Using asterilelooporapplicatorstick,scrapeasmanycoloniespossiblefrom growth standards). with capcontaining6–10,2mm,glassbeads(orusetubethesamesizeaslab’s McFarland Add 4mlofMiddlebrook 7H9Broth (orBBLMGITbroth) toa16.5x128mmsteriletube Complex UsingaChromatographic Immunoassay). Culture: LowensteinJensen(LJ)Mediaand M. tuberculosis or failstogrow DST canbeperformedwithgrowth from positiveLJslantsiftheMGITtubeiscontaminated If theculture hasbeenpositivelongerthanfi subsequently produced. Flow Chart4.Monitorthistechniqueverycloselytoensure thatanexcessofx400errors isnot n u a l

_ • • • • • • • • 2 c Use newtubeforDSTtestsfrom onetoﬁ Enter tubeintoMGIT960instrumentandmonitoruntilitbecomespositive. Cap tubetightlyandmixwellbyinvertinggentlyseveraltimes. Inoculate newMGITtubewith0.5mlofthe1:100dilutedspecimen. Mix tubewellbyinvertinggentlyseveraltimes. MGIT tubeintosterilesalineor7H9broth. Remove inoculumfrombroth thesupernatant andmakea1:100dilutionofpositive Supplement anewMGITtubewith0.8mlGrowth Supplement to settle. Vortex MGITbroth welltomixthoroughly. Leave5-10minutestoallowanylargeclumps Ifalaboratoryroutinely experiencesx200errors whenperformingDSTinthelocal o u l . i n d d

5 (ZNpositive,MPT/MTB64antigenormoleculartestpositive; 8 M. tuberculosis uid (itshouldbesmooth,free ofanyclumps)toathird 16.5 x 128 mm uid toanother16.5x128mmsteriletubewithcap(avoid . Theculture mustbeidentifi Section vedays,subculture intoafresh MGITtube: vedaysofpositivityasdescribedabove. 13:RapidIdentifi undiluted asafi ed asapure growth of cation of rststep,asdescribedin without Section M. tuberculosis PANTA. 11:Solid 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 59 l i _ m y c o b a c t e r i o l o g y NOTE: to right: Place thetubesinappropriate carrierset,ensuringtheyare inthecorrect sequencefrom left 12.7 recap thetubes.Mixtubesbygentlyinverting3-4times. Tightly 3. 2. 1. InoculationofTubes Containing Test Drugs 12.6 4. MixtheGrowth Control suspensionthoroughly bygentlyinverting3-4times. 3. 2. 1. Forpreparation ofPZAGCTube 12.5.2 4. MixtheGCsuspensionthoroughly bygentlyinverting3-4times. 3. 2. 1. Forpreparation ofSIREandsecond-lineDrugGCTube(s) 12.5.1 GrowthControlTube PreparationandInoculation 12.5 DST toensure accurateresults andavoidPZAASTseterrors. _ l a b _ “GC” forthatspecimen,usingamicropipettor andsterile aerosol resistant tips. disturb thesediment. and sterileaerosol resistant tips–aseparatetipmustbeusedforeachtube.Take care notto containing tubes(STR,INH,RIF, EMB,PZA,any2ndline drugtubes),usingamicropipettor Aseptically pipette0.5mloftheorganismsuspensionfromintoalldrug- thesupernatant mix well;letsuspensionsettlefor5-10minutes. Vortex originalMGITtube,dilutedculture, ordilutedinoculumfrom LJasapplicable,to Inoculate 0.5mlofthe the Aseptically pipette0.5mloftheorganismsuspensioninto4.5sterilesalinetoprepare mix well;letsuspensionsettlefor5-10minutes. Vortex originalMGITtube,dilutedculture, ordilutedinoculumfrom LJasapplicable,to specimen, usingamicropipettor andsterileaerosol resistant tips. Inoculate 0.5mlofthe1:100GCsuspensionintoallMGITtubeslabeled“GC”forthat the 1:100GCsuspension(1%growth control). Aseptically pipette0.1mloftheorganismsuspensioninto10sterilesalinetoprepare suspension settlefor5-10minutes. Vortex originalMGITtube,dilutedculture, ordilutedinoculumfrom LJandmixwell;let m a Itisimportanttouseanappropriately prepared n 1:10 Entering andRemovingASTSetsintheMGIT960InstrumentforSIREPZA • • u a For PZA,usethe2tubeASTcarrier:GC,PZA applicable) For SIRE,usethe5tubeASTcarrier:GC,STR,INH,RIF, EMB(orthe8tubecarrier, if l

_ 2 Growth Control suspension. c o u l . i n d d

5 9 1:10 Growth Control suspensionintothe 1:10 dilutionforthe“GC”tubePZA PZA MGIT tubelabeled 111/04/2014 17:40 1 / 0 4 / 2 0 59 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 60 l i _ m 60 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b containing vialsare evaluated. control reaches 400within4-13days(SIRE)or4-21(PZA),theGUvaluesofdrug- Control tubeisusedtodeterminesusceptibilityresults. Whenthegrowth unit(GU)ofthegrowth Analysis ofﬂ The BACTECMGIT960instrumentcontinuallymonitorsalltubesforincreased ﬂ InterpretationofDSTResultsforSIREandPZA 12.8 BACTEC MGIT960System’s ASTmanual,Chapters 4and6,forassistanceinresolving thiserror. NOTE: Open desired drawerinMGITinstrument,andfollowinstructionsbelow. Invalid testsare reported whencertainconditionsoccurthatmay affect testresults: Set” report Printing an“UnloadedAST on thedrawer) sets (indicatedbyared“+” Removing completedAST Entering newASTsets _ m a n u a Ifastationerror hasoccurred, the“!”indicatorwillilluminateYELLOW. Refertothe l

_ • • • 2 ucinDirections Function c R = S = a pure culture oftheisolate. affect thetest.Ifpossible,determinecauseoferror. Inanycase,repeat testingwith X ###=Error orIndeterminateresults; reported whencertainconditionsoccurthatmay − Otherconditions,suchaspowerfailure. − − o u uorescence inthedrug-containingtubescompared tothefl specimen. Pleaserefer toFlowChart5forfurtherinstructions. turbidity andsubculture toabloodagarplateruleoutcontaminationofthe and doesnotprovide aninterpretation oftheASTsetresults. Checkthetubefor X400 =Systemdetectsindicationsofpossiblecontaminatedoroverinoculated tube, growing drug-resistant strain.Pleaserefer toFlowChart4forfurtherinstructions. AST setresults. Oftenaresult oftoolittleinoculum,nonviableorganisms,oraslow- tube inthespecifi X200 =Systemcannotdetectsuffi l . i Susceptible Resistant n d d

6 0 =theGUofdrugtubeis100ormore =theGUofdrugtubeislessthan100 2. Press< 1. Pressthe< 5. 4. Repeatsteps2–3toremoveadditionalASTsets. 3. 2. 1. 7. 6. 5. 4. 3. 2. 3. Match the AST sets with the printed report, andresolveanydiscrepancies. 3. MatchtheASTsetswithprintedreport, 1. Pressthe< Place completedASTsetsinthetuberack. drawer, andscanitsbarcodelabel.TheLEDsatthisstationextinguish. Remove thecarrier, withthecompletedsetclosesttofrontof starting indicators. The fi rst completedASTsetstationsilluminatewithFLASHINGGREEN Open thedesireddrawer. Pressthe< status. CorrectanyerrorsbeforetheMGIT960beginstesting. setintoerror AST sets,thequickscancandetectthisandputaffected a“quickscan”ofdrawercontents.Ifyouhavemisplacedany performs When fi nished, closethedrawerandwaitamomentwhile Repeat Steps1–5foreachoftheASTsetsyouwanttoenter. the tubesANDcarriersarefullyseatedindrawer. Control tubeislocatedintheleftmostindicatedstation.Makesurethatall thetubesetintoindicatedstations,ensuringthatGrowth Insert The stationLEDsofalltheassignedstationsforsetilluminateGREEN. is displayedcorrectlyfortheASTsetcurrentlybeingentered. The displayshowsthedefaultcarrierset.CheckthatASTsetdefi nition < Scan theaccessionbarcode,ifavailable;notpresent,press it isalongerprotocol. For PZAtesting,selectasthedrugintwotubecarrierdefi nition as AST set,howmanytubesareintheandset’s sequencenumber. Scan theASTcarrier’s barcodelabel.Thecarrierindicatesan ed protocol time,anddoesnotprovide aninterpretation ofthe accession barcodenotavailable unloaded ASTsetsreport printer tube entry > soft key to access report selection. > softkeytoaccessreport cient indicationofgrowth intheGrowth Control > softkey. > softkey. > softkey. remove completedASTsets uorescence intheGrowth uorescence. > softkey. 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 61 l i _ m y c o b a c t e r i o l o g y instrument, andfollowinstructionsbelow. one extrastepofchangingtheASTsetdefi Entering andremoving ASTcarriersetsisthesameasdescribedaboveinSection12.7,with unloaded ASTreports mustbewritten andperformedbyallapplicablestaff: For bothmanualandinstrumentinterpretation, thefollowingprocedures forcorrecting theMGIT 2. 1. line drugtestinginMGIT. There are twooptionstoperformsecond-lineDST: MGIT system.Therefore, asite-specifi There isnomeansfordesignatingthedrugnameorconcentrationsofsecond-linedrugsin EnteringandRemovingASTSetsforSecond-lineDrugs 12.9 3. 2. 1. NOTES: _ l a b _ instrument willinterpret results automaticallywhenthetestiscomplete. protocol. Eachsecond-linedrugiscodedwiththenameofonefi Instrument interpretation −UseASTsetcarrierandenterintheinstrumentasafi unloaded ASTsetreport. drugs. Inthiscase,interpretation ofresults isdonemanuallyusingtheGUvaluesfrom the Manual interpretation −UseASTsetcarrierandenterintheinstrumentas“Undefi the fi patient, review theresults andQCrepeat thetest.Ifrepeat result isdiscrepant with If DSTresults forisoniazidorrifampicinare inconsistentwithprevious results forthesame Note toFileshouldbewrittendocumenttheDSTfailure. be obtainedafterthesecondattempt,testshouldreported as‘TF’(testfailed)anda DST forSIREandPZAshouldonlyberepeated onceifthefi growth isnotduetocontaminantsorMOTT. isolate wasnotpreviously resistant tothedrug,testtube(s)withZNandBAPensure that when drugresistance isobservedandthepatient’s isolatehasnotbeentestedbefore, orifthe the instrument.PerformaZNstainonanysuspicioustubeandsubculture toaBAP. Inaddition, Observe all‘resistant’ tubesvisuallyforevidenceofcontaminationwhenfi m a n • • • • • u rstresult, repeat thetestathird timeandrecord thethird testresult asthetiebreaker. a Process forclarifyingthedrugnamesonMGITprintout Approved abbreviations forthefulldrugnames Confi AST carrier(s)used the workingsolutions Instructions forpreparing theantibioticstocksolutionsanddilutionsneededtoachieve − − − l

_ 2 have beenupdated Initial anddatethecorrected MGITreport afteralldrugnamesandconcentrations simply writeintheconcentrationtestedforeachdrug. appropriate concentrationfortheactualdrugtested.If“undefi Cross outeachconcentrationlistedforthefi next toit. drug name,andaddtheapplicable2ndlineorapproved abbreviation, Cross outeach“undefi c o gurationofthedrugsinASTcarrierset(s) u l . i n d d

6 1 ned” orﬁ c procedure mustbedevelopedfordocumenting second nitions (ifapplicable).Opendesired drawerinMGIT rst-line drugnameusingonelinethrough theprinted rst-line drug,andreplace itwiththe rst testfails.Ifavalidresult cannot ned” protocol isused, rst-line drugs.The rst removed from rst-line drug ned” 111/04/2014 17:40 1 / 0 4 / 2 0 61 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 62 l i _ m 62 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b manually forthe“undefi however, susceptibilityisnotinterpreted bytheinstrument.Theinterpretation mustbemade and interprets theresults. Forundefi the GCtubereaches ≥400withinthetimedprotocol, theinstrumentmarksASTsetcomplete For allsetconfi 12.10 Interpretation ofDSTResultsforSecond-LineDrugs • • NOTES of care. Enviomycin intheMGITsystem.However, somelabsare testing thesedrugsaspartofstandard WHO doesnotcurrently recommend criticalconcentrationsforGatifl 12.11 DST forEnviomycin,Gatifl oxacin, andKanamycin 3. 2. 1. NOTES: Set” report Printing an“UnloadedAST on thedrawer) sets (indicatedbyared“+” Removing completedAST sets Entering new2nd-lineAST _ m manual listsallpossibleASTsetconfi For furtherinformation,pleaseseeinstructionsintheBDMGITDSTmanual;appendixAof drug sequence(growth control, undefi Remember toloadtubesintothecarrierseteachtimeinsameorder, i.e.,withthesame a the tiebreaker. is discrepant withthefi results forthesamepatient,review theresults andQCrepeat thetest.Ifrepeat result If DSTresults foranyofthefl failures. in theDST“Comments”sectionofAppendixB,andwriteaNotetoFile documenttheDST cannot beobtainedafterthesecondattempt,report thetestas“TF”,provide abriefcomment DST forsecondlinedrugsshouldonlyberepeated onceifthefi 12.9 above. the ASTsetconfi Errors, e.g.,x200andx400,are generatedthesameasforfi n u a l

_ • • 2 ucinDirections Function c R = S = o u l . i Susceptible Resistant n d gurations exceptthe“undeﬁ d

6 2 guration usedfortesting,andnecessitaterepeating asdescribedin =theGUofdrugtubeis100ormore ned”optionaccording tothefollowingcriteria: =theGUofdrugtubeislessthan100 rst result, repeat thetestathird timeandrecord thethird testresult as 4. 3. 2. Press< 1. 5. PlacecompletedASTsetsinthetuberack. 4. Repeatsteps2–3toremoveadditionalASTsets. 3. 2. 1. 5. 2. Pressthe< 1. Followsteps1-4in“EnteringNewASTsets”above. 3. Match the AST sets with the printed report, andresolveanydiscrepancies. 3. MatchtheASTsetswithprintedreport, Pressthe< Press the< set isindicatedbyacheckmarkinthemainbodyofdisplay). available defi nitions forthescannedcarriersize(Asyouscroll,default Use theUPARROWorDOWNkeytoscrollthrough drawer, andscanitsbarcodelabel.TheLEDsatthisstationextinguish. Remove thecarrier, withthecompletedsetclosesttofrontof starting indicators. The fi rst completedASTsetstationsilluminatewithFLASHINGGREEN key. Open thedesireddrawer. Pressthe< Follow steps5-7in“EnteringNewASTsets”above. being tested. “undefi ned drugs”mustcorrelatewiththenumberofsecond-linedrugs uoroquinolones orinjectable agentsare inconsistentwith previous ned drugsonly, GUsare recorded ontheunloadedsetreport; unloaded ASTsetsreport gurations. printer okay change ASTsetdefi neddrug#1,undefi > softkeytoselectthehighlightedset.Thenumberof ned drug”protocol, whenthegrowth unit(GU)of > soft key to access report selection. > softkeytoaccessreport nition > softkey. neddrug#2,etc.). > softkey. remove completedASTsets rst-line drugs depending upon rst testfails.Ifavalidresult oxacin, Kanamycin,or > soft Section 111/04/2014 17:40 1

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 63 l i _ m y c o b a c t e r i o l o g y patient records. the labrequisition form(ifapplicable).Also,keeptheMGITprintoutsforallDSTresults withthe Record “ 12.12 ofDSTResultsforAllDrugs Reporting on solidmedia. Section recommendations, aswellanyappropriate qualitycontrol andassuranceactivitiesdescribedin solidmediumDSTprocedures,The labshouldfollowtheirinternal anyapplicablemanufacturer’s DST results willbeacceptedfor: 4. 3. . Reportsusceptibilitytestingresults onAppendixBforthespecimentestedasfollows: 2. 1. 12.14 Study DataReporting F -DSTQCform. receipt ofnewdruglots,oratregular intervals,theseresults shouldberecorded onAppendix one ormore ofthesecond-linedrugs)forqualitycontrol ofdrugsusceptibilitytesting,eitherupon NOTE: Record theseresults onAppendixF-DSTQCForm.See testing mustbeperformed: It isextremely importanttoperformqualitycontrol onthedrugsensitivitytestingprocedure. This 12.13 QualityControl Internal _ l a b _ inconsistent withprevious results forapatient(especiallyifthedrugprofi For INH,RIF, anyfl Appendix B. non-tested specimenas“NotApplicableorNotRequired byProtocol” ontheassociated If theotherspecimen(#1or#2)forsamevisitwasalsopositive forMTB,markthe “Not ApplicableorNotRequired byProtocol” onAppendixB. If aspecimenwasnotpositiveforMTBorthevisitintervaldidrequire DSTtesting,mark m a Ifthelaboratorychoosestoincludeotherstrains(suchasclinicalisolateswithresistance to n 16:QualityAssurance.AllDSTreporting guidelinesoutlinedbeloware applicabletoDST • • • • • • • • • u Susceptible a Enviomycin onOgawamediaatthecriticalconcentrationof20μg/ml. any validationtestingperformed. submits awrittenrationaleforthecriticalconcentrationuseddrug,aswell Gatifl DST “Comments”section. If anydiscrepant results are seen,orretesting isperformed,notethisoccurrence inthe each drugtested. Record thetestingmethod,dateresult wasobtained,andthecriticalconcentrationof Tested ( Mark eachdrugresult as“Susceptible( Using apan-susceptiblestrain,suchasH37Rv, whichissensitivetoallofthetestdrugs eachbatchofpatientisolates,whenDSTisperformedlessfrequently.With Weekly, inaDSTrunwhenpatienttestsare runweekly For eachnewbatchofreagents (MGITdrugkits,otherdrugs,media,etc.) l

_ 2 c o oxacin and/orKanamycininMGITorsolidmedia,solongasthelaboratory u l . i n ND d d

)”

”, “ 6 uoroquinolone oranyinjectabledrugonly, ifasubsequenttestresult is 3 Resistant ”, or“ Test Failed S )”, “Resistant( ” on the internal labworksheet/bookand ” ontheinternal Section 16:QualityAssurance. R )”, “Test Failed( le changes from TF )” or“Not 111/04/2014 17:40 1 / 0 4 / 2 0 63 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 64 l i _ m 64 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 5. _ m a “Comments” section. susceptibility results fortheindividualdrugsas“ND”andaddanexplanationinDST was unavailablefortesting,e.g.,culture contaminatedandcouldnotbepuriﬁ If aspecimenwaspositiveforMTBcomplexattimepointrequiring DST, buttheisolate Appendix B. test result forthecurrent visit,repeat thetesta3rd timeandrecord thisastheﬁ “R” to“S”),review dataandrepeat thetest.Ifrepeat result isdiscordant withthe1st n u a l

_ 2 c o u l . i n d d

6 4 nalresult on e, report ed, 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 65 l i _ m y c o b a c t e r i o l o g NOTE: y Flow Chart4 _ l a • Asepticallyadd • ReportDSTfollowingroutine studyprocedures. • Investigateandresolve anydiscrepancies. • • ZN/subculture tube(s)asapplicable. • Checktubesvisuallyforanysignsofcontamination. • Instrumentsignalspositiveinappropriate timeframe. • Entertubesintotheinstrument,andmonitoruntilinstrumentsignalsascomplete. • • Mixbyinvertinggently3-4times. • Recaptubestightly. • • • Vortex 3-5dayoldculture tubewell. Set-up newDST: • Asepticallyadd • Removedrugsfrom freezer; letthawinrefrigerator whilelabelingtubesandaddingsupplement. Prepare newcontrol anddrug-containingmedia: • Ifpurityveriﬁ positive,screen• Whentubeturns forpuritywithZNandBAP. positive. • Enterintoinstrumentandmonitoruntiltubeturns • Captubetightly;mixwellbygentlyinverting3-4times. • Inoculate0.5mlofthe1:100dilutedsampleintonewMGITtube. • SupplementanewMGITtubewith0.8mlGrowth Supplement • Makea1:100dilutionoftheculture tubeusingsalineor7H9broth. • Vortex tubewell;letsit5-10minutes. Prepare newMGITculture from primaryculture tube: b this patient(ifavailable). Compare DSTproﬁ procedure; i.e.,1:10dilutionforPZA;1:100SIREandsecond-linedrugs. Inoculate GCtube(s)with0.5mlofanappropriately diluted MGITculture broth asperroutine DILUTE Using transferpipette,asepticallyadd sediment. broth withoutdisturbingthe Let tubesit5-10minutestosettlebigclumps.Usethesupernatant _ m a All workmustbedoneinthebiologicalsafetycabinet. n u a l

culture. _ 2 c Invalidx200Errors from MGITDST o u ed,re-incubate tubeuntilitis3-5daysold;record GUvalueonlabworksheet. l . i n S cetbeDSTNotAcceptable DST Acceptable 0.1 ml 0.8 ml d d

lewithprevious testresults from

6 5 (100ul)ofeachdrugtoitsappropriately labeledtube. SIREorPZAsupplementtoeachGCanddrugtubeasappropriate. 0.5 ml ofMGITculture broth toeachdrugtube; • • • Machinegivesanothererror. to thesite. errors, andsendacopyoftheNTF Write aNotetoFileexplainthe note inthe“Comments”section. appendix Bandwriteexplanatory Mark result as‘TF’(testfailed)on without PANTA. DO NOT 111/04/2014 17:40 1 / 0 4 / 2 0 65 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 66 l i _ m 66 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a NOTE: b Flow Chart5 _ • • Try tousethisMGITtubewithin1-2daysofinstrumentpositivity. Avoid over-inoculation ofculture. Set-up newDST positive,screen• Whentubeturns forpuritywithZNandBAP. positive. • Enterintoinstrumentandmonitoruntiltubeturns • Captubetightly;mixwellbygentlyinverting3-4times. • Inoculate0.5mlofthe1:100dilutedsampleintonewMGITtube. • SupplementanewMGITtubewith0.8mlGrowth SupplementwithoutPANTA. • Makea1:100dilutionoftheculture tubeusingsterile salineor7H9broth. • Vortex tubewell;letsit5-10minutes. Prepare newMGITculture from primaryculture tube MTB, andrepeat thetest. original culture wasMTB,purifyif AFB withZNfrom BAP. Verify Action: AFB andcontaminated. Conclusion: BAP –growth ZN –positive Control isvisuallyturbid. m • ReportDSTfollowingroutine studyprocedures. • Investigateandresolve anydiscrepancies. • • ZN/subculture tube(s)asapplicable. • Checktubesvisuallyforanysignsofcontamination. • Instrumentsignalspositiveinappropriate timeframe. 1:100 dilutionforSIREandsecond-linedrugs.SeeSection12.3details. Follow procedure forMGITDST, withappropriate dilutionofGCtube(s);i.e.,1:10for PZA; a this patient(ifavailable). Compare DSTproﬁ n u a All workmustbedoneinthebiologicalsafetycabinet. l

_ Rule outrapidlygrowing 2 c o u l Culture positivefor . i Invalid x400Errors from MGITDST n d d

S cetbeDSTNotAcceptable DST Acceptable

6 6 lewithprevious testresults from Prepare ZNandBAPforpuritycheck. Visually observethecontrol.Visually Action: Conclusion: BAP –nogrowth ZN –positive contamination. Control hasnovisual below. necessary; followasoutlined Conﬁ Over-inoculation. rmMTBif • • • Machinegivesanothererror. to thesite. errors, andsendacopyoftheNTF Write aNotetoFileexplainthe note inthecommentssection. appendix Bandwriteexplanatory Mark result as‘TF’(testfailed)on test, ifappropriate. culture wasMTB,andrepeat Action: Conclusion: BAP –growth ZN –negative turbid orclear. Control iseithervisually Discard. Verify original Contaminated 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 67 l i _ m y c o b a c t e r i o l o g y Materials Procedure RAPID IDENTIFICATION OFM.TUBERCULOSISCOMPLEX 13 _ l a number ofstudiesconductedinclinicalsettings. These testshavebeenshowntobehighlysensitive(>95%)andspecifi species ofMTBcomplex: control isincludedtovalidateproper testperformance.Thewilldetectthefollowing colloidal goldparticlesandisvisualizedasapink(orpurple)toredpositive line.Aninternal MPT64/MPB64 antigenispresent inthesample,acolorreaction isproduced bythelabeled where itiscaptured byasecondspecifi This antigen-antibodycomplexthenmigratesacross theteststriptoreaction area, to colloidalgoldparticlespresent ontheteststrip,forminganantigen-antibodycomplex. the testdevice,MPT64/MPB64antigenbindstoanti-MPT64/MPB64antibodiesconjugated protein thatisspecifi The BDassaydetectsMPT64antigen,whileCapiliaMPB64amycobacterial MGIT TBcIDandTauns’ Capilia TBare bothlateralfl A rapidimmunochromatographic assaywillbeusedtodifferentiate MTBandMOTT. BD’s Principle positive MGITandLJcultures. will beused.Defi Dickinson (BD)MGITTBcIdentifi treatment ofthedisease.To ensure consistencyacross allparticipatingsites,onlytheBecton To differentiate MTBfrom mycobacteriaotherthantuberculosis (MOTT)fortheeffective and LJAFB-positivecultures withoutspecialinstrumentsorequipment. To rapidly(<1h)andaccuratelydetect Purpose b _ m a • • • • • • • • n USING ANIMMUNOCHROMATOGRAPHIC ASSAY • • u 200 μLsterileaerosol resistant tips 200 μLmicropipette Timer Vortex mixer Distilled water Weigh boats Analytical balance Clean cylinderandmediafl a buffer (KH Capilia extractionbuffer (commercially available)orin-houseprepared TBcIDextraction TBc IDTest deviceorCapilia TBTest device l

_ 2 c o u l . i n d d 2 PO

nitive identiﬁ nitive

6 cally secreted from MTBcellsduringculture. Whenasampleisaddedto 7 4 ,NaClTween 80) M. tuberculosis,bovis,africanum cation willbeperformedateverytimepointfrom allAFB- cation Test (TBcID)ortheTauns CapiliaTB-NeoTest (Capilia) askorbottle c MPT64/MPB64antibodyﬁ Mycobacterium tuberculosis ow immunochromatographic assays. , and xed tothemembrane.If complex (MTB)inMGIT M. microti c (>95%)ina . 111/04/2014 17:40 1 / 0 4 / 2 0 67 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 68 l i _ m 68 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g 9. Store buffer at2-8°Cforupto6months. 8. Sterilizefor15minutesat121°C,15psi. 7. 6. Mixthoroughly andbringtoaﬁ 5. 4. Dissolvepowdersin500mLdistilledwateravolumetricﬂ 3. Weigh out8.5gofNaCl. 2. y _ l a . Usingweighboatandanalyticalbalance,out1.36gofKH 1. To prepare1Lofbuffer b Forms . Vortex thetightlycappedMGITtubefor30secondstoensure thesuspensioniswell-mixed. 2. Ideally, testAFBsmear-positive MGIT tubeswithin5daysofinstrumentpositivity. 1. From positive MGITtubes: 13.2.1 Specimen preparationandsubsequentstepsmustbeperformedinaBSC, usingfullPPE. 13.2 Specimen Preparation Buffer formula PreparationofTBcIDExtractionBuffer 13.1 room temperature. Before eachuse,checkbuffer visuallyforsignsofcontaminationordegradation,andbringto the buffer name,dateprepared, expirydate,batchlotnumber, andinitialsofpreparer. Aliquot buffer intosmallervolumes,e.g.,250ml,before autoclaving.Labelcontainerswith all Tween material. Using amicropipette, add100μLofTween 80;pipetteup-and-downrepeatedly todislodge _ m a n u a l • •

• • • • • • • _ • 2 Tween 80 -0.01% NaCl -0.145M Appendix B–StudySource Document Appendix G-Identiﬁ Lab-speciﬁ Permanent marker Waste receptacle withbiohazard bagandtuberculocidal disinfectant 10 μLsteriledisposableloops Sterile 2mLcryovials c KH o u 2 l PO . i n d 4 d -0.01M

6 8 cformsforrecording IDtestresults cation QCFormorusesite-speciﬁ nalvolumeof1Lwithdistilledwater, usingacylinder. ask. c formifavailableandequivalent) 2 PO 4 . 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 69 l i _ m y c o b a c t e r i o l o g y interpreted inthesamemanner. The followingpictures are specifi InterpretationofResults 13.4 2. Ifdevicesare refrigerated, bringtoroom temperature inthefoilpouchpriortotesting. 1. the devicewithyourhands. devices shouldnotbeopeneduntiltestistoperformed.Avoid touchingthespecimenwellon sunlight, excessivehumidity, andhightemperatures shouldbeavoided.Foilpouchescontaining The rapidIDtestdeviceshouldbestored at2-30°C(preferably refrigerated at2-8°C).Direct InoculationofIDTest Device 13.3 2. 1. Subculture toanewLJslantobtainadequategrowth andrepeat testingasnecessary. NOTES: b. a. Insuffi ForTBcID,besure thesuspensionturbidityisadjustedtoapproximately 0.5McFarland. 5. Vortex thecryovialfor30secondstocreate auniformsuspension. 4. 3. Add200μLofTBcIDextractionbuffer orCapiliaextractionbuffer toasterilecryovial. 2. Test 2-4weekoldgrowth. 1. From positiveLJslants: 13.2.2 3. 6. Starttimerfor15minutes. 5. 4. _ l a b _ Place thedeviceonafl one yearwhenstored at–20°Cor2to8°C. Positive cultures inliquidmediaandcoloniesonsolidcanbetestedwithCapiliaupto –20°C to8°C. positivity; testingcanbeperformedfortwoadditionalmonthswhentubesare stored at Positive MGITtubescanbestored at2-37°Candtestedinthe any solidmediumand/orcontaminantspresent. Using asterile10μLloop,scrapeloopfulofseveralcoloniesandmixwithbuffer, avoiding information describedin Label onedeviceforeachspecimentobetestedwithastudylabelcontainingtheidentifying pouch immediatelybefore testing. interpret testafter60minutes. Examine thereading area ofthetestdeviceafter15minutesandrecord testresults. specimens. Section Place 100μLofspecimen,eitherMGITculture orbacterialsuspensionfrom LJslant(see m a n u a l

_ 13.2.2above),intothespecimenwelloftestdevice.Changepipettetipsbetween 2 c o cientdensityinthesuspensioncanleadtofalsenegativeresults. u l . i n d d

6 9 at surfaceinsidetheBSC.RemoverapidIDdevicefrom itsfoil Section c totheBDTBcIDkit.However, theTAUNS CapiliaTBtestis 6.2:LoginofSputumSpecimens. TBc ID forupto10daysafter Do not 111/04/2014 17:40 1 / 0 4 / 2 0 69 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 70 l i _ m 70 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g . Record observationsforinvalidoruninterpretable results; e.g.,a contaminatedculture. 6. y _ . Ifthetestisinvalid: 5. l a Mycobacterium CMorGenoType b 3. However, grossly contaminatedcultures maycauseinterference; interpret withcaution. 2. 1. NOTES 3. 2. 1. Table 13 complex”, or“invalidtest”onthelaboratoryworksheet. Record results, includingallrepeat tests;as“positiveforMTBcomplex”,“negative ofIdentifi Reporting 13.5 4. _ Specimen Area Reading Area m a positive results inalllateralfl Staphylococcus aureus slight contaminationwithbacteriadoesnotinterfere with thetest. It ispreferable totestpure cultures withoutcontamination,althoughobservationsshowthat on AppendixB.Inaddition,noteintheMGIT“Comments”sectionthattestwasrepeated. ID testafter48hours.IfMTBisnowdetected,marktheresult as“positiveforMTBcomplex” isolate are consistentwithMTB,re-incubate theMGITtubeat37°C(±1°C)andrepeat rapid If therapidIDtestisnegative,butAFBsmearandmorphologicalcharacteristicsof new device. damage hasoccurred. Inthiscase,thetestshouldbeconsidered invalidandrepeated usinga Invalid not [T]. Negative the device. Positive: to Flowchart2Aforadditionaltestingalgorithmsusingamolecularassay(HAINGenoType 64 gene.IfMTBcishighlysuspected,andidentifi A negativetestdoesnotalwaysruleoutMTB,asmutationsare knowntoariseintheMPT/MPB n u a • • • l

_ molecular assay(HAINGenoType If testremains invalid,refer toFlowchart2Aforadditionaltestingalgorithmsusinga Repeat thetest. heavily contaminatedculture. Investigate causesfortheinvalidresult andtrytoresolve; e.g.,decontaminatinga • • Interpretation ofResults 2 c contaminants andrepeat testingifnecessary. For LJcultures, attempttosubculture afewcolonieswell-separatedfrom the For MGITcultures, decontaminatethe culture andrepeat testingifnecessary. o : Ifnolineisobservedonthereading area labeled[C],technicalerrors orproduct u l Pink/purpletored linesformonthereading areas labeledTest [T]andControl [C]of . i : Apink/purpletored lineformsonthereading area labeled[C]ofthedevice,but n d d

7 0 isknowntoproduce protein A,whichmayinterfere and/orcausefalse cation Results cation owassays. ® MTBDRplus). ® MycobacteriumCMorGenoType cation results persistinbeingnegative,refer ® MTBDRplus). ®

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 71 l i _ m y c o b a c t e r i . Record anycommentsintheMGITorLJculture sectiononAppendixB. 4. o l o g y . IftherapidIDtestisnegativeforMTBcomplex: 3. . Ifthetestconfi 2. section onAppendixB. 1. If identifi StudyDataReporting 13.7 Refer to be runweekly, oralongwitheachbatchofpatientisolates,whentestsare setuplessfrequently. received andwitheachnewbatchofextractionbuffer prepared. Similarly, thesecontrols must A positiveandanegativecontrol mustbetestedwitheachnewlotorshipmentofkits QualityControl Internal 13.6 _ l a b _ m − a n • • • to ruleoutMTB(HAINGenoType complex” intheMGITorLJculture sectiononAppendixB.SpecifytheIDtestused If MTBisnotdetected,record theIdentifi u Section a Proceed withinstructionsformoleculartestinginFlowchart2A. section onAppendixB. Record thetestmethod(CapiliaorTBcID)andanycommentsinMGITLJculture culture sectiononAppendixB. Record theIdentifi − l

_ 2 cation isnotperformedbecauseculture isnegative,mark“N/A”intheappropriate to conﬁ complex” intheMGITorLJculture sectiononAppendixB.SpecifytheIDtestused If MTBisdetected,Record theIdentiﬁ c o u l . 16:QualityAssurancefordetails. i n d rmsthepresence ofMTBcomplex: d rmMTB(HAINGenoType

7 1 cationofAFBresult as“PositiveforMTBcomplex”intheMGITorLJ ® ® MycobacteriumCMorGenoType MycobacteriumCMorGenoType cation ofAFBresult as“NegativeforMTB cation ofAFBresult as“PositiveforMTB ® ® MTBDRplus). MTBDRplus). 111/04/2014 17:40 1 / 0 4 / 2 0 71 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 72 l i _ m 72 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 4 LONG-TERMSTORAGEOFMTBISOLATES 14 . Autoclaveat121°Cfor10minutes.Coolto45°Cin a waterbath. 4. Heatgentlyifnecessarytodissolvethemediumcompletely. 3. Add2mlofglycerol; rinsepipetteafewtimeswithbroth todislodgeallmaterial. 2. Suspend4.7g7H9Broth Basein900mldistilledwater. Mixthoroughly. 1. smaller batchisneeded speciﬁ Note: Thefollowinginstructionsare forpreparing mediausingDifcodehydratedbase.Follow PreparationofMedia 14.1 Materials Procedure Forms _ m frozen foreachMTBisolate. that atleastfouraliquotsfrom baselinevisits,andtwoaliquotsfrom subsequentvisits,be may needtobeperformed(eitherin-houseoratacentrallaboratory).Itisrecommended 7H9 broth plusglycerol topreserve themforanyrepeat oradditionalmicrobiology teststhat for MTB,orasubculture from theoriginalLJculture, ifMGITisunavailable)willbefrozen in MTB isolatesfrom allpositivecultures (usingtheLJsubculture from aMGITtubepositive Principle isolates. This sectionprovides detailedinstructionsontheproper preparation andfreezing ofMTB Purpose a n u c instructionsifanothermanufacturer’s basemediumisusedandadjustquantitiesifa a l

_ • • • • • • • • • • • • • 2 c Positive LJslantsfrom Appendix J–Freezer StorageLogfor MTB Isolates Appendix E–NewReagent/MediaQC Appendix B–StudySource DocumentWorksheet Study-speciﬁ Cryobox andrack Permanent marker Sterile loop,disposableapplicatorstick,orcottonswab Sterile transferpipetteswithgraduationsmarkingvolume(individuallywrapped) ADC orOADCenrichmentbroth 7H9 mediawithglycerol Water bath threaded2 mlsterilecryotubeswithscrew top,externally o u l . i n d d

7 2 clabels Section 11:SolidCulture: LowensteinJensen (LJ)Media 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 1. Freezing ofMTBIsolatesmustbeperformedinaBSC,usingfullPPE. 14.2 9. 8. 7. 6. 5. 3. 2. 1. NOTES 6. 5. 4. 3. 2.

sputum specimen#,datecryotubeisprepared, andlabaccessionnumber. Seeexamplebelow. Label cryotubewithstudylabelcontainingscreening and/orsubjectID,visitnumber, aliquotor Discard anyprepared mediathatshowssignsofcontamination,discoloration,or evaporation. Store at2-8°Cupto3months; protect from anydirect light. Test mediaforsterilityandperformance.SeeSection16.2.1.2.2. Label containerwithmedianame,dateprepared, expirydate,batchlotnumber, andinitials. Aseptically add100mlofADCorOADCenrichment.Mixgently, butthoroughly. • first cryobox.Considerthefollowingexample: visit/sputum specimen (#1,#2)should be stored inaseparatecryoboxthatexactly mirrors the Cryoboxes maycontainisolatesfrom more thanonepatient,butthesecondaliquotfrom each specimen number (#1or#2),in order toretrieve these isolates for furthertesting or shipping. Freezer boxesmustbeorganizedtoeasilyidentifytheisolatesbypatientand by sputum a replacement cryotubemustbeprepared and frozen. If afrozen isolateisusedinthelabforanyreason (excludingshippingtocentrallaboratory), (±10°C) freezer. Place cryotubeincryoboxandfreeze boxinarackorspecifiedlocation-70to-80°C capandthoroughly mixsuspensionusingavortex. Tighten emulsify inbroth. (containing fresh, pure growth – sterile salineorbroth, transferseveralsweepsof Using asterileloop,disposableapplicatorstick,orcottonswabwettedwith growth from theslanttocryotube. Check thelabelonLJtubeandcryotubetoensure theymatchbefore transferring Dispense 1.0-1.2mlof7H9broth intocryotube usingasteriletransferpipette. Subj ID1001 − − Freezing MTBIsolates • • appearance. • Usecoloniesshowinggood,confluentandpure growth within10-15daysoffirst Visit 3 hasapositivecultureVisit from one specimen. 2hasapositive cultureVisit from one specimen. Do notscrapeoff anyculture medium;thiswillgivefalseturbidity. Density ofsuspensionshouldbegreater than a1.0McFarlandstandard. Older cultureswillnotprovidereliable,longtermviability. Date frozen: V#: Aliq/Spec#: Screen ID: Section 10.9:Subculture andStorageof MGIT Cultures) and Lab access#: Subj ID: M. tuberculosis Site#: growth from LJsubculture 73 Microbiology Lab Manual 74 Microbiology Lab Manual 5. 4. Cryobox 1 aliquot 1 V2 Record onAppendixBthat isolatewasbankedforlong-termstorage. Record ontheFreezer StorageLogfor If arackisnotused,record thelocationwhere theboxisstored inthefreezer. applicable), racknumber, boxnumber, andplace/positionnumbersonthefreezer storagelog. date cryotubeisprepared, andlabaccessionnumber. Alsoincludethefreezer number(if and/or subjectID,visitnumber, specimen#(ifapplicable),aliquot #(ifapplicable),totalvolume, • Subj ID1001 Subj ID1002 − − aliquot 2 V2 Visit 5haspositiveculturesVisit from twospecimens-#1 4haspositiveculturesVisit from twospecimens-#1 aliquot 1 V3 aliquot 2 V3 # 1 V4 Subj ID1002 # 2 V4 # 1 V5 M. tuberculosis # 2 V5 Cryobox 2 aliquot 3 V2 Subj ID1001 IsolatesForm(AppendixJ)thescreening aliquot 4 V2 aliquot 3 V3 aliquot 4 V3 and#2 and#2 # 1 V4 Subj ID1002 # 2 V4 # 1 V5 . . # 2 V5 ggli_mycobacteriology_lab_manual _2coul.indd 75 l i _ m y c o b a c t e r i o l o g y Forms Materials . Fileacopyofthe requisition form(s)inthelaboratory binder. 5. Filloutaseparateshippingrequisition formforeachshipmentofisolates. 4. 3. Follow specifi 2. 1. Association (IATA). Category A,infectioussubstancesaffecting humans,asdefi NOTE: PackagingofIsolates 15.2 isolate shippedshouldbemaintainedatthelab. shipped inseparateshipments,toprevent totallossofaparticularvisitisolate.Analiquoteach When shippingisolates,itisrecommended thatduplicatetubesforeachaliquot/specimen#be IsolateShippingSchedule 15.1 SHIPPINGOFFROZENISOLATES 15 _ l a This sectionprovides instructionsontheproper preparation andshippingofMTBisolates. Purpose b damaging theorangelabel. Wrap topofcryovialswithParafi to shipping. Ensure thatisolateshavebeenfrozen at-70to-80°C(±10°C)foraminimumof48hoursprior _ m a Isolatesmustbepackagedbylabstaff withcertifi n • • • • • • • • • • • • • u a Appendix J–Freezer StorageLogforMTB Isolates Appendix B–StudySource DocumentWorksheet Dangerous goodsform Airway bills Shipping labels Shipping requisition form Parafi Shipping containerandboxes(tobeprovided byshipping vendor) Dry Ice(tobeprovided byshippingvendor) Absorbent material Biohazard bags Tape when labeledandwrappedwithParafi Cryoboxes (plastic;sizemustbecompatiblewithdimensionsoflab-specifi l

_ 2 c o lm u l . cinstructionsprovided bytheshippingvendorforfrozen isolates. i n d d

7 5 lm, ensuringtheyﬁ lm) cation inthepackaging andshippingof t comfortablyintothecryoboxwithout ned by the International AirTransportned bytheInternational c cryovials c 111/04/2014 17:40 1 / 0 4 / 2 0 75 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 76 l i _ m 76 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b Appendix B. For allothervisitintervals,whichdonotrequire anisolatetobeshipped,mark“N/A”on 3. 2. IfneitherMGITnorLJculture waspositiveforMTBcomplex,mark“No”. 1. Record onAppendixBwhetherornotanisolatewasshipped. StudyDataReporting 15.3 _ m a “Comments” section. contaminated andcouldnotbepuriﬁ If culture waspositiveforMTBcomplex,butisolateunavailableshipping,e.g.,culture number/isolate numberanddateofshipmentforeachasapplicable. If culture waspositiveforMTBcomplex,andisolateshipped,mark“Yes”, andrecord aliquot n u a l

_ 2 c o u l . i n d d

7 6 ed, mark“No”andaddexplanationintheShipping 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 77 l i _ m y c o b a c t e r i o l o g y Table 16.1 QC/QMSchedule Laboratory 16.1 16 QUALITY ASSURANCE centrifuge temperatures incubator, rooms,and Refrigerator, freezer, controls forAFBsmears Positive andnegative Culture –MGIT) (See MGIT Maintenance _ l a as patientspecimens,e.g.,MGITandLJculture results, colonycounts,etc. and approval from theSponsor. AllQCresults shouldbedocumentedinthesamemanner laboratory hasequivalentformsinuse,thesemaybeusedfordocumentationafterreview for documentingQC,QM,andQIprocedures are included(AppendicesC-N)or, ifthe and resolution ofunacceptableresults, alongwithsupervisorreview. Sampleforms All qualitymonitoringpracticesrequire documentationofresults, corrective action • Quality Improvement(QI) • Quality Monitoring(QM) • • Quality Control(QC) study. Qualityassurancepracticesconsistof: authorities thatthedataproduced are atruerefl laboratoriesparticipatinginclinicalresearch,for international andhelpassure regulatory Good ClinicalLaboratoryPractice(GCLP)standards. GCLPstandards are recommended standards mandatedbyaccrediting agenciesandthequalityassuranceguidelinesin The examinationandmonitoringofmultipleparametersinvolvedintestingare guidedby Principle procedures are performingproperly. keeping andperiodicmonitoringofthedatageneratedwillhelpguaranteethatalllab from thepointofcollectiontoresult reporting anddatabaseentry. Inaddition,goodrecord consistency oflaboratorytestprocesses throughout theexaminationofasputumspecimen – Quality assuranceisacriticalcomponentoflaboratorytesting,asitensures accuracyand Purpose b Daily orwitheach _ Continuous effort toimprove service,function,workfl culture positivityratesthatmayaffect theoutcomeofresearch results Monitoring ofcriticalpractices,instrumentfunction,dataentry, contaminationrates,and reproducibility QC–profiExternal etc. QC–monitoringoftestperformance,mediaquality,Internal reagent activity, DSTaccuracy, Section m a patient run n u a l

LaboratoryQC/QMSchedule _ 10:Liquid 2 c o u l . i n d d

7 7 cultures controls forMGITandLJ Positive andnegative susceptibility testing Reference strainfordrug identiﬁkits cation reagent controlsfor Positive, negative,and ciency testingtoassesstechnicalcompetency, testaccuracyand Weekly orwitheach patient run results to improvemicroscopy qualityassessment Internal strain QC forMTBreference MGIT Time todetection Culture –MGIT) (See MGIT QCReport Monitors form Complete MonthlyData ection oftheresults obtainedduringthe Section otl Newlot#orbatch Monthly ow, customersatisfaction,etc. 10:Liquid reagents forAFBsmears controls fornewstaining Positive andnegative MGIT, LJ,7H9Broth,BAP testing ofculturemedia– Sterility andperformance lots ofidentiﬁ cation kits reagent controlsfornew Positive, negative,and drugs ordrugkits PZA kitsandotheranti-TB new lotsofMGITSIRE, Reference straintestingfor 111/04/2014 17:40 1 / 0 4 / 2 0 77 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 78 l i _ m 78 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y the guidelinesintablebelow: a densitometerorspectrophotometer witha1cmlightpath,assuringparameterscorrespond to Ideally, theaccuracyofdensityaprepared McFarlandstandard shouldbecheckedbyusing 7. Discard after6monthsorsoonerifanyvolumeislost. 6. Store standards inthedarkat20-25°C. 5. 4. Sealthecappedtubeswithwax,parafi 3. 2. _ 3 x10 l a turbidity ofapproximately 1.5x10 of abacterialconcentration.Forexample,McFarlandNo.0.5turbiditystandard isequaltothe According toCLSIguidelineM24-A,eachMcFarlandturbiditystandard isequivalenttotheturbidity using aturbiditystandard, sothatthenumber of bacteriapresent willbewithinagivenrange. Many QCprocedures involvepreparing dilutionsfrom astandardized suspensionoforganisms b 1. precipitate, whichcausesturbidityinthesolution. mixing specifi which lengthentheshelflifeandstabilityofstandard. Alternatively, theycanbemadeby McFarland standards are commercially availableandprepared from suspensionsoflatexparticles, Preparation ofBariumSulfateTurbidity Standards important toadjustinoculumsascloselypossiblethestandard. to anewtube.Aninoculumthatistooheavyorlightwilladverselyaffect theresults soitis i.e., breaking upclumpswithbeads,allowingtosettle,andtransferringthesupernatant more bacteriaiftoolight.However, thelattermustbedoneaftermakingasinglecellsuspension, suspension ofbacteriainsterile7H9broth andadjustedbyaddingmore broth iftooheavy, or Table 16.2 16.2.1.1 16.2.1 Media QC QualityControlActivities 16.2 1. Adjusting Turbidity forInocula * atwavelengthof600nm bobne .3 0.257 55.6 0.132 9.9ml 74.3 0.1ml 9.95 ml 0.05ml Absorbance* % Transmittance* 1.0% Sulfuricacid 1.0% Bariumchloride _ in thetubetoachieveauniformturbidappearance.Replacestandard iflargeparticlesappear. Before eachuse,vortexstandard vigorously, mixingthefi (6 monthsfrom preparation date). Label thestandards withtheMcFarlandnumber, dateofpreparation, anddateofexpiration (BaCl For aMcFarlandNo.0.5standard, mix0.05mlof1.175%bariumchloridedihydrate m a caln tnadN.051 0.5 McFarland StandardNo. (BaCl To prepare aMcFarlandNo.1standard, mix0.1mlof1.175%bariumchloridedihydrate of theMcFarlandstandard tube. Use tubesofthesamesize/diameterforpreparing isolatesuspensionsanddilutionsasthesize n u 8 a CFU.Thestandard iscompared visuallyorusingadensitometer/spectrophotometer toa l

_ 2 2 McFarland Turbidity Standards 2 •2H •2H c Properties ofMcFarlandStandards o u l . 2 2 ed amountsofbariumchlorideandsulfuricacid.Thisformsasulfate i O) with9.95mlof1%sulfuricacid(H O) with9.9mlof1%sulfuricacid(H n d d

7 8 8 CFU/ml;aMcFarlandNo1.0standard equalsapproximately lm,orsomeothermeansofpreventing evaporation. 2 SO 2 SO 4 ). 4 ). ne whiteprecipitate ofbariumsulfate 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 79 l i _ m y c o b a c t e r i o l o g y performance. Media canbereleased forroutine laboratoryuseafterpassingallQCchecksforsterilityand Required procedures forSterilityandPerformanceQCofeachmediatypeare detailedbelow. several goodprepared batches). preferable toestablishlab-specifi 20% aboveorbelowtheestablishedrangemaybeconsidered unacceptable;however, itis yields results outsidetheestablishedrange,itmustbeconsidered unsatisfactory. Colonycounts incubation shouldbecriticallyevaluated.Ifanewlyprepared batchofmedium,whentested, The timetodetectionofgrowth, andnumbersizeofcolonieswithinaspecifi periods shouldbeestablishedfrom results ofseveralwell-prepared batches. time ofincubation).Baselinecriteriaforoptimalgrowth withstandardized inoculaandincubation tested forperformancecharacteristics(abilitytosupportacertainamountofgrowth inaspecifi 1-3% ofeachnewbatchmediummustbeincubatedtotestforsterilityand batches ofmedia(e.g.,everyfourweeksormore frequently) toensure freshness andquality. Lab-prepared mediamustbethoroughly qualitycontrolled. Itisrecommended tomakesmall 16.2.1.2 3. Ifavailable,useaspectrophotometer orsimilarinstrumenttoadjusttheturbidities. 2. _ l a b is unavailable. card asavisualguideforadjustingtheturbiditieswheninstrumentation Use a“Wickerham” _ m a n Laboratory-PreparedMedia u a l

_ 2 c o u l . i n d d

7 9 c criteria(thatis,determinemeanCFUandstandard deviationof ed timeof ed 111/04/2014 17:40 1 / 0 4 / 2 0 79 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 80 l i _ m 80 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a 3. Incubatethetubesat37ºC (±1ºC),andobservegrowth visually. 2. 1. Subculture a Preparation ofMTBCultureSuspensionandWorking Dilution 5. d. c.Investigateandresolve problems, thenprepare newmedia. b. a. 4. 3. 4. b 16.2.1.2.1 _ m 2. 1. Performance QC 5. d. c. b. a. 4. 3. b. a. 2. 1. Sterility Check Use coloniesshowinggood,conﬂ action. results are notacceptable,prepare anAppendixKformtodocumentthecorrective Documentation: Corrective Actions: 20% ofthelaboratory’s ownestablishedreference rangesforeachdilution. AcceptableResults: dilutions). Seeprocedure below. of mediawouldinclude6tubesintotal,2inoculatedwitheach3 working tuberculosis any culture medium(whichgivesfalseturbiditymeasurement). sterile looporwoodenapplicatorstick.Take extreme precaution nottoscrapeoff Remove growth from theslantbycarefully scrapingthecoloniesoff theslantwitha appearance. Oldcultures donotgivereliable results. a n u prepare newmedia. If tubesshownogrowth, notifysupervisorimmediately, discard entire batch,and consideration whenfreezing lowconcentrationsofMTB. MTB suspension,especiallyifprepared from afrozen stock.Lossofviabilityisa If colonycountsare lowerthantheacceptablerange,checkpreparation ofthe and discard entire batch. If growth stillnotinacceptablerangeafterrepeat testing,notifysupervisorimmediately a Controls: Frequency: contamination isseen,prepare anAppendixKformtodocumentthecorrective action. Documentation: Corrective Actions: texture, andhomogeneityofmedium. AcceptableResults Controls: Frequency: l

_ 2 discard entire batch. If >1tubeiscontaminateduponrepeat testing,notifysupervisorimmediatelyand prepare newmedia. If alltubesare contaminated,notifysupervisorimmediately, discard entire batch,and 1-3% ofLJtubesfrom abatch(forexample:forof100tubes,select2tubes) Investigate andresolve allproblems, andthenprepare newmedia. If onetubeiscontaminated,repeat exercise withatleast10additionaltubes. Incubate for14daysat37°C(±1°C). c QC ProtocolforLJMedium o u l . i n (H37RvorH37Ra)in7H9broth (e.g.,testing2%ofabatch100tubes d d M. tuberculosis 1-3% ofLJtubesfrom abatch,testedwith10

Eachnewbatchofprepared medium.

each newbatchofprepared medium. 8 Record results ontheReagent/MediaQCform–AppendixE.If 0 Record results ontheReagent/MediaQCform–AppendixE.If Growth onalltubesisconsistentwith : Nogrowth inspectionconﬁ onanytube.Visual QCstrain(H37RvorH37Ra)ontoseveralLJslants. uent, andpure growth within10-15daysofﬁ –2 , 10 M. tuberculosis –3 and10 –4 dilutionsof rms proper color, proper rms andwithin rst M. 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 81 l i _ m y c o b a c t e r i o l o . Removeonealiquotofeachdilution(10 1. Inoculation andIncubation 11. 10. g y . Compare countswiththelaboratory’s establishedreference range. 6. Incubateat37°C(±1°C),reading weeklyfor21-30days. 5. InoculateLJtubeswith200μlofeachdilution. 4. 3. Whilethawing,bringLJmediumtoroom temperature. 2. 16.2.1.2.2 _ l a up tosixmonths. Aliquot 1.5mlofeachdilutiontotheappropriate cryotubesandfreeze at–70°C(±10°C), date (sixmonthsafterpreparation). Prepare 20-50cryotubesforeachdilution.Labelwithstraintype,dilution,andexpiration broth. 2. 1. Performance QC 5. d. c. b. a. 4. 2. 1. Sterility Check 3. 9. 8. 7. Lettubesitfor30minutesclumpedorganismstosettle. 6. 5. b with thepipettor2-3timestoensure evendistributionof MTB. amicropipettor andsterileaerosolWith resistant tips,mixtheworkingsolution tips(ART) _ m dilutions). Seeprocedure below. 100 mlbatchofmediumis4tubesintotal,2inoculatedwitheach of twoworking a Controls: Frequency: contamination isseen,prepare anAppendixKformtodocumentthecorrective action. Documentation: Corrective Actions: Controls: Frequency: 10 Acceptable Results: Prepare 3serial10-folddilutionsoftheadjustedsolution(10 McFarland No.1standard using7H9broth. Transfer toanewtubeandadjustturbidityofthissuspension thesupernatant sediment) andletthesuspensionstandforanother15minutes. Transfer ﬂ thesupernatant beads (2mmdiameter),andvortexwell. Transfer growth intoascrew captubecontaining4mlofsterile7H9broth and6-10 glass n u –2 entire batch. If >1tubeiscontaminateduponrepeat, notifysupervisorimmediatelyanddiscard prepare newmedia. If alltubesare contaminated,notifysupervisorimmediately, discard entire batch,and Investigate andresolve problems, thenprepare newmedia. If onetubeiscontaminated,repeat exercise withatleast10tubes. a QCfor7H9Broth , and10 l

_ 2 c o u Numberoftubesequivalentto1-3%totalvolumeprepared. l . Number oftubesequivalentto1-3%totalvolumeprepared, testedwith i n Eachnewbatchofprepared medium. Eachnewbatchofprepared medium. d –4 d dilutionsof

8 1 Record results ontheReagent/MediaQCform–AppendixE.If Nogrowth inanytubeafter7daysof incubation. M. tuberculosis uid toanothersteriletube(avoidtransferringanyofthe –2 , 10 H37RvorH37Rain7H9broth (e.g.,2%ofa –3 and10 –4 ) from thefreezer. –2 , 10 –3 , and10 –4 ) in7H9 111/04/2014 17:40 1 / 0 4 / 2 0 81 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 82 l i _ m 82 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 16.2.1.2.3 characteristic growth toappear. Observe alltubesforevidenceofgrowth, andnotetheapproximate numberofdaysfor Incubatetubeswithcapsloosenedat37°C(±1°C),reading weeklyfor7-14days. 4. 3. 2. _ m 2. 1. Sterility Check 3. 2. 1. Performance Check contamination isseen,prepare anAppendixKformtodocumentthecorrective action. 5. c.Investigateandresolve problems, thenprepare newmedia. b. a. 4. . Removeonealiquotofthe10 1. Inoculation andIncubation Prepare dilutionsasabovein Preparation ofMTBCultureSuspensionandDilutions 5. 3. c. b. a. 4. tuberculosis a n Vortex tomix. to ensure evendistributionofMTB,andinoculatetubeswith500μleachdilution. amicropipettor tips,mixtheworkingsolutionwithpipettor2-3times andART With disposable tubes,e.g.,16.5x128mm. into tubes,asepticallydispense4.5ml7H9broth intoappropriate numberofsterile While thawingdilutions,bringbroth toroom temperature. Ifnotalready aliquoted u a Controls: Frequency: Acceptable results: total). Seeprocedure below. orStaphylococcusaureus E. coli Controls: Frequency: Documentation: Corrective Actions: action. results are notacceptable,prepare anAppendixKformtodocumentthecorrective Documentation: Acceptable Results: Corrective Actions: l

_ performance QCwithfresh culture of If sterilitycheckwasacceptableandonlyinoculatedtubesare contaminated,repeat 2 If growth isseenonanyplate,repeat sterilitycheckusing10additionalplates. prepare newmedia. If tubesdonotshowgrowth, notifysupervisorimmediately, discard entire batch,and discard entire batch. If >1plateiscontaminateduponrepeat testing,notifysupervisorimmediately, and or subculture tosolidmedia. If tubesshowoverallturbiditysuggestingbacterialgrowth, conﬁ c QCProtocolforBloodAgarMedium o u l . i n d (ﬂ 1-3%oftotalnumberplatesprepared, incubatedat35-37°Cfor72hrs. 1-3%oftotalnumberplatesprepared, testedwithdilutedsuspensionof d occulent,granular)within1-2weeks.

Eachnewbatchofprepared medium.

Each newbatchofprepared medium. 8 2 Record results ontheReagent/MediaQCForm–AppendixE.If Record results ontheReagent/MediaQCform–AppendixE.If Nogrowth onanyplate. Growth inalltubesisconsistentwiththeappearanceof Section –2 and10 (e.g.,2%ofa100mlbatchmediumis4tubesin 16.2.1.2.1:QCProtocol forLJMedium. –4 dilutionsfrom thefreezer. M. tuberculosis . rm withZNstainand/ M. 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 83 l i _ m y c o b a c t e r i o l o g y . Tubes orplatesare notdamagedorcracked. 3. Expirationdates-notifythevendorofrecurring shortexpirydates. 2. 1. Upon receipt ofeachnewmediashipment,checkthefollowing: Recommendations forNewShipmentsofMedia and LaboratoryStandards Institute(CLSI). or are categorizedasgenerally“exempt”from userqualitycontrol according totheUSClinical Commercial mediaare thoroughly testedbythemanufacturer andrequire eitherlessQCtesting 16.2.1.3 _ 3. l 4. a 3. 2. 1. Inoculation andIncubation 3. c.Freeze at-70to-80°C(±10°C)foruptwelvemonths. b.Aliquot~1.5mlvolumeinto labeledcryovials. a. To maintainafrozen stockofthecontrol bacteria: 2. 1. Preparation ofBacterialSuspensionandWorking Dilutions results are notacceptable,prepare anAppendixKformtodocumentthecorrective action. 5. d. c. b. a.Ifnogrowth isseen,repeat performancecheck. 4. b records. Manufacturer’s QCrecords are provided -theserecords mustberetained withthelab’s QC Using this10 _ Make another100-folddilution,using10μlofthe water. Mixwell. m morphology. Incubate at35-37°Cfor48hours,andcheckgrowth withtypicalcolonysizeand a Acceptable results: Make a100-folddilution(10 dilute tothesamedensity. water toMcFarlandNo.0.5standard. Alternatively, thawthestocksuspensionand Prepare asuspensionofcoloniesfrom asolidbacteriologicmediuminsteriledistilled at35-37°C. other appropriate mediumandincubate overnight thawed, well-mixed To prepare afresh suspension:Twenty-four hourspriortouse,streak onedrop of the microbiology laboratoryorreference strain. The QCstrainof Documentation: Corrective Actions: n u Commercially-Prepared Media and discard entire batch. If performancestillnotasexpecteduponrepeat testing,notifysupervisorimmediately, standard. glycerol (orotherappropriate storagemedium)tothedensityofaMcFarlandNo.1 Prepare asuspensionfrom 24-hourgrowth from BAPinTryptic Soybroth with15% Investigate andresolve problems, thenprepare newmedia. performance checkwithafresh culture ofthestockstrain. If coloniesare nottypicalofthebacteriausedorgrowth ismixed,repeat a l

_ 2 c o u l . i n d –4 d dilution,inoculatetheplatewith10μlandstreak forisolation.

8 3 Record results ontheReagent/MediaQCForm–AppendixE.If Escherichia coliorStaphylococcusaureus Growth oftypicalcolonieswithin48hours. E.coliorS.aureus –2 ) byaddinga10μlsuspensionto1mlsteriledistilled stocksuspensionontoabloodagarplateor –2 dilutionin1mldistilledwater. canbeaclinicalisolatefrom 111/04/2014 17:40 1 / 0 4 / 2 0 83 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 84 l i _ m 84 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b . Tubes/plates are sufﬁ 6. Agarisnotdetachedfrom thesidesoftube/plate,frozen orsoftened. 5. 4. 16.2.1.3.1 Section lot closely, andifcontaminationissuesare suspected,followtheprocedure asrecommended in Contamination cansometimesoccurduringtransitofcommercial BAPmedia.Monitoreach QC ofCommercialBloodAgarPlates QC procedures withnontuberculous mycobacterialstrains. M. tuberculosis outlined in OADC/PANTA mixture, are acceptable.Ataminimum,theSponsorrequires theQCprocedure use), toensure thattheperformancecharacteristicsofmedium,oncesupplementedwith MGIT mediumand960Growth Supplementuponreceipt (before puttingintoroutine Becton Dickinson(BD),themanufacturer ofMGITreagents, recommends QCtestingofBACTEC QC ofCommercialMGITMedium 16.2.1.2.1 QCProtocol forLJMedium. in thelab≥6weeksshouldbetestedaccording tothesterility/performancechecksin growth canbecompromised. Therefore, commercially-sourced LJmediathathasbeenstored Commercial LJmediahasbeendemonstratedtodeteriorateovertime,anditsabilitysupport QC ofCommercialLJMedia as expected. necessary, performcompletequalitycontrol toensure therecovery ofisolatesissatisfactoryand It isimportanttomonitortheperformanceofcommercial mediacloselyduringuse,andif QC ofCommercialMediaInventory from patientuseuntilissuesare resolved. If anyproblems are found,notifysupervisor, contactvendorimmediately, andwithholdmedia Mediaisnotexcessivelymoistordehydrated. 7. _ m 5. i. b. a.IftheTTDisnotwithinspeciﬁ 4. 2. 1. 3. a media, bloodagarhemolyzed,etc. The mediumisnotcontaminatedorchangedinitsappearance;e.g.,colorchangewithLJ n u a action. results are notacceptable,prepare an AppendixKformtodocumentthecorrective Documentation: Corrective actions: Controls: Frequency: Acceptable results: l

_ 16.2.1.2.3above. 2 procedures. and checktheviabilityofinoculum,ageculture, ifstored frozen, andother If teststilldoesnotgivesatisfactoryresults uponrepeat, notifysupervisorimmediately, c

and MGIT960GrowthSupplementKit QC ProtocolforBACTECMGIT960CultureMedium o BD DiagnosticSystemsforassistanceandwithholduseoflot. If allprocedures are withinestablishedspeciﬁ Section u l . i n d strain.RefertotheMGITculture mediapackageinsertforfurtherinstructionson Dilutionsof d

Eachnewlotorshipmentof960mediagrowth supplement.

8 4 16.2.1.3.1belowfornewlots/shipmentsofMGITculture mediumusingan Record results ontheReagent/MediaQCForm–AppendixE.If cientlyandequallyﬁ MGITtubewillﬂ M. tuberculosis uoresce positive(TTD)in6to10days edrange,repeat thetest. (H37RvorH37Ra)in7H9broth. lled. cations, contactTechnical Servicesat Section 111/04/2014 17:40 1

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 85 l i _ m y c o b a c t e r i o l o g y _ l a 3. Incubatethetubesat37ºC(±1°C),andobservegrowth visually. 2. Subculture theMTBstrainontoseveralLJslants. 1. Preparation ofCultureSuspension 6. 5. 4. 11. 10. 9. Let 8. Letthesuspensionstandundisturbedfor20minutes. 7. 2. Removeonealiquotofthestocksuspensionfrom thefreezer andallowtothaw. 1. Preparation ofDilutions routine QCprocedures. 13. Ideally, ensure thatnewfrozen stockpassestestingindicatedbelow before usingin 12. 6. The fi 5. 4. 3. b _ m a Use coloniesshowinggood,confl Vortex beads (6-10beads,2mmdiameter),whichhelptobreak upclumps( Transfer growth intoascrew captubecontaining4mlofsterile7H9broth andglass → sterile looporwoodenapplicatorstick. Remove growth from theslantbycarefully scrapingthecoloniesoff theslantwitha appearance. Younger oroldercultures maynotgivereliable results. Adjust theturbidityofsuspensionin C Carefully from transferthesupernatant → sterile screw capglasstube( Using atransferpipette,carefullyfrom transferthesupernatant → and veryturbid. 15 daysoffi Alternatively, prepare afresh 0.5McFarlandsuspension,usingcolonieswithin10- → → → Tube C → adding more 7H9broth. Mixwell. water orsaline( Mix wellandthendilute1:10againbyadding0.5mlfrom Tube 2to4.5mlofsterile saline ( Dilute 1:10byadding0.5mlofsuspensionfrom Tube 1into4.5mlofsterilewateror suspension to4.0mlofsterilewaterorsaline.Mixwell( Dilute thestocksuspension(freshly prepared orfrozen) 1:5bytransferring1.0mlof card.spectrophotometer orvisuallywithaWickerham n ) withouttakinganysediment. u

a turbidity measurement). Take extreme precaution nottoscrapeoff anyculture medium(whichgivesfalse Avoid pipettinganysediment. Turbidity shouldbegreater thanMcFarlandNo.1standard. Once thawed,donotrefreeze. The frozen suspensionsmaybeusedforuptosixmonths. (±10ºC). This suspensionmaybefrozen insmallaliquots(~1.5ml)cryotubesat-70to-80ºC and adjusttheturbiditytoaMcFarlandNo.0.5standard. If thesuspensionistooturbid,transfersomeoftoanothersteriletube Tube B l

naldilutionof _ 2 c Tube 2 Tube A o is thestocksuspensionforQCtesting. u l . i n standundisturbedfor15minutes. d d rst appearancefrom LJmedia.Carefully adjusttheinoculumwitha

foratleast1-2minutes,makingsure thesuspensioniswelldispensed 8 5 Tube 3 Tube 3 ). Mixwell. is1:500. Tube B uent, andpure growth within10-15daysofﬁ ). Tube B Tube C intoanotherscrew capglasstube( toaMcFarlandNo.0.5standard by Tube 1 ). Tube A Tube A toanother ). Tube rst 111/04/2014 17:40 1 / 0 4 / 2 0 85 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 86 l i _ m 86 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 16.3.1 Acid-fastStains:Fluorescent andZiehl-NeelsenMethods batch number, dateprepared, andexpirydateonallreagent containers. tests andextractionbuffer, anddrugsusceptibilityreagents. Inaddition,record reagent name, reagents, sputumdigestion/decontaminationreagents, immunochromatographic identifi the clinicaltrial,Sponsorrequires thefollowingreagent qualitycontrol procedures forstaining Periodic qualitycontrol ofallreagents iscriticalforensuringconfi ReagentQualityControl 16.3 _ m 3. 2. 1. 1. Inoculation andIncubation from thestockculture usedinSection16.2.1.2.3:QCProtocol forBloodAgarMedium. bacillus) forthenegativecontrol shouldbeused.The (H37Rv orH37Ra)forthepositivecontrol and sediment toprepare thesecontrols, butifnotavailable,asuspensionof unstained inaclosedcontainerdryarea untilused.Itispreferable touseaseededsputum Prepare abatchofpositiveandnegativecontrol smearsinadvance,heat-fi Preparation ofSmears 5. c. b. a. 4. b.Negativecontrols mustbeclearlynegativewithnoacid-fastbacilli present. a.Positivecontrols mustdemonstratethepresence ofacid-fastbacilli. . Inoculate0.5mlfrom 2. . Retrievedatafortimetodetectionfrom theMGITprintout. 5. Removethetubewhenindicatedpositivebyinstrument. 4. EntertheinoculatedtubeinMGIT960instrument. 3. a n u a Acceptable results: (non-acid-fast bacteria)control organisms. Controls: reagents, oreachnewlot/newshipmentofcommercial reagents. Frequency: Supplement MGITmediumwithGrowth SupplementandPANTA asspecifi K formtodocumentthecorrective action. Staining QCForm–AppendixC.Ifresults are notacceptable,prepare anAppendix Form –AppendixE.Foreachbatchofsmears,record results ontheappropriate DailyAFB Documentation: Corrective Actions: 10: LiquidCulture –MycobacteriaGrowth IndicatorTube (MGIT). l

_ When QCresults are acceptable,repeat patienttestsandreport results. 2 Repeat controls; ifacceptable,repeat patienttests. and/or newcontrols, asapplicable,toresolve issue. If results stillunacceptable,notifysupervisorimmediatelyandprepare newreagents c o u l . i n d Smearswithknownpositive d

Eachbatchofpatienttestsandeachnewin-houseprepared

8 6 Record results fornewstainingreagents ontheReagent/MediaQC Correct results asexpectedforpositiveandnegativecontrols. If eithercontrol result isunacceptable,donotreport patientresults. Tube 3 toaMGITtube.Mixwell. M. tuberculosis Escherichia coli E. coli (H37Rv orH37Ra)andnegative suspensionmaybeprepared dence inlaboratoryresults. For (oranothernon-acid-fast M. tuberculosis ed in x, andstore Section cation

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 87 l i _ m y c o b a c t e r i o l o g y 16.3.3 Immunochromatographic Identiﬁ 16.3.2 SputumDigestion/DecontaminationReagents _ l a c. b. reagent a.Internal control indevice. 2. b.Weekly, orwitheachbatchofpatienttests,iftestingisperformedlessfrequently. a.Eachnewlotorshipmentofkitsand each newprepared lotofextractionbuffer. 1. 2. 1. Processing, below. AdditionalQCforthephosphatebuffer isperformedasfollows: Quality control forthesereagents isperformedaspartofQualityMonitoringSputum For eachnewbatchoftheNALC,NaOH,andNacitratereagents, record preparation details. 5. 4. 3. . Followprocedures outlinedin 1. Procedure 5. c. b. a.Repeattestwithnewcontrols; ifacceptable,repeat patienttests. 4. c.MOTTstrainoruninoculated broth mustresult inanegativetest. b. control a.Internal lineisvisible. 3. 2. b _ m a Controls: Frequency: Controls: Frequency: action. If QCresults are notacceptable,prepare anAppendixKformtodocumentthecorrective Documentation: supervisor immediately, andprepare newbatchusingreagent powder, ifnecessary. Corrective Actions: Acceptable results: acceptable, prepare anAppendixKformtodocumentthecorrective action. results fornewkitsonMTBIdentiﬁ Documentation: Corrective actions: Acceptable results: applicable. Perform QCtestsimmediatelybefore testingthepatientbatchandresolve issues,if n u M.tuberculosis avium Negative control: Culture ofaMOTTstrain(e.g.,wellcharacterized results. After investigationiscompleteandQCacceptable,repeat patienttestsandreport broth. Positive control: Culture of potential causesforfailure. If repeat results stillunacceptable,notifysupervisorimmediatelyandinvestigate a l

_ 2 c o u complex) inMGITbroth orbroth from anuninoculatedMGITtube. pHpaperormeter. l . i n Eachnewbatchofphosphatebuffer. d d

8 7 Record reagent detailsandpHonReagent/MediaQCForm–Appendix E. Record results fornewextractionbuffer onAppendixE;Record mustresult inapositivetest. Ifanycontrol result isunacceptable,donotreport patienttests. pHforphosphatebuffer is6.8. IfpHforbuffer cannotbeadjustedto6.8,discard batch,notify Correct results asexpectedforallcontrols. M. tuberculosis Section cation QCForm–Appendix G.IfQCresults are not cation Tests 13totestcontrols. reference strain(H37RvorH37Ra)inMGIT M. 111/04/2014 17:40 1 / 0 4 / 2 0 87 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 88 l i _ m 88 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 16.3.4 DrugSusceptibilityTesting QualityControl _ m 3. 2. c. b. a. 1. 1. from oneofthree options: tested followingthesameprocedures asforpatientisolates.Theinoculumcanbeprepared The inoculummustconsistofapure culture of Inoculum Preparation 5. d. c. b. a. 4. 2. Follow instructionsin Set-up ofDSTQualityControl c.Usethisadjustedsuspensionastheinoculum fordrugtestsandgrowth control. b.Dilute1mlofthesuspension insterilesalineor7H9broth (1:5dilution). a.Removeonealiquotoftheorganismsuspension from thefreezer andallowtothaw. 3. patient isolates. Monitor QCdrugsuntilsignaledcompletebytheinstrumentandinterpret thesameasfor → → → the drugsbeingtested.Importantconsiderationswhenpreparing theDSTQCare: a n Proper dilutionoftheQCorganismforGrowth Control anddrugtubes. Proper preparation ofdrugpowdersuspensions. Proper reconstitution oflyophilizeddrugs. u a Acceptable results: Controls: Frequency: A freshly grown culture ofMTBinMGITmediumfollowingtheguidelines document thecorrective action. AppendixF.Form – IfQCresults are notacceptable,prepare anAppendixKformto Documentation: results forthatparticulardrug(s). Corrective Actions: e.g., SIREandsecond-linedrugswithin4-13days;PZA4-21days. Section A fresh, pure subculture ofMTBgrowing onLJmediumfollowingtheguidelinesin 12.4: InoculumforMGITDST. above. Protocol forBACTECMGIT960Culture MediumandMGIT960Growth Supplement A suspensionofMTBprepared according tosteps1-12in l

_ Weekly orwitheachbatchofpatienttesting. assistance. If unacceptableresults persistwithSIREorPZAkit,consultBDTechnical Servicesfor 2 tubes. Upon receipt ofanewlotorshipmentMGIT960SIREkit,PZA repeat patienttests. Thoroughly review causeforunacceptableresults andrepeat QCtest;ifacceptable, of antibioticpowdersforsecond-linedrugs. Upon receipt ofanewlot,shipment,ornewlyprepared batchofstocksolutions When QCresults are acceptable,repeat patienttestsandreport results. and/or anewcontrol, asapplicable,toresolve issue. If repeat results stillunacceptable,notifysupervisorimmediately;prepare newdrugs c o u l . i n 12.4.2:UsinganInoculumfrom PositiveLJCulture. d d A pan-susceptible

8 8 Record results fornewantibioticsandweeklytestingontheDSTQC Section Susceptibleresults foralldrugswithinthedeﬁ Ifthecontrol results showanyresistance, donotreport patient 12.5:Growth Control Tube Preparation andInoculationfor M. tuberculosis H37RvorH37Rastrain M. tuberculosis (H37RvorH37Ra)andbe Section ned timeprotocol; 16.2.1.3.1: QC Section

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 89 l i _ m y c o b a c t e r i o l o g GT9037±1 37±1 4-12 –70to-80±10 MGIT 960 Refrigerated centrifuge Incubators Freezer (Ultralow) y and maintaindocumentationoftheircalibrationinalabworksheetorlogbook. house orthrough acommercially availableservice.Identifythermometersbyanumberingsystem, (US Bureau ofStandards orequivalent)before puttingintouseandannuallythereafter, eitherin- assessing equipmenttemperatures must becalibratedagainstastandardized/certiﬁ thermometersmustbeusedforallequipmentmonitoring.Allin Internal 16.4.1.2 room containingunrefrigerated centrifuge:≤20°C. c Rangedeterminedbyrequirements ofreagents orinstrumenthousedintheparticularroom; e.g.,MGITroom: 19-30°C; instrumenttemperatureb Internal reading shouldbe±1.5°Cofmanualreading. monitored daily. a Temperature oftherefrigerator where sputumspecimensare stored untiltheyare senttothelaboratorymustbe Table 16.3 16.4.1.1 16.4.1 Temperature Monitoring(Required) Analysisoflaboratorydata 2. ReviewofAFBexaminationcompetency 1. recommended In addition,thoughnotrequired, thefollowingqualitymonitoringactivitiesare Supervisorreview ofQCactivitiesonamonthlybasis 6. Contaminationrateassessment 5. MGITtimetodetection(TTD)monthlyexercise 4. Regularmonitoringofspecimenprocessing 3. 2. Equipment maintenance 1. Temperature monitoring the durationofstudy. Thesepracticesinclude: Monitoring ofcriticalpracticesthatmayaffect theoutcomeofclinicaltrialresults is QualityMonitoringActivities 16.4 Refrigerators _ Room Temperature l a b. a. 1. b _ m a Frequency: n Thermometers u Temperature Reading/Recording day. malfunction duringworkinghours,whichmaynotbedetecteduntilthe following before workcommences.Twice dailyreadings are Daily reading/recording oftemperatures isrequired, preferably intheearlymorning working day. during thosetimes;checktemperatures immediatelythebeginningofnext Weekends/holidays maybeexcludedifnostaff are availabletomonitortheequipment a qimn Temperature Ranges(°C) Equipment l

EquipmentTemperature Ranges a _ 2 c o u l andwillservetostrengthen laboratoryresults: c . i n d d

8 9 varies 2-8 b recommended asequipmentmay ed thermometer required strongly for 111/04/2014 17:40 1 / 0 4 / 2 0 89 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 90 l i _ m 90 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a performance ofroutine cleaningina lab worksheetorlogbook. speciﬁ Suggested equipmentcleaningactivitiesare giveninthetablebelow, howeverthemanufacturers’ essential foraccurateperformanceoflaboratorytestsandmaintainingthelongevityequipment. Keeping laboratoryequipmentcleanandperformingrecommended routine maintenanceare b 16.4.2.1 EquipmentCleaning/Maintenance(Required) 16.4.2 _ m 2. c. b.Documentminoradjustmentsandcorrective actionsonAppendixD. a.Record temperatures onAppendixD:EquipmentTemperature Record Form. 4. i. b. a. 3. a n u c recommendations andprocedures shouldbefollowed.Maintaindocumentationforthe a Acceptable results: Documentation: Corrective actions: equipment/environment. l

_ Equipmentcleaning and resolutions onanAppendixKform. Document allmajorunacceptableresults, e.g.,equipmentfailure, corrective actions 2 to decideonfurthercorrective action. has notreturnedtonormalwithinthenextworkingday, notifysupervisorimmediately Adjust temperature control andmonitoruntilcorrect rangeisachieved;iftemperature If equipmentisnotfunctioning,notifysupervisorimmediatelysoservicecanbeinstituted. c equipment/environment withproper temperature range. Relocate specimens,reagents, etc.,asapplicable,toafunctioningpieceof o u l . i n d d

9 0 Ifthetemperature reading isnotacceptable: Temperatures are withinthedeﬁ ned rangeforthespeciﬁ c pieceof 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 91 l i _ m y c o b a c t e r i o l o g y _ l a Table 16.4 Equipment Cleaning Schedule b _ m a n Equipment Cleaning Daily Weekly Monthly Other u a l

Spray work surfaces thoroughly with tuberculocidal disinfectant; let stand 3 minutes. Wipe dry AM & PM and _

2 with absorbent towel. If indicated, follow with 70% alcohol to remove disinfectant residue that following each task c o u may harm BSC surface. Wipe equipment stored in the BSC with disinfectant soaked absorbent l . i n towel. d d

Biosafety cabinet

Thorough interior and exterior cleaning with appropriate disinfectant(s) At least once/year or as 9 1 needed for any spill of infectious material Clean UV lights with 70% alcohol Every 1-2 weeks Spray interior walls of centrifuge with disinfectant (70% alcohol) and let stand 3 minutes. Wipe X dry with absorbent towel. Clean exterior surfaces with mild detergent, rinse and dry. Centrifuge Remove carriers and soak in warm, soapy water. Rinse thoroughly and place upside down to X drain/dry. Spray thoroughly with appropriate tuberculocidal disinfectant; let stand 3 minutes. Wipe dry AM & PM Counters with absorbent towel. If indicated, follow with 70% alcohol to remove disinfectant residue. Clean fan cover and remove ice buildup X –70 to –80°C Freezer Defrost and clean interior When needed Micropipettors/ Wipe exterior with 70% alcohol Before/after Pipette-Aid each procedure use pH Meter Replace electrode immersion ﬂ uid (pH 7.0 standard). Clean electrode tip As needed Refrigerator Wipe interior surfaces and shelves with damp towel soaked in mild detergent; rinse and dry. X Wipe interior surfaces and shelves with damp towel soaked in mild detergent; rinse and dry. X Incubator Follow with 70% alcohol spray. Autoclave shelves if applicable. Slide warmer Wipe surface with a damp towel soaked in mild detergent; rinse and dry. X Vortex mixer Wipe surface with a damp towel soaked in mild detergent; rinse and dry. X Analytical balance Clean pan surface with brush. X 111/04/2014 17:40 1 / 0 4 / 2 0 91 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 92 l i _ m 92 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a to using. this list.Inaddition,iflargeequipmentismoved/relocated, itmustbere-certiﬁ applicable, isshownbelow. Semi-annualorannualmaintenanceisrequired forallequipmentin A suggestedmaintenancescheduleperformedin-houseorbyoutsidetechnicalpersonnel,as or logbook. a vendorservicecontractMaintaindocumentationofallroutine maintenanceinalabworksheet maintenance (Table 16.5).Maintenancemaybeperformedbyin-housetechnicalpersonneland/or the manufacturer oftheequipment,andascheduledevelopedforperformingapplicable Routine, in-housemaintenancemustbecarriedoutatthetimeintervalsrecommended by b 2nd Edition.World HealthOrganization.Geneva,Switzerland.2008. are performingproperly. aspects oftheculture process (manualandinstrumented),from sputumprocessing toisolation, Running positiveandnegativecontrols onaregular basisassessestechniquesandassures thatall contamination from aheavilyacid-fastpositivespecimentoother, possiblynegative,samples. Careful attentiontotechniquewhenprocessing specimensisessentialtopreventing cross- MonitoringofSputumProcessing (Required) 16.4.3 1 16.4.2.2 Table 16.5 MGIT Incubator Refrigerators Freezer -70to-80°C Air conditioners Microscopes Centrifuge Biosafety cabinet Analytical balance Micropipettors/ Pipette-Aid Autoclave Forfurtherdetailsonequipmentmaintenanceandcleaning,refer to:MaintenanceManualforLaboratoryEquipment, _ m a n u a l

_ Equipment Maintenance 2 qimn aneac eianal Annually Semi-annually Maintenance Equipment c o Minimum EquipmentMaintenanceSchedule u l . i n d d

9 2 etficto required – of relevantcomponents required Calibration tubecheck,thoroughcleaning recommended required maintenance required recommended recommended elements, electroniccomponents;routine required Check doorgasketseal,heatingandcooling recommended required Clean condensersandfans;routinemaintenance recommended Clean fanandblowermotor;routinemaintenance Clean condensers;verifydoorgaskets required mechanics andcheckfi lter recommended Clean condensers,fansandblowermotor;verify provider Inspection, cleaning,andlubricationbyservice required recommended Calibration ofspeed,timer, temperature Certifi cation Calibration Calibration Maintenance byautoclavetechnician 1 eomne required recommended required recommended required recommended eurd– required ed orservicedprior 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 93 l i _ m y c o b a c t e r i o l o g y 644 MGIT960TimeTo Detection MonthlyExercise (Required) 16.4.4 16.4.3.1 _ 5. c. b. checkallreagents ii.Visually used;subculture toBAPifcontaminationissuspected. iii. i. a. ii.Nogrowth inMGIToronLJculture. 4. i.Negativeﬂ iii.Growth onLJculture; colonycountsshouldbecomparablewithsimilartests. b. ii.Growth inMGIT; TTDshouldbecomparablewithsimilartests. Negative i.Positiveﬂ control a.Positivecontrol 3. b. l a a. 2. 2. 1. turbidity standard, dilutingMTBsuspensions,andpipettingskills. assesses consistencyoftechnicalperformanceinpreparing MTBsuspensionsaccording toa (reproducibility) oftheTTDforknownquantities An importantcontrol measure oftheMGITculture systemistoevaluatetheconsistency 1. recommended thatafrozen stock,noolderthansixmonths,beusedtoprepare thedilution. b _ m Appendix Kformtodocumentthecorrective action. MGIT/LJ Culture QCForm–AppendixH.Ifresults are notacceptable,prepare an Documentation: technician repeat exercise withnextprocessing batch. Corrective actions: Acceptable results: a patient specimensandperformroutine microscopy, MGIT, andLJculture. before thenegativecontrol. Process bothcontrol specimensinthesamemanneras Controls: Controls: Frequency: Frequency: n If TTDsand/orcolonycountsonLJvarywidely, investigatepossiblecausefordeviations. Refer to (H37Rv orH37Ra).Suspensionshouldbeprepared using10–15dayoldgrowth onLJ. suspected. reagents. contamination hasoccurred orthere isaproblem withsterilityoftheprocessing If growth isdetectedoneithermediafrom thenegativecontrol, eithercross- used anddilutingtechnique. If nogrowth occursoneithermediafrom thepositivecontrol, checkageofisolate Negative control: 4ml7H9broth. u Quality MonitoringofSputumProcessing with 700μlofa10 Positive control –4ml7H9Broth, oraknownnegativesputumspecimen,inoculated a adding reagents, openingcapstoosoon aftervortexing,etc. contribute tocontamination;e.g.,poororganizationoftubes,splashingwhen Observe thetechnicianwhoprocessed thesespecimensfortechniquesknownto results. Record patientsamplesprocessed atthistimeandmonitorcloselyforexpected l

_ 2 c o u Placecontrols attheendofbatchpatienttests,withpositivecontrol Onedilutionof l section . i n Onceperweek,orwitheachpatientbatch. Notify theSponsorimmediatelyifcross-contaminationofMTBis Once per month, and performed by alternating technicians,ifappropriate. Once permonth,andperformedbyalternating d uorescent smear. d uorescent smear.

Record themicroscopy, MGITandLJculture results ontheWeekly 9 3 IfQCresults are unexpected,notifysupervisorimmediatelyandhave 16.2.1.3.1fordetails. –2 dilutionofaMcFarlandNo.0.5suspension M. tuberculosis (H37Rv orH37Ra)in7H9broth. Itishighly M. tuberculosis organisms. Thisexercise M. tuberculosis

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 94 l i _ m 94 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 1. Inoculation andIncubation positive bytheinstrument.Retrievedatafortimetodetection. EntertheinoculatedtubeinMGIT960instrument.Take thetubeoutwhenindicated 3. InoculateoneMGITtubewith0.5mlofthe1:500 dilution( 2. _ m Follow instructionsin Preparation ofCultureSuspension c. b. a.Record results ontheMGITTTDWorksheet –AppendixL 5. c. b. a. 4. b.Monthtomonthconsistencyinthelab’s establishedTTDrangeforthe1:500dilution. a. 3. of sterilewaterorsaline( sterile waterorsaline( a 1:500dilution,representing aboutlog10 Tube 3 Dilute1:10twomore timesbyadding0.5mlofsuspensionfrom 3. suspension to4.0mlofsterilewaterorsaline.Mixwell( Dilutethestocksuspension(freshly prepared orfrozen) 1:5bytransferring1.0mlof 2. card. with aWickerham appearance onLJmedia.Carefully adjusttheinoculumwithaspectrophotometer orvisually prepare afresh McFarlandNo.0.5suspensionusingcolonieswithin10-15daysofﬁ Removeonealiquotofthestocksuspensionfrom thefreezer andthaw. Alternatively, 1. NOTE: Preparation of1:500Dilution Medium andMGIT960Growth SupplementKittoprepare theculture suspension. a n Supplement MGITmediumwithGrowth SupplementandPANTA asspeciﬁ 10: LiquidCulture –MycobacteriaGrowth IndicatorTube (MGIT). u a Documentation: deviations withinthelaboratory’s ownestablishedrange: by theSponsorlaboratorynetwork.Ifexpectedresults varybymore than±2standard result tothelaboratory’s ownongoingrangeofresults andtoaglobalrangeestablished Corrective actions: Acceptable results: l

_ documents inlaboratoryQCbinder. applicable MGITprintoutsandanyAppendixKforms,totheSponsor. Filecopyof Each monthupontestcompletion,provide completedAppendixLform,alongwith frozen stocks). If TTDissigniﬁ 2 test underobservationifnecessary. Review dataandallprocedures withtechnicianperformingtheexercise andrepeat Manual. The TTDmustfallwithinthe6-10dayrangeasreferenced intheFINDMGITProcedure Itishighlyrecommended toprepare thedilutionfrom afrozen stocksuspension. corrective actions. If QCresults are notacceptable,prepare anAppendixKformtodocumentthe repeat exercise withfresh culture. If TTDissigniﬁ willbeusedfortheinoculationandincubationofMGITtube.Thistuberepresents c o u l . i n d d

9 4 cantly decreased, checkculture forcontaminationand,ifcontaminated, cantly increased, checkviabilityofculture andageofculture (ifusing TTDresults willbecloselymonitored bycomparingeachmonthlyTTD Tube 2 Section Tube 3 ). Mixwell,andthenagainadd0.5mlfrom 16.2.1.3.1:QCProtocol forBACTECMGIT960Culture ). 5 organisms. Tube 1 Tube 3 ). ). Tube 1 Tube 2 ed in into4.5mlof Section to4.5ml rst

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 95 l i _ m y c o b a c t e r i o l o g y 646 AFBSmearReviewforTechnical Competency(Strongly recommended) 16.4.6 ContaminationRateAssessment(Required) 16.4.5 _ l a 4. b. a. 3. b.Positivesmearsshouldnotvarybymore thanonequantiﬁ a.Negativesmearsshouldberesulted asnegativebyalltechnicians. 2. 4. b. a. 3. b. a. 2. . Calculatetheon-goingcumulativemeanrateforeachmedium. 5. 4. 3. RepeatStep1forLJcultures. 2. 1. Procedure 1. assure consistencyamongexaminers. Periodic smearreview isrecommended toassessandimprove technicianproﬁ 1. trends ofincreasing ordecreasing contaminationandshouldbemonitored. A monthlyassessmentoftheratescontaminationinMGITandLJcultures demonstrates b _ Corrective Actions: m a Documentation: Corrective actions: Acceptable results: Documentation: Results: previous monthandthelab’s average. Record the%contaminatedforeachmediumandcompare results withdatafrom the collected specimensforbothMGITandLJcultures. Repeat steps1and2,separatingpatient-collectedspecimenshospital/clinic- provide consistentindicatorsfortheoverallperformanceoflabprocedures. exactly withthecultures reported inthatsamemonth,butstatisticsovertimewill NOTE: contaminated (number÷numberreported ×100=%contamination). of contaminatedcultures reported inthatmonth.Calculatethepercentage (%) Count thetotalnumberofMGITcultures reported inthemonthandnumber Frequency: Frequency: n u agreement. Review discrepant results withtechnician(s)andre-examine smearsthatare notin and troubleshooting instructions. If MGITcontaminationrateis>10%,notifytheSponsorimmediatelyforguidance overgrowth. mucoid orhavebeenimproperly stored duringtransportencouragingbacterial When specimensare inadequatelydecontaminatedbecausespecimensare heavily Provide additionaltrainingasindicatedtoimprove performance. troubleshooting instructions. If LJcontaminationrateis>6%,notifytheSponsorimmediatelyforguidanceand Some highlyresistant bacterialspeciesare unaffected bydecontamination. a l

_ 2 c Using thismethod,thecultures processed inanygivenmonthmaynotcorrelate o Contaminationofliquidandsolidcultures occurs: u l . i n Onceamonth Onceamonth d d

9 5 Saveresults inQCbinderandtechnicians’personnelﬁ Report ﬁ Reasonableratesofcontaminationare unavoidable. ndingsonMonthlyDataMonitorsForm–AppendixI. cationlevel. les, ifappropriate. ciency and ciency 111/04/2014 17:40 1 / 0 4 / 2 0 95 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 96 l i _ m 96 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b 16.4.7 AnalysisofLaboratoryData(Strongly recommended) _ m c. b. a. 3. c. b.50%ofculture positive specimensmaybefrom smear-negative specimens. a. 2. e.Calculatethecumulativemeanpositivityrateforeachmedium. d. c.RepeatStepsa.andb.forLJcultures. b. a. 1. Culture PositivityRate Frequency: procedures mustbereviewed andcorrective measures instituted. If there issigniﬁ Monitors Forms–AppendixIisprovided forrecording thesedata. or normaltrend foranindividuallaboratoryworkloadcanbedetermined.MonthlyData properly. Afterinitiallyanalyzingtherecords forthree tosixmonths,anoverallaverage that specimenprocessing procedures, aswellmycobacterialisolationrates,are performing It isstrongly recommended thatlaboratorydataberecorded andanalyzedtohelpassure Reviewresults forconsistencyandanydiscrepancies amongtechnicians. 3. Asktechnicianstoread andrecord results inablindedmanner. 2. 1. Procedure d. a n u a Corrective Actions: Results: Procedure: negative toscanty, 1+,2+,and3+. Select 10slidesfrom thoseread intheprevious monthandthatrepresent arangefrom l

_ homemade), followingtheguidelinesin of specimens,performQCprocedures fortheapplicablelotofLJ(commercial or If theLJisolationratediffers by>20%oftheMGITisolationrateforsameset very fewAFB. liquid medium,theisolationrateonLJmaybeslightlylower, especiallyifsmearsshow Rates shouldberelatively consistentforeachmedium.Duetothehighersensitivityof 2 media types,review decontaminationprocedures. If there isasignifi > 90%ofsmearpositivespecimensshouldbeculture positive. the monthx100). rate from smear-positive specimens(smearpositive÷totalculture positive number ofpositiveMGITcultures. UsethesevaluestocalculatetheMGITisolation Count thenumberofpositiveMGITcultures thatare smearpositive.Countthetotal following mediaQCguidelinesabove. If there isasignifi lab’s average. Record isolationratesandcompare results withdatafrom theprevious monthandthe reported positivity from smearnegativespecimens(smear÷totalculture positive number ofpositiveMGITcultures. UsethesevaluestocalculatetheMGITculture Count thenumberofpositiveMGITcultures thatare smearnegative.Countthetotal intervals examined,review allmicroscopy procedures anddiscusswithpersonnel. If there isasignifi c o u l . Eachparametershouldbecalculatedmonthly. i n d d

inthemonthx100).

cant changeordeviationfrom normalresults inanyoftheparameters,all 9 6 cant decrease inonlyonemediatype,checkthegrowth performance, cant decrease inoverallculture positivity(isolation)rateforboth cant changeinsmearpositivity/negativity, unrelated tothevisit Section 16.2.1.2.1:QCProtocol forLJMedium. reported in 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 97 l i _ m y c o b a c t e r i o l o g y _ l a c.Reviewsettingsandmaintenancerecords forcentrifuge. a. 1. Isolation RateofMTBandMOTT b. a. 4. b. a. 3. 2. d.CalculatethecumulativeaverageTTDsforeachmediumtype. c. b. a. 1. Time toDetection b. a. 4. 3. 2. e. d. c. b. b _ m a Procedure: Documentation: Corrective Actions: may belessthanoptimal. established range,processing ofspecimensmaybetooharshorcentrifugespeed/time indicates thatprocessing hasimproved. IfthemonthlyaverageTTD>20%oflab’s increase incontaminationrateorisolationofrapidlygrowing mycobacteria),thislikely Results: Procedure: Documentation: processing, culture media,etc.),usingsupervisorobservationwhenevernecessary. patient colonization,thoroughly review allprocedures (e.g.,safety, cleaning,specimen If there isasignifi Corrective Actions: environmental contaminantorcross-contamination event. with MOTT(acommonobservation).However, thismayalsoindicatethepresence ofan it maybeduetoincrease innumbersoffollow-upspecimensfrom subjectscolonized should remain fairlyconstantovertime.Ifthere isasuddenincrease inisolationofMOTT, Results: n u and lab’s average. Record results forbothmediaandcompare results withdatafrom theprevious month Repeat stepsa.andb.forLJcultures. cultures Count thenumberofMTBisolatesandcalculatepercentage usingthetotalMGIT lab-specifi Record dataontheMonthlyDataMonitorsForms–AppendixIorothercomparable procedures anddiscusswithpersonnel,usingsupervisorobservationifnecessary. If there isasignifi negative specimensthatare determinedtobe Calculate thetimetodetectionforMGITcultures from smearpositiveand comparable document. Record dataontheMonthlyDataMonitorsForms–AppendixIorotherlab-specifi Calculate thecumulativemeanpositivityrateforeachmycobacterialspecies. decreases inTTDmayalsoprompt review ofapplicableprocedures. the activities. If anycorrective measures are necessary, prepare anAppendixKformtodocument Although animprovement inprocessing isgenerallyconsidered benefi Repeat stepa.forLJcultures. the activities. If anycorrective measures are necessary, prepare anAppendixKformtodocument month andlab’s average. Record isolationratesforbothspeciesandcompare results withdatafrom theprevious MGIT cultures Count thenumberofMOTTisolatesandcalculatepercentage usingthetotal a l

_ 2 c o IsolationofMTBcomplexorganismsinrelation toothermycobacterialspecies If themonthlyaverageTTDis<20%oflab’s establishedaverage(withoutan u l . i n reported d cdocument. d

9 7 reported cant increase inMOTTisolation,seeminglyunrelated toindividual inagivenmonth(MTBculture ÷totalculture positivex100). cant increase inTTD(unrelated tovisitinterval),review processing inagivenmonth(MOTTculture ÷totalculture positivex100). MTB . cial, signiﬁ cant c 111/04/2014 17:40 1 / 0 4 / 2 0 97 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 98 l i _ m 98 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b to correct theproblem. Theformincludesthefollowingcomponents: observed, theuseofastandardized form,suchasAppendixK,mustdocumenttheactiontaken Quality Control testsfailtogivetheproper results and/orwhendeviationsfrom baselinedataare The Sponsorrequires thedocumentationofefforts toimprove thequalityoflab.When issues ofconcern. with QCandQM,toelicitsuggestionsforimprovement, andencourageinputfrom staff onany of allpersonnel.Regularstaff meetingsshouldbeheldtoshare anddiscussqualityissuesfound workﬂ Continuous efforts toimprove theoverallqualityoflaboratory, byimproving service,function, 16.5 QualityImprovement _ m a. 3. 2. b.Record thelabaccessionnumbersofspecimensprocessed ineachbatch. a. 1. the normalrange. contamination occurs,orwhenoverallpositivityratesandotherparametersdeviatefrom responsibilities ishelpfulintheeventthatanunusualepisodeofcontaminationorcross- responsible forprocessing specimens.Tracking labpersonnelalongwiththeirspecimen All parametersdescribedaboveshouldremain similaramongdifferent technologists Technologist Assessment b. a. 4. b. a. 4. a n u Procedure: ow, personnel,andcustomersatisfactionare necessaryandrequire theinputandsupport a b. Corrective Actions: deviations from standard processing protocols. check foranycorrelation withaspeciﬁ Results: Documentation: Documentation: l

_ • Ex:FormNumber:01JAN11-a • a frequent (quarterly)basis,andespeciallybefore eachchangeinrotation. Ideally, review eachstepofthespecimenprocessing SOPwiththetechnologist(s)on 2 If reviews implicateaspecifi processing worksheet. Record thenamesoftechnologistsperformingprocedures onthelaboratory’s comparable document. Record dataontheMonthlyDataMonitorsForms–AppendixIorotherlab-specifi specifi Document alllabtrainings,re-trainings andcompetencyassessmentsonthelaboratory- the activities. If anycorrective measures are necessary, prepare anAppendixKformtodocument the activities. If anycorrective measures are necessary, prepare anAppendixKformtodocument related deviations. Perform annualcompetencyassessmentsofalltechnologiststocontrol fortechnician- c here. Description oftheproblem/improvement -Thedateandashortdescriptionare written the dateisincorporatedintothisnumber. Form Number-numberassignedtoeachformalloweasyfi 30JAN11. 2011.Problemdiscovered Ex: MonthlyTTDQCexercisenotperformedinJanuary o u l . i n Whenconductingreviews onidentifi cform. d d

9 8 cstaff member, re-train thetechnologistasappropriate. c technologist(s),whichcouldindicatepossible ed deviationsintheparametersabove, ling andretrieval. Often c 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 99 l i _ m y c o b a c t e r i o l o g y _ l a b _ m a n • • • • • u a Ex: TTDQCexerciseperformedimmediatelyon30JAN11. he hasbeennotifi and thelabsupervisor, shouldreview theformandsigndocument,attestingthats/ All personsinvolvedintheincident,e.g.,personidentifyingissue,QAdelegate explain howtheeffectiveness ofthepreviously mentionedactionisevaluated. Person(s) responsible formonitoringtheeffectiveness ofaction-Thissectionshould should address theroot causeoftheproblem. differs from theactiontakentofi Preventative actionrequired toeliminateroot causeofproblem -Thisactionoften immediate issueiswrittenhere, alongwiththe expecteddateofresolution. Corrective actiontofi and theroot causewritteninthissection. problem thatcausesaQCfailure ordeviationtooccur. Theissueshouldbeanalyzed, Investigation intotheroot causeoftheproblem -Oftentimes,there isabiggerunderlying February andMarch.21MAR11. February laboratorian signedbytheirnameafterperformingtheTTDQCexerciseforboth Ex: Ms.PimwasassignedtocheckthescheduleinMarch,andverifyappropriate 01FEB11. Ex:AschedulewaspreparedtoensureeachMGITuserperformstheTTDexercise. Ex: TTDQCexercisewasnotassignedtoaparticularlaboratorian.30JAN11. l

_ 2 c o u l . i n d d

9 9 edoftheissueandagree totheactionstaken. x thecurrent problem -Theﬁ x theimmediateproblem (mentionedabove),and rst actiontakentoresolve the 111/04/2014 17:40 1 / 0 4 / 2 0 99 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 100 l i _ m 100 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b line Anti-tuberculosis Drugs.WHO/HTM/TB/2008.392.Geneva,Switzerland,2008. 18. World HealthOrganization.PolicyGuidanceonDrugSusceptibilityTesting (DST)ofSecond- Geneva, Switzerland.2008.www.who.int/diagnostics_laboratory 17. World HealthOrganization.MaintenanceManualforLaboratoryEquipment,SecondEdition. 2004 16. World HealthOrganization.LaboratoryBiosafetyManual,Third Edition.Geneva,Switzerland, 2009. 15. World HealthOrganization.GoodClinicalLaboratoryPractice(GLCP).Geneva,Switzerland, tics, July2006. 14. Siddiqi, SHandRüsch-Gerdes, S.MGITProcedure Manual.FoundationforInnovative Diagnos- HIV ClinicalResearch SupportContract No.N01-AI-50022.Bethesda,Maryland,2008. National InstitutesofHealth.DAIDSGuidelinesforGoodClinicalLaboratoryPracticeStandards.13. PrintingOffi Health andHumanServices,USGovernment Disease Control, DivisionofLaboratoryTraining andConsultation.Atlanta,GA,USDepartmentof 12. Kent PT, KubicaGP. PublicHealthMicrobiology, aGuidefortheLevelIIILaboratory. Centersfor Society forMicrobiology (Publisher),Washington, D.C.,2004. 11. Isenberg HD(Editor).ClinicalMicrobiology Procedure Handbook,Vol.1, 2ndEdition.American Bacteriology ServicesinLow-IncomeCountries,2ndEdition”Paris,France,2007. 10. UnionAgainstTuberculosisInternational andLungDisease.Priorities forTuberculosis 2008; 46(1):18–29. Research ofPharmaceuticalandBiomedicalAnalysis. andClinicalResearch Laboratories.Journal Ezzella,J.etal.GuidelinesonGoodClinicalLaboratoryPractice: BridgingOperationsbetween 9. Wayne, PA, 2004. Model forLaboratoryServices;Approved Guideline–Third Edition.NCCLSDocumentGP26-A3. ClinicalandLaboratoryStandards Institute.Application ofaQualityManagementSystem 8. Mycobacteria; Proposed Guideline.CLSI DocumentM48-P. Wayne, PA, 2007. Clinical andLaboratoryStandards Institute.LaboratoryDetectionandIdentifi 7. Wayne, PA, 2004. Microbiological Culture Media;Approved Standard –Third Edition.NCCLSDocumentM22-A3. ClinicalandLaboratoryStandards Institute.QualityControl forCommercially Prepared 6. M24-A2. Wayne, PA, 2011. and OtherAerobic Actinomycetes;Approved Standard– SecondEdition.NCCLSDocument ClinicalandLaboratoryStandards Institute.SusceptibilityTesting ofMycobacteria,Nocardiae, 5. M23-A3. Wayne, PA, 2008. Criteria andQualityControl Parameters;Approved Guideline–Third Edition.CLSIDocument ClinicalandLaboratoryStandards Institute.DevelopmentofIn-Vitro SusceptibilityTesting 4. Printing Offi Microbiological andBiomedicalLaboratories.HHSPublicationNo.(CDC)21-1112.USGovernment CentersforDiseaseControl andPrevention. NationalInstitutesofHealth.Biosafetyin 3. MA-0126. Sparks,Maryland,2007/01. Becton,DickinsonandCompany. BACTECMGIT960ASTInstructions.BDDocumentNumber 2. Number MA-0117.Sparks,Maryland,2004/06. Becton,DickinsonandCompany. BACTECMGIT960 SystemUser’s Manual.BDDocument 1. REFERENCES _ m a n u a l

_ 2 c o ce.Washington, 2009. u l . i n d d

1 0 0 ce,1985. cto of cation 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 101 l i _ m y c o b a c t e r i o l o completing thisform Name ofperson collected) specimen (or receiving,ifpatient- Name ofpersoncollecting (circle one) Mode oftransport to laboratory Date*/Time ofdispatch g y This sectionshouldbecompletedbythelaboratorytechnicianreceiving thespecimen. Laboratory Section This sectionshouldbecompletedbythedriver/courier. Transport Section This sectionshouldbecompletedbyPrincipalInvestigatorordelegate. Dispatch Section sample submitted.Oncetheformiscompleted,itshouldremain inthelabﬁ patient enrolled intheSponsorstudytolaboratory. UseaseparateAppendixAformforeach Attach astudylabeltothispage.Thisformshouldaccompanyanyspecimengeneratedby APPENDIX A:SPECIMENTRANSFERFORM _ Comments: dd/mmm/yyyy *Date format (tick one) kept oniceuponreceipt? Specimens refrigeratedor received Date*/time specimen kept onice?(tickone) Specimens refrigeratedor fn,gv eal Isstudylabelmissing? If no,givedetails (tick one) proper temperature? no leakage,specimenat and properlylabeled, i.e. containerisintact Sample ingoodcondition, of courier Driver nameor aeo ehiinSignature Name oftechnician (circle one) Specimen collectedby: N/A ifVisit 2or3) (circle one-onlycircle Specimen Number SubjectID# collected) (or received,ifpatient- Date*/time collected Visit #(circleone) Screening ID# Site name Time atroomtemp l a b _ m a n u a l

_ 2 c o u l . 1 i n d d

1 0 1 ( 1 Record 0 hr 0 min if immediately Record 0hrminifimmediately 27 28293031a 31b 32a 15 1617181920212223242526 32b 33 34ET 2 345678910 11 UNS 12 13 14 ¨ ¨ ¨ refrigerated orshipped) in-person …… hrmin patient ¨ ¨ 1 Ys Yes Yes ¨ ¨ Yes 2 ¨ ¨ ¨ ¨ …… /:(24hclock) sitestaff …/… … ……:(24hclock) …… / NASputuminduced? N/A No No …… /:(24hclock) ¨ ¨ No Signature Attach studylabelhere: courier Site # pcmnvlm ……ml Specimen volume Signature Lab accessionnumber ¨ Unknown ¨ labmessenger (3 characters) PT initials ¨ les. ¨ ¨ Yes Ys Yes other:………… …… ¨ No ¨ No 111/04/2014 17:40 1 / 0 4 / 2 101 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 102 l i _ m 102 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y (tick onebox) Smear Result: Comments: _ ptmidcd Yes Patient Sputum collectedby: Sputum induced? Comments: ID Test Method Identiﬁ (tick onebox) ZN Result: l Date ofMGITresult: Comments: a b MGIT 960systemculture ofsputum AFB microscopy from sputumspecimen Sputum specimen APPENDIX B:STUDYSOURCEDOCUMENTWORKSHEET Storage ofSpecimens AFB =AcidFastBacilli;BAPBloodAgarPlate;ZNZiehl-Neelsen uevsrsgaue ………… Date:……/ accurate forthisspecimen. My signatureconﬁ Supervisor signature: …………………… _ cenn D#SbetI Visit # SubjectID# Site# Sputum Specimen Screening ID# Lab Accession# storage onLJmedium? Was specimenbankedforshort-term Final dateofMGITculturecompletion Did specimenrequirere-decontamination? (tick onebox) MGIT CultureResult: (dd/mmm/yyyy) (dd/mmm/yyyy) Date ofinoculation: (tick onebox) Type ofsmear: m (dd/mmm/yyyy) cation ofAFB a n u Date a l

_ 2 c eevdProcessed Received o u l . i n rms thatalldatahavebeenreviewedforaccuracyandpre-ﬁ d Negative d ¨ ¨ ¨ ¨ ¨ ¨ ¨ ¨

Acridine Orange Auramine/Rhodamine Auramine O

(24hr clock)

Other:………… Capilia TBcID N/A PositiveforAFB NegativeforAFB PositiveforMTBcomplex ¨ No TBgrowth,butpositive for othermycobacteria 0 Time ¨ 2 NegativeforMTBcomplex (Only tickN/AforV2orV3) 1 ¨ ¨ ¨ 2 No ¨ (dd/mmm/yyyy) (dd/mmm/yyyy) Site Rare ¨ Date ¨ ¨ ¨ ¨ ¨ ¨ N/A Yes ¨ ¨ N/A PositiveforMTBcomplex NegativeforMTBcomplex ¨

¨ /Tech initials: dd/mmm/yyyy Technician initials: Date: No Ys Yes ¨

Original

read: read: Date smear from MGIT (24hr clock) Few

printout: ¨ ¨ (tick one Time Result: N/A Afﬁ box) BAP ¨ specimen labelhere:

¨ x acompletedstudy TTD No long-term storageat–70°C? long-term Was specimenbankedfor ¨ ¨ ¨ asHours Days Many N/A Nogrowth Contaminatedand/orMOTT Volume ¨ (ml) ¨ ¨ ¨ Tech initials/date Unknown Contaminated PositiveforMTBcomplex and contaminated (dd/mmm/yyyy) (dd/mmm/yyyy) Tech initials/date ain ntas…… Patient Initials Purulent Liquid /Saliva initials: Technician (TTD) original Final or revised (tick allboxesthatapply) TNTC lled informationonthisformis to Result ¨ ¨ Viscou Time Character ¨ Blood ¨ Yes (Comment ¨ asHours Days required) NotDone

No ¨ ¨

¨ N/A ¨

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 103 l i _ m y c o b a c t e r i o eo xcnMI 2.0 2.0 0.25 1.0 2.5 1.0 5.0 1.0 0.1 MGIT Initials/Date: MGIT MGIT 1st lineDSTTech 100 Enviomycin Sulfate MGIT MGIT Gatifl oxacin Levofl oxacin Moxifl oxacin MGIT Ofl MGIT oxacin Capreomycin MGIT Amikacin MGIT Kanamycin Streptomycin Ethambutol Pyrazinamide MGIT Rifampicin Isoniazid l ( Result: (tickonebox) o S g =Sensitive; y uevsrsgaue ………… Date:……/ accurate forthisspecimen. My signatureconfi Supervisor signature: …………………… Shipping Drug susceptibilitytesting LJ Culture ofSputum MTB complex MTB complex _ Negative for Negative for Method: ID Test Identifi a/eeioaes hpe?Yes Comments: (dd/mmm/yyyy) Date ofshipment: Aliquot #1 For V2andV3shippingonly: Was/were isolate(s)shipped? Comments: (dd/mmm/yyyy) (dd/mmm/yyyy) Date ofinoculation: Comments: (dd/mmm/yyyy) Final dateofLJculturecompletion: Confi 1 Sputum Specimen ID # Screening Isolate ¨ l a b _ m rmatory ZNStainResults:(Tick onebox) rmatory cation ofAFB a Drug n u a l (1-9 colonies)

_ TB growth 2 R c =Resistant; o ¨ ¨ rms thatalldatahavebeenreviewedforaccuracyandpre-ﬁ u l . TcI TBcID i n d d (MGIT, other-specify)

Testing Method

1 TB growth TB growth colonies) 0 (10-100 (10-100 (Only tickN/AforV2orV3) 3 ¨ ID # Subject ¨ TF 1 Capilia ¨ =Test Failed;

100 colonies) 2 (more than (more than TB growth TB growth ¨ ¨ ¨ 2 (dd/mmm/yyyy) Initials/Date: 2nd lineDSTTech N/A Date ofResult (dd/mmm/yyyy) ………… ¨ Other: ¨ (innumerable (innumerable or conﬂ TB growth TB growth N/A ND growth) ¨ ¨ ¨ ¨ =NotTested) NegativeforMTB (dd/mmm/yyyy) Date ofshipment: Aliquot #2 (dd/mmm/yyyy) If Yes, dateofshipment (dd/ Date ofLJresult: (dd/mmm/yyyy) NegativeforAFB ¨ uent uent ii Site# Visit # Tech initials/date:

Not ApplicableorRequired perProtocol complex (dd/mmm/yyyy) mycobacteria but positive but positive ¨ for other for other growth, growth, No TB No TB (OnlytickN/AforV2orV3) ¨ (ug/ml) Conc. Tech Initials /yyyy) Contami- nated ¨ ¨ ¨ ¨ PositiveforMTB PositiveforAFB lled informationonthisformis contaminated ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ ¨¨¨¨ complex complex and FND TF R S for MTB for MTB Positive Positive ¨ No ¨ Unknown ¨ N/A ¨ ¨ N/A N/A ¨ 111/04/2014 17:40 1 / 0 4 / 2 103 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 104 l i _ m 104 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l Comments: Signature: Date** Reviewed by: a preparation Slides dateof Control Positive ot:……………… Year: …………… Date** Expiration Batch orLot#/ Orange Acridine Note: Month: …………………………… b APPENDIX C:DAILY QCSTAINING FORMS *Conﬁ _ positive control 1 Day MTB straintested/ m 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 a 9 8 7 6 5 4 3 2 1 rmation required by another technician or prepare another smear, stain and read. **Date format: dd/mmm/yyyy **Dateformat: requiredbyanothertechnicianorprepare smear,rmation stainandread. n If QC is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor IfQCisoutofrange,inform u a l

Positive control _ 2 c Required*, Scanty, 1+,2+,3+) o u Results (Neg,Conﬁ l . i n d d

1 0 4 1 Appendix C:AcridineOrangeDailyQCStainingForm Expiration Date** Batch orLot #/ expiration date of control slide Positive 2N H Negative control rmation rmation 2 SO 4

2 CPs Ys o Technician Initials QC Pass(Yes, No) control 2 Strain tested/negative preparation date of control slide Negative Expiration Date** Batch orLot #/ Acid Alcohol expiration date of control slide Negative 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 105 l i _ m y c o b a c t e r i o l o Comments: Signature: Date** Reviewed by: g ot:……………… Year: …………… Date** Expiration Batch orLot#/ Auramine O Note: Month: …………………………… preparation Slides dateof Control Positive y *Conﬁ _ positive control 1 Day MTB straintested/ l a b 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 _ rmation required by another technician or prepare another smear, stain and read. **Date format: dd/mmm/yyyy **Dateformat: requiredbyanothertechnicianorpreparesmear,rmation stainand read. If QC is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor IfQCisoutofrange,inform m a n u Positive control a l Required*, Scanty, 1+,2+,3+)

_ 2 Results (Neg,Conﬁ c o u l . i n d d

1 1 0 5 Expiration Date** Batch orLot #/ Decolorizer Acid Alcohol expiration date of control slide Positive Negative control Appendix C:AuramineODailyQCStainingForm rmation rmation 2 CPs Ys o Technician Initials QC Pass(Yes, No) control 2 Strain tested/negative preparation date of control slide Negative Expiration Date** Batch orLot#/ Permanganate Potassium expiration date of control slide Negative 111/04/2014 17:40 1 / 0 4 / 2 105 0 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 106 l i _ m 106 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l Comments: Signature: Date** Reviewed by: a b ot:……………… Year: …………… Date** Expiration Batch orLot#/ Rhodamine Auramine/ Note: Month: …………………………… preparation Slides dateof Control Positive *Conﬁ _ positive control 1 MTB straintested/ Day m 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 a 9 8 7 6 5 4 3 2 1 rmation required by another technician or prepare another smear, stain and read. **Date format: dd/mmm/yyyy **Dateformat: requiredbyanothertechnicianorprepare smear,rmation stainandread. n If QC is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor IfQCisoutofrange,inform u a l

_ Positive control 2 c Required*, Scanty, 1+,2+,3+) o u Results (Neg,Conﬁ l . i n d d

1 0 Appendix C:Auramine/RhodamineDailyQCStainingForm 6 1 Expiration Date** Batch orLot #/ Decolorizer Acid Alcohol expiration date of control slide Positive Negative control rmation rmation 2 CPs Ys o Technician Initials QC Pass(Yes, No) control 2 Strain tested/negative preparation date of control slide Negative Expiration Date** Batch orLot#/ Permanganate Potassium expiration date of control slide Negative 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 107 l i _ m y c o b a c t e r i o l o Comments: Signature: Date* Reviewed by: g ot:……………… Year: …………… preparation Slides dateof Control Positive Date* Expiration Batch orLot#/ ZN Stain Note: Month: …………………………… y *Date format: dd/mmm/yyyy *Date format: _ positive control 1 Day MTB straintested/ l a b 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 _ If QC is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor IfQCisoutofrange,inform m a n u Positive control a l

_ 2 Results (Positive,Negative) c o u l . i n d d

1 1 0 7 expiration date of control slide Positive Expiration Date* Batch orLot #/ Decolorizer Acid Alcohol Appendix C:Ziehl-NeelsenDailyQCStainingForm Negative control 2 CPs Ys o Technician Initials QC Pass(Yes, No) control 2 Strain tested/negative preparation date of control slide Negative Expiration Date* Batch orLot#/ Methylene Blue expiration date of control slide Negative 111/04/2014 17:40 1 / 0 4 / 2 107 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 108 l i _ m 108 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a Comments: Signature: Reviewed by: Date* ot:……… Year: …………… Month: …………… Note: –70°C ±10°C –80°C Temperature Range(circleone): ±10°C Equip Number:…………… b APPENDIX D:EQUIPMENTTEMPERATURE RECORDFORMS *Date format: dd/mmm/yyyy *Date format: _ a Temp 1 Day m 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 a n If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform u a l

_ 2 c o u l . i n d d

1 0 8 Time Taken/ Appendix D:FreezerTemperature RecordForm Initials Temp 2 Thermometer #:…………… Thermometer Location (room#orequivalent):…………… Time Taken/ Initials Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 109 l i _ m y c o b a c t e r i o l o g Comments: Signature: Reviewed by: Date* ot:……… Year: …………… Month: …………… Note: Temperature Range:2°C-8°C Equip Number:…………… y *Date format: dd/mmm/yyyy *Date format: _ a Temp 1 Day l a 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 b _ If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform m a n u a l

_ 2 c o u l . i n d d

1 0 9 Appendix D:RefrigeratorTemperature RecordForm Time Taken/ Initials Temp 2 Thermometer #:…………… Thermometer Location (room#orequivalent):…………… Time Taken/ Initials Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 109 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 110 l i _ m 110 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a Day Comments: Signature: Reviewed by: Date* ot:……… Year: …………… #:…………… Thermometer Freezer Temp Range:–20°C±2°C Month: …………… Note: #:…………… Thermometer Refrigerator Temp Range:2-8°C Equip Number:…………… b 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 *Date format: dd/mmm/yyyy *Date format: 9 8 7 6 5 4 3 2 1 _ m a n Refrigerator If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform u a Temp 1 l

_ 2 c o u l . i n d d Freezer Temp 1

1 1 0 Time Taken/ Appendix D:Refrigerator/FreezerRecordForm Initials Refrigerator Temp 2 Location (room#orequivalent):…………… Freezer Temp 2 Time Taken/ Initials Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 111 l i _ m y c o b a c t e r i o l o g Comments: Signature: Reviewed by: Date* ot:……… Year: …………… Month: …………… Note: Temperature Range:37°C±1°C Equip Number:…………… y *Date format: dd/mmm/yyyy *Date format: _ a Temp 1 Day l a 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 b _ If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform m a n u a l

_ 2 c o u l . i n d d

1 1 1 Time Taken/ Appendix D:IncubatorTemperature RecordForm Initials Temp 2 Thermometer #:…………… Thermometer Location (room#orequivalent):…………… Time Taken/ Initials Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 111 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 112 l i _ m 112 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ Comments: Signature: Reviewed by: Date* 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 l CO Temperature Range:37°C±1°C Equip Number:…………… 9 8 7 6 5 4 3 2 1 a b *Date format: dd/mmm/yyyy *Date format: a Temp 1 Day ot:……… Year: …………… Month: …………… Note: _ m a 2 n Range:5-10% u If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform a l

_ 2 c o u l . i CO n #1 d d 2 %

1 1 Time Taken/ 2 Initials Appendix D:CO Temp 2 Thermometer #:…………… Thermometer Location #(roomorequivalent):…………… #2 CO 2 2 IncubatorRecordForm % Time Taken/ Initials pressure CO 2 tank Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 113 l i _ m y c o b a c t e r i o l o g y Comments: Signature: Reviewed by: Date* ot:……… Year: …………… Month: …………… Note: Temperature Range:4°C-12°C Equip Number:…………… *Date format: dd/mmm/yyyy *Date format: _ l a Temp 1 Day a 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 b 9 8 7 6 5 4 3 2 1 _ m If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform a n u a l

_ 2 c o u l . i n d Appendix D:RefrigeratedCentrifugeTemperature RecordForm d

1 1 3 Time Taken/ Initials Temp 2 Thermometer #:…………… Thermometer Location (room#orequivalent):…………… Time Taken/ Initials Adjustments Action/ Maintenance/ Notes 111/04/2014 17:40 1 / 0 4 / 2 113 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 114 l i _ m 114 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a Comments: Signature: Reviewed by: Date* ot:……… Year: …………… Month: …………… Note: Temperature Range:**seebelow…………… Location (room#orequivalent):…………… b centrifuge: room;e.g.,MGITroom: 19-30°C;roomcontainingunre **Based onrequirementsofreagentsorinstrumenthousedintheparticular dd/mmm/yyyy *Date format: _ Day m 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 a n If temperature is out of range, inform the supervisor andrecordanyactiontakenbelow. thesupervisor Iftemperatureisoutofrange,inform u a l

≤ _ 20°C. 2 i a ntasMnMxInitials Max Min Initials Max Min c o ep1Tm ae ep2Time Taken Temp 2 Time Taken Temp 1 u l . i n d d

1 1 4 Appendix D:RoomTemperature RecordForm Thermometer #:…………… Thermometer Adjustments Action/ Maintenance/ Notes frigerated 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 115 l i _ m y c o b a c t e r i o l o g Acridine Orange Component y Number of Dt omt=d/m/yy **Conﬁ *Date format=dd/mmm/yyyy, APPENDIX E:NEWREAGENTS/MEDIAQCFORMS Lot/Batch _ Prepared HCL, concentrated Ethanol Sodium barbital/Tricine Benzalkonium chloride Sulfuric acid Sodium carbonate control positive tested/ 1 l MTB strain a Stain b _ m a n u a l

_ Prepared* Reagent 2 c Date o u l . i n d Appendix E: New Reagents/Media QC Form: AcridineOrange Appendix E:NewReagents/MediaQCForm: d

control negative tested/ 1 2 Strain 1 Date of Batch* Expiry Expiry 5 (Manufacturer)

Brand rmationrequired byanothertechnicianorprepare anothersmear, stainandread. New Lot/BatchStainingReagents Prepared Quantity Lot Number Date Tested*/ Tech Initials Received Date* (date*/initials) Review Supervisor Monthly Required**, Scanty, 1+,2+,3+) Results (Neg,Conﬁ Control Positive Expiry Expiry Date* 1 Negative Control Received Quantity rmation rmation 2 Opened* QC pass? (Yes/No) Date 111/04/2014 17:40 1 / 0 4 / 2 115 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 116 l i _ m 116 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a tao Potassium permanganate HCL, Ethanol Phenol concentrated Auramine O Component b Number of Dt omt=d/m/yy **Conﬁ *Date format=dd/mmm/yyyy, Lot/Batch Prepared _ control positive tested/ 1 MTB strain m Stain a n u a l

_ 2 c o Prepared* u Reagent l . Date i n d d

1 1 6 Appendix E: New Reagents/Media QC Form: AuramineO Appendix E:NewReagents/MediaQCForm: control negative tested/ 2 Strain Date of Batch* Expiry Expiry (Manufacturer)

Brand rmationrequired byanothertechnicianorprepare anothersmear, stainandread. New Lot/BatchStainingReagents Prepared Quantity Lot Number Date Tested*/ Tech Initials Received Date* Required**, Scanty, 1+,2+,3+) (date*/initials) Review Supervisor Monthly Results (Neg,Conﬁ Control Positive Expiry Expiry Date* 1 Negative Control Received Quantity rmation rmation 2 Opened* QC pass? (Yes/No) Date 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 117 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix E: New Reagents/Media QC Form: Auramine O (Commercial) _ m

a Results (Neg, n u Auramine O Acid Alcohol Potassium Permanganate Conﬁ rmation Required**, a l Scanty, 1+, 2+, 3+) _ QC Pass 2 c o Rec’d Rec’d Rec’d (Yes, No)/ u

l Negative . Tech Initials

i Brand/Lot Date*/ Quantity Date* Brand/Lot Date*/ Quantity Date* Brand/Lot Date*/ Quantity Date* Positive n 1 2 d control

d Number Expiration Rec’d Tested Number Expiration Rec’d Tested Number Expiration Rec’d Tested control

Date* Date* Date* 1 1 7

MTB strain tested/ 2Strain tested/negative Monthly Supervisor positive control1 control Review (date*/initials) *Date format = dd/mmm/yyyy, **Conﬁ rmation required by another technician or prepare another smear, stain and read. 111/04/2014 17:40 1 / 0 4 / 2 117 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 118 l i _ m 118 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a tao Potassium permanganate HCL, Ethanol Phenol concentrated Auramine O Component b Number of Dt omt=d/m/yy **Conﬁ *Date format=dd/mmm/yyyy, Lot/Batch _ Prepared control positive tested/ 1 MTB strain m Stain a n u a l

_ 2 c o Prepared* u Reagent l . i Date n d d Appendix E: New Reagents/Media QC Form: Auramine/Rhodamine Appendix E:NewReagents/MediaQCForm:

1 1 8 control negative tested/ 2 Strain Date of Batch* Expiry Expiry (Manufacturer)

Opened* QC pass? (Yes/No) Date 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 119 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix E: New Reagents/Media QC Form: Auramine/Rhodamine (Commercial) _ m

a Results (Neg, n u Auramine/Rhodamine Acid Alcohol Potassium Permanganate Conﬁ rmation Required**, a

l QC Pass Scanty, 1+, 2+, 3+) _

2 (Yes, No)/ c o Date*/ Date*/ Date*/ Negative u Brand/Lot Quantity Date* Brand/Lot Quantity Date* Brand/Lot Quantity Date* Positive Tech Initials l 1 2 . Expiration Expiration Expiration i n Number Rec’d Tested Number Rec’d Tested Number Rec’d Tested control control d Date* Date* Date* d

1 1 9

Mycobacteriology Laboratory Manual

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 120 l i _ m 120 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a tao Methylene blue HCL, Ethanol Phenol concentrated Fuchsin Component b Number of *Date format=dd/mmm/yyyy, Lot/Batch Prepared _ control positive tested/ 1 MTB strain m Stain a n u a l

_ 2 c o Prepared* u Reagent l . Date i n d d

1 2 Appendix E: New Reagents/Media QC Form: Ziehl-Neelsen Appendix E:NewReagents/MediaQCForm: 0 control negative tested/ 2 Strain Date of Batch* Expiry Expiry (Manufacturer)

Brand New Lot/BatchStainingReagents Prepared Quantity Lot Number Date Tested*/ Tech Initials Received Date* (date*/initials) Review Supervisor Monthly Control Positive (Positive, Negative) Expiry Expiry Date* 1 Results Negative Control Received Quantity 2 Opened* QC pass? (Yes/No) Date 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 121 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix E: New Reagents/Media QC Form: Ziehl-Neelsen (Commercial) _ m

a Results n Carbol Fuchsin Reagent Acid Alcohol Methylene Blue u (Positive, Negative) a QC Pass l

_ (Yes, No)/ 2 Date*/ Date*/ Date*/ c Brand/Lot Quantity Date* Brand/Lot Quantity Date* Brand/Lot Quantity Date* Positive Negative o Expiration Expiration Expiration 1 Tech2 Initials u control

l Number Rec’d Tested Number Rec’d Tested Number Rec’d Tested control . i Date* Date* Date* n d d

1 2 1

MTB strain tested/ 2Strain tested/negative Monthly Supervisor positive control1 control Review (date*/initials) *Date format = dd/mmm/yyyy, 111/04/2014 17:40 1 / 0 4 / 2 121 0 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 122 l i _ m 122 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a AC Monopotassium phosphate Disodium Sodium Sodium phosphate NALC citrate hydroxide Component b *Date format=dd/mmm/yyyy, _ m Reagent Name a n u a l

_ 2 c o u Number of l Lot/batch . Prepared i Reagent n d d

Appendix E: New Reagents/Media QC Form: ProcessingReagents Appendix E:NewReagents/MediaQCForm:

1 2 2 (Manufacturer) Prepared*

Reagent Date Brand New Lot/BatchReagents Expiry Date* Expiry Lot Number Prepared Quantity Received Date* initials) (date*/ Review Supervisor Monthly Buffer only) Buffer Phosphate pH (for Expiry Expiry Date* Date Putinto Received Quantity Use* Opened* Initials Date Tech 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 123 l i _ m y c o b a c t e r i o l o g (Manufacturer) Component/Brand D-MI ltbs BD -MGIT7mltubes BD -MGIT7mltubes BD -MGIT7mltubes y *Date format=dd/mmm/yyyy, _ tested 1 Number MTB strain l a b Lot _ m a n u a l

_ Supplement 2 c Number o MGIT u l . i n d d

1 2 3 (if tested) strains 2 Appendix E: New Reagents/Media QC Form: MGIT Appendix E:NewReagents/MediaQCForm: Other Tested* Lot Number Date New Lot/BatchMedia Initials Tech Received Date* Results MTB 6-10 days 1 Expiry Expiry Date* Results: initials) (date*/ Review Supervisor Monthly Strains Other 2 Tech Initials Received Quantity Result*/ Date QC pass? (Yes/No) Date Put in Use* 111/04/2014 17:40 1 / 0 4 / 2 123 0 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 124 l i _ m 124 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a opnn LotNumber Component BD MGITPANTA BD MGITSupplement BD MGITPANTA BD BD BD MGIT MGIT MGIT Supplement PANTA Supplement b *Date format=dd/mmm/yyyy, _ tested 1 Lot Number Supplement MTB strain m a n u a l

_ 2 c o u l . i n MGIT Media Lot Number d d

1 Appendix E: New Reagents/Media QC Form: MGITSupplement Appendix E:NewReagents/MediaQCForm: 2 4 (if tested) 2 Other strains Tech Initials Tested*/ Date New Lot/BatchReagents Received Results MTB 6-10 days Date* 1 initials) (date*/ Review Supervisor Monthly Expiration Results: Strains Other Date* 2 Tech Initials Result*/ Received Quantity Date QC pass? (Yes/No) Date Put in Use* 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 125 l i _ m y c o b a c t e r i o 1 l o g lcrl KH Glycerol L-J Media Base Component Malachite green Sodium glutamate y *Date format=dd/mmm/yyyy, Number of _ Prepared/ control positive tested/ Lot/Batch MTB strain l a Media b 4 _ PO m a 4 n u a l

_ 2 Date Media Prepared*/ Tech Initials c o u l . i n d Appendix E: New Reagents/Media QC Form: LowensteinMedia Appendix E:NewReagents/MediaQCForm: d

1 2 5 media culated 2 Unino- Date of (Manufacturer) Batch* Expiry Expiry

Brand Prepared Quantity Media New Lot/BatchMedia Number Lot Tested*/Tech Initials Date Received Date* 10 (date*/initials) Review Supervisor Monthly –2 MTB growth 10 Results: –3 Expiry Expiry Date* 10 1 –4 Control Sterility Results Received Quantity 2 Date*/Tech (Yes/No)/ QC pass? Initials Date Put in Use* 111/04/2014 17:40 1 / 0 4 / 2 125 0 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 126 l i _ m 126 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b (Manufacturer) Dt omt=d/m/yy **Explainincommentssection *Date format=dd/mmm/yyyy, _ actions: corrective Comments/ m a Brand n u a l

_ 2 Appendix E: New Reagents/Media QC Form: LowensteinJensenMedia(Commercial) Appendix E:NewReagents/MediaQCForm: c o u l . i n Number d d

Lot

1 2 6 Received Date* Expiration Date* Acceptable Received Quantity Appearance Acceptable Not ** (date*/initials) Review Supervisor Monthly Put into Date* Use QC pass? (Yes/No) Initials/ Date* Tech 111/04/2014 17:40 1 / 0 4 / 2 0 1 4

1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 127 l i _ m y c o b a c t e r i o l 1 o g lcrl ADC Glycerol medium Middlebrook 7H9basal or OADC supplement Component y *Date format=dd/mmm/yyyy, _ Prepared tested Lot/Batch Number MTB strain l a Broth b _ m a n u a l

_ Prepared*/ 2 Tech Initials Date Broth c o u l . i n d d

1 Appendix E: New Reagents/Media QC Form: 7H9Broth Appendix E:NewReagents/MediaQCForm: 2 7 tubes culated 2 Unino- Date of

Batch* Expiry Expiry (Manufacturer) Brand Prepared Quantity New Lot/BatchMedia Number Lot Tech Initials Tested*/ Date Received Date* 10 Review (date*/initials) Monthly Supervisor Days MTB –2 Results: growth 10 1 –4 Expiry Expiry Date* Control Sterility Results 2 Received Quantity Date*/ Initials Tech QC pass? (Yes/No) Opened* Date 111/04/2014 17:40 1 / 0 4 / 2 127 0 1 4

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1 7 : 4 0 ggli_mycobacteriology_lab_manual _2coul.indd 128 l i _ m 128 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a S ae TSA base Component b *Date format=dd/mmm/yyyy, _ performance for growth 1 Lot/Batch Prepared Strain tested m Number Media a n u a l

_ 2 c o u Number l Source . Blood i n d d

1 2 Prepared* 8 plates culated 2 Unino- Media Initials /Tech Date Appendix E: New Reagents/Media QC Form: BAP Appendix E:NewReagents/MediaQCForm: (Manufacturer) Brand Date of Batch* Expiry Expiry New Lot/BatchMedia Prepared Quantity Number Lot Tech Initials (Initials/Date*) Review Monthly Supervisor Tested*/ Date Received Date* Growth Positive Results: Expiry Expiry Date* 1 Control Sterility Results: 2 Received Quantity Date*/ Initials Tech QC pass? (Yes/No) Opened* Date 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 129 l i _ m y c o b a c t e r i o l o g y (Manufacturer) Dt omt=d/m/yy **Explainincommentssection *Date format=dd/mmm/yyyy, _ actions: corrective Comments/ l a b _ Brand m a n u a l

_ 2 Appendix E: New Reagents/Media QC Form: BloorAgarMedia(Commercial) Appendix E:NewReagents/MediaQCForm: c o Number u l . i Lot n d d

1 2 9 Received Date* Expiration Date* Acceptable Received Quantity Appearance Acceptable Not ** (date*/initials) Review Supervisor Monthly Put into Date* Use Date/Tech Initials QC pass? (Yes/No) 111/04/2014 17:41 1 / 0 4 / 2 129 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 130 l i _ m 130 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a Tween 80 Sodium chloride Monopotassium phosphate Component b Number of *Date format=dd/mmm/yyyy, Lot/Batch _ Prepared tested strain 1 MTB m Buffer a n u a l

_ 2 c Prepared*/

Tech Initials o Date Buffer Date Buffer u l . i n d d Appendix E: New Reagents/Media QC Form: TBcIDExtractionBuffer Appendix E:NewReagents/MediaQCForm:

1 3 0 control negative tested/ 2 Strain Date of Batch* Expiry Expiry

(Manufacturer) Brand Prepared New Lot/BatchExtractionBuffer Quantity Number Date Tested*/ Lot Tech Initials Received Date* Review (date*/initials) Monthly Supervisor Lot Number TBc IDKit Expiry Expiry Date* (Positive, Negative) Control Positive 1 Results Received Quantity Negative Control 2 Opened* QC pass? (Yes/No) Date 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 131 l i _ m y c o b a c t e r i o l o g Manufacturer/ Drug BD -PZA BD BD BD BD - - - - Ethambutol Rifampicin Isoniazid Streptomycin y Drug Tested (SIRE, PZA) *Date format=dd/mmm/yyyy, APPENDIX F:DSTQC/INVENTORY FORMS _ tested 1 l MTB strain a b _ m a n u a l

_ Media Lot 2 Number c MGIT o u l . i n d d

1 SIRE orPZA Supplement 3 1 Number Appendix F: DST QC Form -BDMGITDrugs Appendix F:DSTQCForm Drug Lot Number (if tested) 2 Date Tested*/ Other strains Tech initials Drug Information Received Date* hours) (days, TTD Results: MTB Expiration (S/R) result Drug 1 Date* hours) (days, TTD (date*/initials) Review Supervisor Other Strains Results: Received Quantity (S/R) result Drug 2 QC pass? (Yes/No) Opened* initials/ Date* Tech Date 111/04/2014 17:41 1 / 0 4 / 2 131 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 132 l i _ m 132 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a uniy Date Opened* Quantity Ofl Moxifl oxacin Levofl oxacin Expiration oxacin Kanamycin Gatifl oxacin Capreomycin Received Amikacin Drug Date* Source Date* Drug Lot (Brand) Number b Drug Tested *Date format=dd/mmm/yyyy, _ tested 1 MTB strain m a n u a l

2 c o u Media Lot l Number . i n MGIT d d

1 3 2 Supplement Appendix F: DST QC Form -MGITSecondLineDrugs Appendix F:DSTQCForm Number SIRE (if tested) 2 Date Tested*/ Other strains Tech initials Drug Information hours) (days, TTD Results: MTB (S/R) result Drug 1 hours) (days, TTD (date*/initials) Review Supervisor Other Strains Results: (S/R) result Drug 2 QC pass? (Yes/No) initials/ Date* Tech 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 133 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix F: DST QC Form - Drugs Tested on Solid Media _ m Results: QC pass? a Results n Drug Drug Lot Date of Expiry Date Drug Date Drug Stock 1 2 Media Lot u Drug MTB strain Other strain (Yes/No)/ Tech a Source Number Manufacture* Date* Opened* Solution Made* Number l

(S/R) (S/R) initials/Date* _ 2 c o u l . i n d d

1 3 3

Supervisor 1MTB strain 2Other strains Date tested*/ Review tested (if tested) Tech Initials: (date*/initials)

*Date format = dd/mmm/yyyy, 111/04/2014 17:41 1 / 0 4 / 2 133 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 134 l i _ m 134 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b rgNm rgNm DrugName DrugLotNumber Purity(Potency) DrugLotNumber Purity(Potency) DrugName Drug LotNumber Purity (Potency) Drug Name *Date format=dd/mmm/yyyy, _ m Date* a n u a l

_ 2 c o Amount u l Used . i n d d

1 3 Appendix F: MGIT Second-Line Drugs - Inventory Worksheet -Inventory Appendix F:MGITSecond-LineDrugs 4 Remaining Amount Date* Amount Used Remaining Amount Date* Amount Used Remaining Amount 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 135 l i _ m y c o b a c t e r i o l o g y *Date format=dd/mmm/yyyy, APPENDIX G _ control: tested/positive 1 Lot Number l MTB strain (Manufacturer) a b _ m Brand a n u a l

_ 2 c o Date Tested*/ Appendix G:MTBIdentiﬁ u Tech Initials l . i n d d

Number

1 3 Lot 5 control: negative 2 Strain tested/ Negative) (Positive, Internal Internal Control Results: Received -ICASlideTestcation QCForm MPT64/MTB64 Date* Negative) (Positive, Control Positive Results: 1

Expiry Date* (Date*/Initials) Review Supervisor Negative) (Positive, Negative Control Results: 2

Received Quantity QC Pass? (Yes/No) Date*/ Tech Initials Opened* Date 111/04/2014 17:41 1 / 0 4 / 2 135 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 136 l i _ m 136 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b _ m a APPENDIX H n u a l

_ 2

c Appendix H: Weekly MGIT/LJ Culture QC Form o u 1,2 l Smear Result . Results: i

n QC pass? d Specimen MGIT or LJ (Neg, d Date Tested*/ Lab Accession Expiry (Yes/No)/

Type Media Lot Conﬁ rmation Comments Growth MGIT: TTD LJ: number 1 Tech initials Number Date* Tech initials/ 3 (Pos/Neg) Number required, Scanty, 6 (Pos, Neg) (days, hours) of colonies Date 1+ 2+ 3+) Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive 1MTB strainNegative 2Negative control (neg sputum, Supervisor Review tested 7H9 broth, etc.) (Date*/Initials) *Date format = dd/mmm/yyyy, 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 137 l i _ m y c o b a c t e r i o l o g y _ l a b APPENDIX I: MONTHLY DATA MONITORING FORMS _ m a n u a Appendix I: MONTHLY DATA MONITORS - AFB MICROSCOPY l

_ 2 c Year: ………… Supervisor Review (Date (dd/mmm/yyyy)/initials): ……………………………… o u l . i n SPECIMEN TALLY* AFB MICROSCOPY* d d

Received Unacceptable Completed Smear Positive Smear Negative

1 Total 3 7 Month/ Pt. Site Pt. Site Pt. Site Smears Total Total Total Total Percent Total Percent Tech Initials collected collected collected collected collected collected Read Jan

Feb

March

April

May

June

July

Aug

Sept

Oct

Nov

Dec *Calculate numbers based upon cultures received in the month. 111/04/2014 17:41 1 / 0 4 / 2 137 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 138 l i _ m 138 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b _ m Appendix I: MONTHLY DATA MONITORS - MGIT CULTURES a n u a Year: ………… Supervisor Review (Date (dd/mmm/yyyy)/initials): ……………………………… l

_ 2 c

o Culture Positive/ Smear Culture Positive/Smear Culture Negative/ Culture Negative/

u Positive Cultures* Negative Cultures* l

. Positive* Negative* Smear Positive* Smear Negative* i n d d Total Reported*

Month/ Average Average 1 3

8 Tech Pt. Site Total Percent Total Percent TTD Total Percent TTD Total Percent Total Percent Total Percent Total Initials collected collected (Days) (Days) Jan

Feb

March

April

May

June

July

Aug

Sept

Oct

Nov

Dec *Calculate numbers based upon cultures reported in the month. 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 139 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix I: MONTHLY DATA MONITORS - LJ CULTURES _ m a Year: ………… Supervisor Review (Date (dd/mmm/yyyy)/initials): ……………………………… n u a l

Culture Positive/ Smear Culture Positive/Smear Culture Negative/ Culture Negative/ _ Positive Cultures* Negative Cultures* 2

c Positive* Negative* Smear Positive* Smear Negative* o u l . Total Reported* i n

d Month/ Average Average d

Tech Total Percent Total Percent TTD Total Percent TTD Total Percent Total Percent Total Percent

Pt. Site 1 Total 3 Initials collected collected (Weeks) (Weeks) 9 Jan

Feb

March

April

May

June

July

Aug

Sept

Oct

Nov

Dec *Calculate numbers based upon cultures reported in the month. 111/04/2014 17:41 1 / 0 4 / 2 139 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 140 l i _ m 140 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b _ m Appendix I: MONTHLY DATA MONITORS - CONTAMINATION RATES a n u a Year: ………… Supervisor Review (Date (dd/mmm/yyyy)/initials): ……………………………… l

_ 2 c MONTHLY MGIT CULTURES* MONTHLY LJ CULTURES* o u l . i Contamination Rate n

d Number of Contamination Rate (# Number of d Number MGIT Cultures Number LJ Cultures for LJ Cultures

Cumulative Cumulative LJ

Contaminated MGIT Contaminated/ Contaminated LJ

1 Month/ Reported* Reported* (# Contaminated/

4 Cultures* # Reported)* MGIT Cultures* Contamination

0 Tech Contamination # Reported)* Rate Initials Pt. Site Pt. Site Pt. Site Rate (Total %) Pt. Site Pt. Site Pt. Site (Total %) Total Total Total Total Total Total coll. coll. coll. coll. coll. coll. coll. coll. coll. coll. coll. coll. Jan

Feb

March

April

May

June

July

Aug

Sept

Oct

Nov

Dec *Calculate numbers based upon cultures reported in the month. Pt. coll = Patient-collected specimens Site coll = site-collected specimens 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 141 l i _ m y c o b a c t e r i o l o g y _ l a

b Appendix I: MONTHLY DATA MONITORS - ISOLATION RATES MTB and MOTT _ m a Year: ………… Supervisor Review (Date (dd/mmm/yyyy)/initials): ……………………………… n u a l Isolation of Mycobacterium tuberculosis Complex* Isolation of MOTT*

_ 2 c MGIT LJ MGIT LJ o u l . i n Total # Total # Percent Average Percent Average Total # Total # Percent Average Percent Average d Month/ Total # LJ Total # Total # LJ Total # d

MGIT MTB pos specimens MTB specimens MTB MGIT MOTT specimens MOTT specimens MOTT

Tech cultures MTB pos LJ cultures MOTT pos 1 cultures MGIT yielding isolation yielding isolation cultures pos MGIT yielding isolation yielding isolation 4

1 Initials reported cultures reported LJ cultures reported cultures MTB rate (%) MTB rate (%) reported cultures MOTT rate (%) MOTT rate (%) Jan

Feb

March

April

May

June

July

Aug

Sept

Oct

Nov

Dec *Calculate numbers based upon cultures reported in the month. 111/04/2014 17:41 1 / 0 4 / 2 141 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 142 l i _ m 142 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b ree :…… Location(room#orequivalent):……………………………… Freezer #:………… APPENDIX J:STORAGELOGFORMS ** Dateformat:dd/mmm/yyyy other than2or3). * Sputumspecimennumbershouldbe#1or#2(orN/Aiffrom visits2or3).Aliquot#shouldbe1-4(orN/Aiffrom visits Number _ m Rack Rack a n u a l

Number _ 2 Box Box c o u l . i n d Position # Position # d in Box

1 4 Appendix J:FreezerStorageLogfor 2 Screening ID Screening ID Number Subject ID Subject ID Number Number Visit Visit Accession Accession Number Number M. tuberculosis Lab Lab Specimen Specimen Number* Sputum Sputum (Date**/Initials) Review Supervisor Isolates Aliquot Aliquot #* Collection Date of Date of ** Tech Initials Frozen**/ Date Date Date shipped** Date shipped** 111/04/2014 17:41 (if applicable) 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 143 l i _ m y c o b a c t e r i o l o g y ** Dateformat:dd/mmm/yyyy * Sputumspecimennumbershouldbe#1or#2(orN/Aiffrom visits2or3). Rack Number Rack Number (if applicable) _ l a b _ m a n u a l

_ Number 2 c Box Box o u l . i n Appendix J:StorageLogfor d Position # Position # d in Box

1 4 3 Screening ID Screening ID Number Subject ID Subject ID Number M. tuberculosis Number Visit Visit Accession Accession Number Number Lab IsolatesonLJMedia Specimen Specimen Number* Sputum Sputum (Date**/Initials) Review Supervisor Collection Date of Date of ** Date Stored**/ Tech Initials Discarded**/ Tech Initials 111/04/2014 17:41 1 Date Date / 0 4 / 2 143 0 1 4

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1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 144 l i _ m 144 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ Date:*……………… Name/Signature/Title ofSupervisor…………………………………………………… Date:*……………… Name/Signature/Title ofResponsiblePerson………………………………………….. Date:*……………… Name/Signature/Title ofPersonIdentifyingIssue……………………………………… Date:*……………… ……………………………………………………………………………………………………………………… of thesepolicies/practices: Identify theindividual(s)responsibleformonitoringeffectiveness ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… occurrences (ifapplicable): Describe policies,practices,etc.tobeimplementedasaresultofthisinvestigationpreventfurther SECTION IV: PREVENTION Attach documentationtothisreport ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… Results acceptable? Comments: If Yes, date*ofre-test:…………………… Was retestingnecessary? ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… If Yes, describe: Did thisincidentimpactanypatientresults? Expected date*forresolution:…………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… Describe correctiveactiontakentoresolvethisissue: SECTION III:CORRECTIVEACTIONFORTHISINCIDENT: ……………………………………………………………………………………………………………………… Describe therootcauseofproblem: SECTION II:INVESTIGATION If Yes, number:…………………… includeQIform Has thissameissueoccurredpreviously? ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… Describe theproblem,issue,preventableerror, etc.: Date* ofoccurrence:…………………… SECTION I:SUMMARY OFISSUE Type ofIssue: l a b APPENDIX K:CONTINUOUSQUALITYIMPROVEMENTFORM * Date format: dd/mmm/yyyy * Dateformat: _ QI Number:………………………… m a n u a l

_ 2 c o u l . ¨ ¨ i n d QCfailure Clericalerror d

1 4 4 ¨ Yes

¨ ¨ ¨ No ¨ Supplyproblem Equipmentproblem Yes ¨ Yes ¨ N/A ¨ No ¨

Date* Form Prepared:…………………….... Date* Form ¨ ¨ Unknown ¨ Yes ¨ ¨ N/A Other(list):…………………….... Proceduralerror ¨ No 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 145 l i _ m y c o b a c t e r i o l o g y ** Dateformat:dd/mmm/yyyy * EstimatedConcentrations:Tube 3-log10 APPENDIX L:MGITTTDWORKSHEET _ MGIT): Type ofsuspensionused(frozenorfreshsubculture; stock): (transfers fromoriginal # ofsubcultures for dilutions: MTB strainused aeIouae* 1:500(Tube 3*) Date Inoculated** l a b _ m a n u a l

_ 2 c o u l . i n d d

1 4 5 5 TTD (daysandhours) TTD Results susceptible? (Yes/No) Is thisstrainpan- WHO, etc.): Source ofstrain(ATCC, Initials Tech Supervisor Review Supervisor (Date**/Initials) 111/04/2014 17:41 1 / 0 4 / 2 145 0 1 4

Mycobacteriology Laboratory Manual

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 146 l i _ m 146 y c o Mycobacteriology Laboratory Manual b a c t e r i o l o g y _ l a b _ m a APPENDIX M: EARLY MGIT POSITIVES/ EARLY CONTAMINATED CULTURES TRACKING WORKSHEET n u a l

_ 2 c o u l Screening/Subject ID Visit # Sputum Specimen # Lab Accession # . i n d d Date* of Positivity: TTD ZN Result: BAP Result:

4 Reincubated: ¨ Yes ¨ No Date: Number of Days: Location: ¨ MGIT ¨ Off-line 6 Results for 1st Reincubation TTD from MGIT Date* of Positivity: ZN Result: BAP Result: printout (if applicable): Reincubated: ¨ Yes ¨ No Date*: Number of Days: Location: ¨ MGIT ¨ Off-line Results for 2nd Reincubation TTD from MGIT Date* of Positivity: ZN Result: BAP Result: printout (if applicable): Reincubated: ¨ Yes ¨ No Date*: Number of Days: Location: ¨ MGIT ¨ Off-line Results for 3rd Reincubation TTD from MGIT Date* of Positivity ZN Result: BAP Result: printout (if applicable): Reincubated: ¨ Yes ¨ No Date*: Number of Days: Location: ¨ MGIT ¨ Off-line End of 42 Day Protocol Date of MGIT result TTD ZN Result: BAP Result: at 42 days: ¨ Pos MTB Sponsor Notiﬁ ed Tube Turbid? ¨ Yes ¨ No ID Result: LJ Subculture Date*: ¨ Neg MTB 1 (Date*/Tech): Final Results ¨ Negative MTB ¨ Positive MTB ¨ Pos MTB Supervisor Final Date of Final Culture ¨ Positive MTB and contam Final ID result: ¨ Neg MTB Final TTD: Review (Initials/ MGIT result: Result: ¨ No TB growth, positive MOTT ¨ N/A Date*): ¨ Contam ¨ Unknown COMMENTS: *Date format = dd/mmm/yyyy TTD = time to detection ZN = Ziehl-Neelsen BAP = Blood Agar Plate Contam = contaminated 1 Final results section to be completed after the reincubation step that provides sufﬁ cient data to result the culture. 111/04/2014 17:41 1 / 0 4 / 2 0 1 4

1 7 : 4 1 ggli_mycobacteriology_lab_manual _2coul.indd 147 l i _ m y c o b a c t e r LabDirectorName:……………………………………………………… …………………………………………………………….………………………………….. Address /Location:…………………………………………………………….…………………………………... LabName:……………………………………………………………….. Protocol No.:……………………… i o l o g y ** Date format: dd/mmm/yyyy ** Dateformat: APPENDIX N:LABORATORY VISITORLOG _ l day ofvisitona a separate line) b Date* ofVisit (Record each _ m a n u a l

_ 2 c o u l . i n

Printed Nameof d d

1 Visitor 4 7 Title of Visitor Purpose ofVisit Signature of Visitor Printed Name of Laboratory of Laboratory Personnel Page []of Initials 111/04/2014 17:41 1 / 0 4 / 2 147 0 1 4

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1 7 : 4 1