M b t iMycobacteri
Increasing transmantimicrobic resista
& introduction of newrequire adjustments
iservice
i l S iial Services
mission and ance of Mtbcw technologies s in laboratory es
Safety in the MySafety in the My
Acti e TB in M cobaActive TB in Mycoba
100 x General Popula100 x General Popula3 x Other Laborato
Skin Test Conversion8 30%8-30%
ycobacteria Labycobacteria Lab
acteriologistsacteriologists
ationationory Workers
ns in TB Labs
Safety in the MycoSafety in the Myco
Non-behavioral risk facPoor directional airflowPoor directional airflowEquipment failure
– Biological safety cabinetg y– Centrifuges– Autoclaves
Specimens – Number of positives pro– Organism concentration
obacteria Labobacteria Lab (continued)
ctors for infection
ts
cessed
Safety in the MycoSafety in the Myco
Behavioral risk factorLack of adequate safetyLack of adequate safetyNoncompliance with guAerosol productionAerosol production
– Pouring, pipetting & mix– Homogenizing & cuttingg g g– Dropping or spilling cultu– Preparing smears
Needle sticks
obacteria Labobacteria Lab (continued)
rs for infectiony guidelinesy guidelines
uidelines
xing liquidsg tissuesgure flasks
Laboratory BioLaboratory Bio
Biosafety Level - 2
Personnel trained in haLab directed by competAccess to lab is limited Extreme precautions wAerosol-producing procbiologic safety cabinet
osafety Levelsosafety Levels
andling pathogenstent scientistduring work periodsith “sharps”
cedures conducted in
Laboratory BiosaLaboratory Biosa
Biosafety Level - 3
Training in handling age& able to cause lethal dSupervised by scientistwith such agentsP3 l b t f ilitiP3 laboratory facilities a
afety Levelsafety Levels (continued)
ents acquired by inhalation diseasets experienced in working
d dand procedures
Levels of LaboATS RATS Recom
Level IC ll t & tCollect, smear & transp
Level IISmear & culture; ID & s
Level IIISmear & culture; ID & smycobacterial isolates
ratory Serviced timendations
t iport specimens
sensi testing of Mtb
sensi testing of all
Level I LaLevel I La
Tasks
C ll t d iCollect good specimens
Prepare & examine smp
Ship to Level II/III for cu
Smear proficiency test
aboratoriesaboratories
s
ears
ulture & sensitivities
scores of > 80%
Level I LaboLevel I Labo
CharacteristicsReceive minimum of 10Receive minimum of 10Use fluorochrome stainQuantitate smear AFBQuantitate smear AFB Use certified biologic saSend specimen for cultSend specimen for cultMeet Dept. of HHS Biorequirementsrequirements
oratoriesoratories (continued)
0 AFB smears/week0 AFB smears/week n& report within 24 hr& report within 24 hr.afety cabineture within 24 hrure within 24 hr.safety Level 2 (BL-2)
Sputum SpecimSputum Specim
Collection of good specimen isFirst morning expectorationRinse mouth with waterProduce specimen by deepE t t di tl i t l bExpectorate directly into labIf specimen inadequate, ind
Dispatch immediately to labDispatch immediately to labContainers to be closed anRefrigerate if not processedg p
men Collectionmen Collection
s criticaln
p coughingb id d t bb-provided tubeduce sputum
d sealedd immediatelyy
AFB SAFB S
Level I LaboratoriesRecommended: specimRecommended: specim
– Open in BSC & sterilize – Stain & read on open be– Specimen cannot be use
Probably safe - not curr– Open, prepare, dry & he– Stain & read on open be– Dispatch sealed contain
SmearsSmears
men sterilizationmen sterilizationwith Clorox x 15 min
enched for culturerently recommendedeat fix x 2h in BSCench er to Level II/III lab
Level II LaLevel II La
TasksPerform all procedures donpIsolate organisms in pure cIdentify M. tuberculosis comPerform susceptibility testinRefer MOTT isolates for IDR t i d i l t fRetain recovered isolates foProficiency testing of isolatproceduresprocedures
aboratoriesaboratories
ne by Level Iyculturemplexng of Mtb
D & sensitivities6 thor 6 months
ion, ID and sensitivity
Level II LaboLevel II Labo
Characteristics
R i i i f 20Receive minimum of 20 speUse Class II or III biologic sDetect growth in liquid medDetect growth in liquid medID with DNA probes, BACTwithin 2-3 weeksDetermine sensis of Mtb isoMeet Dept of HHS Biosafet
oratoriesoratories (continued)
i / kecimens/weeksafety cabinetdia in 10 daysdia in 10 daysTEC NAP or mycolic acid profile
olates in 3-4 wty Level 3 (BL-3) requirements
AFB SAFB S
Concentrated Smear
D i L l III l bDone in Level III laboraLiquify & decontaminateCentrifuge > 3000G x 1Prepare smear, dry, heStain and read within BFlourochrome (auramin
StainsStains
for Stain & Culture
t BSCatory BSCe
15min in sealed cupseat fix x2 hBL-3 facilityne) stains preferred
Level III LaLevel III La
Tasks
Id tif ll b t iIdentify all mycobacteri
Perform susceptibility tep y
Proficiency testing on M
Work concentrated in v
aboratoriesaboratories
um
ests on MOTTs
MOTT identifications
very few labs
Level III LaboLevel III Labo
Characteristics
P f 20 tibPerform ~ 20 susceptibUSE DNA probes, HPLmethodsmethodsDo not rely only on NAM t D t HHS Bi fMeet Dept HHS Biosaferequirements
oratories ( i d)oratories (continued)
biliti thbilities per monthLC or other rapid ID
A amplification for IDt L l 3 (BL 3)ety Level 3 (BL-3)
New TechNew Tech
Fluorescent microscopRadiometric culture syRadiometric culture syNAP test, DNA probesR di t i tibiRadiometric susceptibiDirect amplification of macid from specimens
hnologieshnologies
pystemsstems
s, HPLCilit t tiility testingmycobacterial nucleic
New TechnoDi A lifiDirect Amplifica
AMTD approved for sspecimensp
Requires 4-5 hours forDetects only M. tubercuDetects only M. tubercuSpecificity approaches Sensitivity 90-100%; lesSensitivity 90 100%; lesspecimens
ologies (continued)
i T h iation Techniques
smear + respiratory
r completionulosis complexulosis complex100%ss in smear negativess in smear negative
AFB SAFB S
Sensitivity on expectorSensitivity on expectorM. tuberculosisMOTTMOTT
C S iti it i iCum Sensitivity in serieM. tuberculosis
StainsStains
rated sputumrated sputum65%20%20%
f 3 tes of 3 sputa80%
AFB SAFB S
Cum sensi of smears iyield both a (+) culturey ( )one or more positive sm
First specimenFirst specimenSecond specimenThird specimenThird specimenFourth specimen
StainsStains
n culture series that for M. tuberculosis and mears
86%86%95%95%95%
100%
AFB StaAFB Sta
Specificity
Abs. False (+)
Rel False (+)Rel. False (+)
Specificity
PPV
ains (continued)ains (continued)
0.05
1 81.8
99.9
98.1
CultCult
Specimen %Mtbc
Sputum 3pBody Flds <1Bloods 0Tissues 1Other 2
tureture
Culture (+)MOTT Totals
8 11<1 <116 16 2 3 6 8
CulturCultur
Media Sensitivity
Bactec 90
7H11 45
LJ 30
re ( i d)re (continued)
y (%) Time to Pos. (d)
12
26
34
CDC IsolatioCDC Isolatio
Initiation of IsolationMember of ‘high risk’Member of high risk
– Contact with infectious – Born in area of high TB – Medically under-served– HIV+, elderly, homeless– Alcoholics, IV drug userAppropriate clinical f
on Guidelineson Guidelines
populationpopulationTB patientprevalence populationss, imprisonedrsfindings
CDC Isolation GCDC Isolation G
Initiation of Isolation
M b f ‘hi h i k’Member of ‘high risk’ po
Appropriate clinical fi– Cough > 3 weeks– Bloody sputum, – Anorexia, weight loss– Fever, night sweats
GuidelinesGuidelines (continued)
l tiopulation
ndings
CDC IsolatioCDC Isolatio
Di ti ti f IDiscontinuation of IsPatients with proven inf
– Appropriate Rx (optima– Clinical Improvement
3 consecutive negative– 3 consecutive negative – Pt discharged to suitabl
Patients suspected of hPatients suspected of h– Alternate diagnosis con– Pt determined to be nont dete ed to be o
on Guidelineson Guidelines
l tiolationfectious TBlly 2-3w)
smears (q2w)smears (q2w)le localehaving infectious TBhaving infectious TBnfirmedninfectiousect ous
Availability ofAvailability of
CDC recommendatio
Results available within
No longer refer to “STANo longer refer to STA
f AFB Smearsf AFB Smears
ns:
n 24 hours
AT” smearsAT smears
The
Ques
End
stions
Timeline ComparisonNew ver
S iSpecimenReceived
Week 0 1 2 3 4 5 6 7______________________________________________
Mycobacteria growth Bactec 460 System
MTD
Bactec 460 System
TB DNA Probe ID (after growth)
Bactec Sensitivities (after growth)(after growth)
HPLC Atypical Mycobacteria ID
Conventional Mediagrowth
TB Conventional BiochemicalID (after growth)
Conventional PlateSensitivitie
Conventional Biochemicals
n of TB Methodologyrses Old
7 8 9 10 11 12 13 14 15 16 17 18 _______________________________________