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 _______________________________________