New Tools for Recognizing TB (Molecular Testing) (Dr. Mark Perkins)

8/7/2019 New Tools for Recognizing TB (Molecular Testing) (Dr. Mark Perkins)

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Hope in the Pipeline:

New Molecular Tools for Recognizing TB


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Evolution ofTB

diagnostics in

the publicsector

Fundamental

diagnostic: 1882

Fundamental

diagnostic: 2007


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3 - 9 months


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No of countries

implementing

DOTS

DOTS EXPANSION HAS NOT RESULTED IN

BETTER CASE DETECTION RATES

0

50

100

150

200

250

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

0

10

20

30

40

50

60

70

80

90

100

Year

Total number of countries

Global case

notification rate

(All forms of TB)

Countries

Global CNR

ource: WHO Report 2003: Global Tuberculosis Control: surveillance, planning, financing. WHO, 2003.


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WHO/TDR 2001

Microscopy and case detection in

Peru

0

2 0 0 0 0 0

4 0 0 0 0 0

6 0 0 0 0 0

8 0 0 0 0 0

1 0 0 0 0 0 0

1 2 0 0 0 0 0

1 4 0 0 0 0 0

1 9 91 1 9 9 2 1 99 3 1 9 9 4 1 9 9 5 1 9 9 6 19 9 9

S m e a r s e x a m i ne d S m e a r -p o s i tiv e c a


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The slow road to microscopy diagnosis of TB

AFB/ml

Implement molecular test with

sensitivity similar to culture


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10,000,000

AFB/ml

Where delay contributes greatest to

morbidity, mortality, transmission

Abbreviating delay through better sensitivity or better access

AFB/ml

Implement dipstick with

sensitivity equal to microscopy

Implement molecular test with

sensitivity similar to culture


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Smear-positive patients are those most

contagious

Amerindian Causasian

Sputum Status

of source case Intimate Casual Intimate Casual

Positive Smear 44.7 37.4 34.7 10.1

Pos. Cx only 27.7 15.6 8.9 2.4

Negative Cx 25.7 18.7 7.2 3.3

Grzybowski, et al. Bull Int Un Tuberc 1975;50:90


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Years

Mortality

Smear-positive

patients

Smear-negativepatie

nts

1 3 4 7652

Cumulative TB mortality in Sanitorium patients


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Annual TB Deaths

0

1

2

3

4

5

1850 1900 1950 2000 2050

Dea

thsin

millions

Adapred from: Pilheu Int J Tuberc Lung Dis 2:696

Discovery

of

TB Bacillus

1882

Sanatoria

Movement

Starts

1900

Discovery of

First TB

Drug 1945

WHO Declares

Global

Emergency

1993

The Second

Epidemic

Asia + AfricaThe First Epidemic

Europe and Americas

Current toolsinadequate to avert

this.


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Windows of opportunity in public health

Smallpox vaccinated

HIV-infected

individual.

Eradication of virus

just prior to HIVpandemic.


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TB notification in Zambia, 1974 - 1999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998

incidence in Lusaka >900/100,000Disproportionate increase in smear-negative disease


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US MDR outbreaks in 1990-1992 in Florida,

New York, and New Jersey


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TB case fatality rates: Afri

HIV+ = 3.5 x HIV-

0

5

10

15

2025

30

35

40

45

BFA

TAN

DRC

ZAM

CAR

CDI

KEN

MAL

MAL

SAF

SAF

SAF

DRC

countr

CFR(%)

HIV+

HIV-

all form smear-positive

source: Ya Diul 2000


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995 (65%) Cx -

221(41%) MDRTB 323 (59%) DS

53 XDR-TB

1539 specimens

544 (35%) Cx+

Tugela Ferry DSTSurvey: 1/05-3-06


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POSITIVE

NEGATIVE

Tests that revolutionize

patient care or disease control POC smear replacement

POC culture replacement

2-day high-TP sensitive lab

test for case detection +/-

DST for urban centers

2-day lab-free culture

replacement

Specific predictor of

progression from LTBI

Tests that are significant

incremental improvements

over existing tools

Improved microscopy

Simplified or speeded culture

Simplified or speeded DST

2005 20092004 200820072006

FIND board Feb 2004

P ti fL l f t h l


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Current diagnostic service

Solid culture 30d


Microscopy 60% sensitive


None

District Laboratory

Peripheral Lab

Clinic (true POC)

30%

Reaching new patients

Portion of

population served

faster than culture

more sensitive than smear

simpler than microscopy

Increas

eddela

y,morbidity,co

st

In

creased

acce

ss,ear

lierdetection

Level of technology
http://groups.msn.com/_Secure/0TgDvAr0XD6yo*Q5ALVOQJ6jnqe1DX9pj*WJsnOyjhK*EnBitOZ11wZw!3o!kNvIKVeI!vep1EE4XN8ht2q6GjWmQwpZCjPiMx6!a65jiCnUrxfgGR9*n!Q/Picture-718.jpg


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Strength of

health system
http://www.oraifite.com/city-center/


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Addressing equity: Making EME standard

accessible in DEC

Price negotiations on MGIT

Licensing agreement for MPT64

Development for lower cost version

Large demonstration projects (>100,000 pts)

Customer support plan


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GGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTG TCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTG

507

rpoB

533* ** * * *** **** * *

81 base pair core region* * *** ** ****

*** ****

Insertion

TTC

Insertion

TTCATG

Deletion

CCATTC

Deletion

GGCACC

DelAAC

Deletion

CAGAAC

Deletion

GACCAG

Deletion

AATTCATGG

Deletion

GAACAA

Rifampin resistance

Adapted from: Musser. 1995. Clin. Microbiol. Rev. 8:496.

utations map to a single core region of the rpoB gene

ccounts for ~ 95% of clinical rifampin-resistance.


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Steps in Hain test for molecular MDR screening

with PCR and line probe hybridization

Process specimen, extractDNA, amplify DNA targets

with PCR

Hybridize amplified DNAto oligonucleotide probes

on strips


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Addressing MDR crisis: Proving PCR and

line-probe hybridization in HBCs

May 2004 Peru study of 5 methods


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Addressing MDR crisis: Proving PCR and

line-probe hybridization in HBCs


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Automated solution: Cepheid


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Molecular

Beacon

Target

Hybrid

rpoB Molecular Beacon Assay


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Inactivation procedure


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Overall performance: per patient

analysis

UPCH, Peru

%

S iti it & ifi it f i l di t


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Sensitivity & specificity of a single, direct

Xpert; 1462 patients

36

Decentralization of molecular diagnostics


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37

Decentralization of molecular diagnostics

2008 2010 2011 2015

LPA

1st generationMDR

LAMP

1st generation

manual detection

2nd generationmanual detection

POC testXpert

2nd generationautomated MDR

Lesscomplexity,morerobus

tness


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Closed system

IsothermalRapidMultiprimer

Visible readout


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Basic principle of the LAMP method - Starting structure producing step -

B3 Primer

65

DNA polymerase with strand

displacement activity

(5)

(6)

(7)

F3c B1F2c F1c B2 B3 5

F3 F1F2 B1c B2c B3c5

3

F1F2

(4)

F1c F2 B1c B2c B3c5 3F1

F1c

B1c B2c B3c3

F1c

B1c

F2 F1

B2c B3c5 3

F1 B1F2c F1c B2 B33 5

F1c B1

B2

B1cF1

F2c

3

5

5

3

B1

B2B1c

3

3

3

B1 B2(2) F3c F2c F1c 5

5 3F1c

F1 B1c B2c B3c

B3

(8)

F3 F1F2 B1c B2c B3c

5F3c F2c F1c B2 B33

5Target DNA

(3)3 F3c B1F2c F1c B2 B3 5

F3 PrimerF1c

5

3F2 B1c B2c B3cF1

(1) B15

F3c F2c F1c B2 B33

FIP5

F1cF2

F2

3

5BIP


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B2

B2c

B2

F2

F2

F2c

Loop

Primer F

FIP

BIP

Loop

Primer B

Loop

Primer B Loop

Primer F

B 1

B 2B 1 cF 1

F 2 cF 1 c

3 5 F 2

F 1 c

B 1

B 2 cB 1 cF 1

F 2F 1 c 3 5

B 2

B 1 c

Loop Primer B

Loop Primer F

FIP

BIP

Loop

Primer F

Loop

Primer B

B2cF2

B2

BIP

Annealing position of Loop Primers


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Improved analytic sensitivity of LAMP with new primers

MTB gDNA /Tube MTB gDNA /Tube

0

10

20

30

40

50

60

100cps 10cps 5cps 1cps

Tt(min)

0

10

20

30

40

50

60

100cps 10cps 5cps 1cps

Tt(min)

Old New


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Detection using the real-time turbidimeter

SARS CoV RNA

Time (min)

Turbidity


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Spiked sputum samples


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Hain

MGIT

Capilia2007-8

2009-11

Approved

iLED

XpertTB

LAMP

2012-15Ag ?

Ab ?


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No new TB tests in public sector for many years

No WHO approval mechanism

No dedicated laboratory strengthening initiative

No mechanism to link policy change to scaled-

up implementation

No DEC pricing mechanism for existing tools

No public sector platform for discovery and

development of new TB tests

Situation in 2004


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Multiple new TB tests in public sector use

WHO approval mechanism established

Global laboratory initiative established/Maputodeclaration

UNITAID, PEPFAR, GFATM funding scale-up

Negotiated pricing in place

Multiple discovery and development activities

led or partnered with the public sector

Situation in 2008


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Thank you


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What action would primary care TB

testing sponsor?

Immediate referral for

initiation of treatment

(high NPV)

Referral for furthertesting, syndromic

management of

negatives (high PPV)

P i t f t ti


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Point of care testing

Antigen detection

Feasibility studies of Ag

detection in sputum

Evaluations of commercial LAM

Development of new LAM

reagentsMS characterization of LAM

species in urine

Proteomic discovery from urine,

sputum, blood

Feasibility studies of moresensitive POC platforms

Antibody detection

Screening entire proteome

Alternate expression systems

Peptide profiling

Molecular testingFeasibility assessment of POCmolecular

Feasibility studies of trans-renal

DNA

Volatiles detection

Feasibility studies of eNose

VOC discovery projects

New approaches

FIND RFA for POC 2008


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Detection options for POC testing


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Metabolites

VOCs

Protein signatures

RNA signatures

Whole organism

DNA in

sputum

Reward

Reward

Risk

Antibodies toTB

TB antigens

DNA in urine


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S iti it f l t d ti t 95%


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Antigen Europe, HIV(n=71)

Africa, HIV(n=79)

Africa, HIV+(n=77)

TB9.7 35% 79 % 91%

CFP10:ESAT6* 25% 64% 49%

TB10.2 21% 45% 48%TB15.3 41% 75% 65%

TB16.3 55% 81% 88 %

TB 51 31% 76% 48%

TB51.7 57% 83% 78%aCry:MPT83 26% 83% 58%

38 kDa 19% 29% 15%

Sensitivity of selected antigens at >95%

specificity level compared to healthy controls

Wh l t i f


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Whole proteome screening of

M. tuberculosis for diagnostic antigens

D l ti f ti f


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Down-selection of antigens for a

TB serologic test

High density array with crude expression product

4000 proteins

Moderate density bead array with purified antigens

60 antigens

Low-density quantitative glass slide array and ELISA

15-50 antigens

Lateral flow qualitative assay


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multiplexed serodiagnostic


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Antigen detection

Targets,

Matrices

Detection

platforms


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A ti d t ti LAM i i


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Densitogram

0.1 1 10 1000

2000

4000

6000

8000

Sample 1

LAM concentration (ng/ml)

R = 0.85P < 0.0001

P-of-P in experimentally

infected mice

Initial clinical data in Swedish

and Ethiopian patients

Antigen detection: LAM in urine

FINDs approach to AG/AB Rapid Detection


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FIND s approach to AG/AB Rapid Detection

BetterBetter

platform?platform? NovelNovel

antigens?antigens?

Partners APartners A Partner BPartner B Partner CPartner C Partner DPartner D

Suboptimal detection system

LAM in urine New AG/AB sets & cocktails LFI reader AG/AB discovery

ImprovedImproved

reagents?reagents?

Suboptimal reagents

Do existingDo existing

reagents work?reagents work?

Suboptimal antigen/antibody


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Proteins in urine

Small molecules in urine

Proteins in sputum

Proteins in blood

Pl tf l ti


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TOAD

Dual Path Platform

ESE reader

RAMP

Matrix sensor

Platform evaluations


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POC molecular

Matrix obstaclesTr-DNASputum processing research

Technology obstaclesXpert developmentLAMP POC feasibility


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zNose

eNose

MS

Cricetomys gambianus

DIMS


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Whole cell detection


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Urease -

lactamase

NMR

Fluorescent detection of Mtb -lactamase


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activity

Documents

New Tools for Recognizing TB (Molecular Testing) (Dr. Mark Perkins)