Hope in the Pipeline:

New Molecular Tools for Recognizing TB

 Evolution of
TB
diagnostics in
the public
sector

Fundamental Fundamental
diagnostic: 1882 diagnostic: 2007
3 - 9 months
 DOTS EXPANSION HAS NOT RESULTED IN
BETTER CASE DETECTION RATES

No of countries 250 100 Global case

implementing 90 notification rate
DOTS Total number of countries (All forms of TB)
200 80

70

150 60

50

100 40

30

50 20 Global CNR
10 Countries

0 0
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999Year
2000

Source: WHO Report 2003: Global Tuberculosis Control: surveillance, planning, financing. WHO, 2003.
 Microscopy and case detection in
1400000 Peru
1200000
1000000
800000
600000
400000
200000
0
1991 1992 1993 1994 1995 1996 1999
Smears examined Smear-positive cases WHO/TDR 2001
 The slow road to microscopy diagnosis of TB

Implement molecular test with

sensitivity similar to culture

AFB/ml
 Abbreviating delay through better sensitivity or better access

Implement dipstick with

sensitivity equal to microscopy

10,000,000
AFB/ml

Implement molecular test with

sensitivity similar to culture

AFB/ml

Where delay contributes greatest to

morbidity, mortality, transmission
 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

 Cumulative TB mortality in Sanitorium patients

p at ients
- pos itive
ar
Sme

eg a tiv e patients
Smear- n
Mortality

1 2 3 4 5 6 7
Years
 Annual TB Deaths

5 Current tools
inadequate to avert
4 this.

3
Deaths in
millions

2
The “Second”
1 Epidemic
The “First” Epidemic Asia + Africa
Europe and Americas
0
185 0 19 00 1950 2000 205 0

Discovery Sanatoria Discovery of WHO Declares

of Movement First TB Global
TB Bacillus Starts Drug 1945 Emergency
1882 1900 1993

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

Windows of opportunity in public health

Smallpox vaccinated
HIV-infected
individual.
Eradication of virus
just prior to HIV
pandemic.
 TB notification in Zambia, 1974 - 1999
50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

0
1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998

•incidence in Lusaka >900/100,000

•Disproportionate increase in smear-negative disease
•<20% notified pulmonary TB pts are smear-pos
US MDR outbreaks in 1990-1992 in Florida,
New York, and New Jersey
 TB case fatality rates: Africa
HIV+ = 3.5 x HIV-
45
40 all forms smear-positive
35 HIV+

30 HIV-
CFR (%)

25
20
15
10
5
0 KEN
ZAM
DRC

DRC
CAR

SAF
SAF

SAF
MAL

MAL
BFA

TAN

CDI

source: Ya Diul 2000 country

Tugela Ferry DST
Survey: 1/05-3-06 1539 specimens
544 (35%) Cx+ 995 (65%) Cx -
221(41%) MDRTB 323 (59%) DS
53 XDR-TB
 FIND board Feb 2004

Tests that revolutionize

patient care or disease control
Tests that are significant
incremental improvements
over existing tools
• Improved microscopy
• Simplified or speeded culture
• 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

POSITIVE
• Simplified or speeded DST

NEGATIVE

2004 2005 2006 2007 2008 2009

Level of technology
District Laboratory Portion of
population served

“faster than culture”

Current diagnostic service

Solid culture – 30d

Peripheral Lab

Increased access, earlier detection

Increased delay, morbidity, cost
“more sensitive than smear”
Current diagnostic service
Microscopy – 60% sensitive

Clinic (true POC)

Reaching new patients

“simpler than microscopy”

Current diagnostic service

None 30%
Referral for 3.8b people in 22 HBC
325

1500
Reference center

District hospital

27,000
Microscopy center

270,000 Health post

1,52m?
 Strength of
health system
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
 Rifampin resistance
Mutations map to a single “core region” of the rpoB gene
Accounts for ~ 95% of clinical rifampin-resistance.
rpoB

Deletion Deletion Deletion

AATTCATGG GACCAG GAACAA
Deletion Deletion
CCATTC CAGAAC
Deletion Insertion Insertion Del
GGCACC TTC TTCATG AAC

GGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTG TCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTG

* *** *** **** * *** * * * **** **** *

507 81 base pair core region **** * 533
Adapted from: Musser. 1995. Clin. Microbiol. Rev. 8:496.
 Steps in Hain test for molecular MDR screening
with PCR and line probe hybridization

Process specimen, extract Hybridize amplified DNA

DNA, amplify DNA targets to oligonucleotide probes
with PCR on strips
Addressing MDR crisis: Proving PCR and
line-probe hybridization in HBCs

May 2004 Peru study of 5 methods

Addressing MDR crisis: Proving PCR and
line-probe hybridization in HBCs
Automated solution: Cepheid
rpoB Molecular Beacon Assay

Molecular
Beacon

Target

Hybrid
 Concentrates bacilli &
removes inhibitors

Sample is Ultrasonic lysis of filter-

End of hands on work automatically captured organisms to
4
3 filtered & washed release DNA

DNA molecules are

mixed with dry PCR
reagents

Transfer of 2 ml GeneXpert
after 15 min
 Inactivation procedure

TB inactivation by sample treatment buffer

 Overall performance: per patient
analysis Sensitivity Sensitivity Sensitivity Specificity
All C+ S+C+ S-C+ Non-TB
UPCH, Peru
% 99.1 100.0 83.3 100.0
(Correct / total) (209 / 211) (199 / 199) (10 / 12) (102 / 102)
[95 CI] [96.6 – 99.7] [98.1 – 100.0] [55.2 – 95.3] [96.4 – 100.0]
Borstel & STI, Azerbaijan
% 96.6 100.0 92.8 97.1
(Correct / total) (144 / 149) (80 / 80) (64 / 69) (68 / 70)
[CI] [92.4 – 98.6] [95.4 – 100.0] [84.1 – 96.9] [90.2 – 99.2]
UCT, South Africa
% 95.9 99.0 90.4 98.4
(Correct / total) (142 / 148) (95 / 96) (47 / 52) (186 / 189)
[CI] [91.4 – 98.1] [94.3 – 99.8] [79.4 – 95.8] [95.4 – 99.5]
SAMRC, South Africa
% 95.6 100.0 86.7 97.3
(Correct / total) (43 / 45) (30 / 30) (13 / 15) (213 / 219)
[CI] [85.2 – 98.8] [88.6 – 100.0] [62.1 – 96.3] [94.2 – 98.7]
Hinduja, India
% 98.4 100.0 88.5 97.2
(Correct / total) (185 / 188) (162 / 162) (23 / 26) (35 / 36)
[CI] [95.4 – 99.5] [99.7 – 100.0] [71.0 – 96.0] [85.8 – 99.5]
Total (Three Xpert MTB/RIF)
% 97.6 99.8 90.2 98.1
(Correct / total) (723/741) (566/567) (157/174) (604/616 )
[CI] 96.2 – 98.5 99.0 – 100.0 84.9 – 93.8 96.6 – 98.9
Sensitivity & specificity of a single, direct
Xpert; 1462 patients

36
Decentralization of molecular diagnostics
Le
ss
1st generation co
MDR mp
lex
ity
, mo
2nd generation re
automated MDR rob
us
1st generation tne
manual detection
ss

2nd generation
manual detection

LPA Xpert LAMP POC test

2008 2010 2011 2015

37
• Closed system
• Isothermal
• Rapid
• Multiprimer
• Visible readout
 Basic principle of the LAMP method
- Starting structure producing step -

(4)
F3c F2c F1c B1 B2 B3 5’ 3’ F3c F2c F1c B1 B2 B3 5’
3’
Target DNA
5’ 3’ 5’
F3 F2 F1 B1c B2c B3c F3 F2 F1 B1c B2c B3c 3’
65℃ ＋
(5)
(1) 5’
F3c B1 B2 B3 F1c F2 F1 B1c B2c B3c 3’
3’ F2c F1c 5’
F1c
F2 3’ DNA polymerase with strand
5’ B1c B2c B3c
FIP
displacement activity (6) F1 3’
F2
5’ B3 Primer
F1c 3’ B2
(2) 5’ B1c
3’ F3c F2c F1c B1 B2 B3 5’ BIP
(7) F1c F2 F1
5’ B1c B2c B3c 3’
5’ 3’
F2 F1 B1c B2c B3c
F1c 3’ F1 F2c F1c B1 B2 B3 5’
(3) 3’ ＋
F3c F2c F1c B1 B2 B3 5’ (8) F1c B1
F2c B2
5’
F3 Primer F2 F1 B1c B2c B3c 3’ 3’
F1c F1 B1c
5’
 Annealing position of Loop Primers
BIP
Loop Primer B
Loop
F2c F1c B1 B2c Primer F
F2
F2 Loop
FIP 5’
F1 3’ B 1c B2 Primer B
F1c B2

BIP Loop
Loop Primer F
Primer F
F1 B 1c B 2c
B 2 BIP B2c
5’ 3’B 1
F2 F1c F2
B1c
Loop FIP
Primer B B2
F2c
B2
Loop
Primer B F2 Loop
Primer F
Improved analytic sensitivity of LAMP with new primers 　

Old New

60 60

50 50

40 40
Tt (min)

Tt (min)
30 30

20 20

10 10

0 0
100cps 10cps 5cps 1cps 100cps 10cps 5cps 1cps

MTB gDNA /Tube MTB gDNA /Tube

Detection using the real-time turbidimeter

SARS CoV RNA

Turbidity

Time (min)
Spiked sputum samples
 Approved

MGIT

Capilia
2007-8
Hain

iLED

2009-11 XpertTB

LAMP

Ag ?
2012-15
Ab ?
Situation in 2004
• 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 2008
• Multiple new TB tests in public sector use
• WHO approval mechanism established
• Global laboratory initiative established/Maputo
declaration
• UNITAID, PEPFAR, GFATM funding scale-up
• Negotiated pricing in place
• Multiple discovery and development activities
led or partnered with the public sector
Thank you
What action would primary care TB
testing sponsor?
Immediate referral for
initiation of treatment
(high NPV)

Referral for further

testing, syndromic
management of
negatives (high PPV)
 Point of care testing
• Antigen detection
− Feasibility studies of Ag
detection in sputum
− Evaluations of commercial LAM
− Development of new LAM
reagents
− MS characterization of LAM
species in urine
− Proteomic discovery from urine,
sputum, blood
− Feasibility studies of more
sensitive POC platforms
• Volatiles detection
− Feasibility studies of eNose
− VOC discovery projects
• Antibody detection
− Screening entire proteome
− Alternate expression systems
− Peptide profiling
• Molecular testing
− Feasibility assessment of POC
molecular
− Feasibility studies of trans-renal
DNA
• New approaches
− FIND RFA for POC 2008
Detection options for POC testing
 RNA signatures
Metabolites
Protein signatures DNA in urine
VOCs
TB antigens
Risk

Antibodies to
TB

DNA in
Whole organism sputum

Reward
Reward
 Sensitivity of selected antigens at >95%
specificity level compared to healthy controls

Antigen Europe, HIV– Africa, HIV– Africa, HIV+

(n=71) (n=79) (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%
 Whole proteome screening of
M. tuberculosis for diagnostic antigens
 Down-selection of antigens for a
TB serologic test

High density array with crude expression product

1200 pts
4000 proteins

Moderate density bead array with purified antigens

2500 pts 60 antigens

Low-density quantitative glass slide array and ELISA

5000 pts 15-50 antigens

Lateral flow qualitative assay

<5 antigens
mBio Diagnostics (division of Precision Photonics): low-cost,
multiplexed serodiagnostic
 Antigen detection

Targets, Detection
Matrices platforms
Detection options for POC testing
 Antigen detection: LAM in urine
Initial clinical data in Swedish
and Ethiopian patients

P-of-P in experimentally
infected mice
Densitogram

8000

6000
R = 0.85
P < 0.0001

4000
Sample 1

2000

0
0.1 1 10 100
LAM concentration (ng/ml)
FIND’s approach to AG/AB Rapid Detection

Do existing
reagents work? Improved
reagents? Better
Suboptimal reagents platform? Novel
antigens?
Suboptimal detection system

Suboptimal antigen/antibody

Partners A Partner B Partner C Partner D

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

Proteins in sputum
Small molecules in urine
 Platform evaluations

TOAD

ESE reader

Matrix sensor

Dual Path Platform RAMP

Detection options for POC testing
 POC molecular

Technology obstacles Matrix obstacles

•Xpert development •Tr-DNA
•LAMP POC feasibility •Sputum processing research
Detection options for POC testing
zNose MS
eNose Cricetomys gambianus

DIMS
Detection options for POC testing
 Whole cell detection

Urease -lactamase NMR

Fluorescent detection of Mtb -lactamase activity