New Approaches for the Diagnosis of Tuberculosis

1
New Approaches for the Diagnosis of Tuberculosis

• Richard OBrien
• Consultant, FIND and Chair, GLI
• 16th Annual Conference of the
• Union North America Region
• San Antonio, TX, 24 February 2012

2
Disclosure
I have no personal financial conflicts of
interest to report . However, my former
employer, FIND, has had contractual relationships
with a number of companies whose products I will
mention in my presentation. These called on FIND
to support development, evaluation and
demonstration studies of these tests in
low-income settings. In turn, the companies were
to provide the tests at favorable prices to the
public sector in developing countries.
3
Outline of Presentation

• Summary of WHO process for endorsing new TB
  control tools and strategies
• Overview of the Stop TB Partnership Global
  Laboratory Initiative Working Group
• Update on Line Probe Assays, GeneXpert MTB/RIF
  and LAMP

4
Global Laboratory Capacity Gap
To reach MDG targets, a global capacity need of
120 million smears, 60 million cultures and 6
million DST investigations must be met by 2015,
requiring at least 6.1 billion USD by 2015
Establish 5 000 new microscopy laboratories
Establish 2 000 new culture and DST laboratories
Train 9 000 new technicians in smear microscopy
Train 23 000 new technicians in culture and DST.
of tests required (mln)
USD funding required (mln)
200 150 100 50
2500 2000 1500 1000 500
Urgent
MDG Target
Required expansion of Smear capacity from 80 to
200 million
Required expansion of Culture capacity from 10
to 60 mlllion
2010
2012
2015
2008
5
TB Diagnostics Pathway
Stop TB New Diagnostics Working Group A
blueprint for the development of TB Diagnostics,
2009
6

Identifying the need for policy change
WHO TB diagnostics policy formulation process

• WHO monitoring of country needs
• Partners (researchers, industry, etc)
• Body of evidence available

• Commissioning of systematic reviews
• QUADAS/other diagnostic accuracy tool
• Meta-analyses (where feasible)

Reviewing the evidence

• Experts, methodologists, end-users
• GRADE process for evidence synthesis

Convening an Expert Group

• Strategic and Technical Advisory Group
• Endorsement/revision/addition
• Advise to WHO to proceed/not with policy

Assessing policy proposal and recommendations

• Guidelines Review Committee
• Dissemination to Member States
• Promotion with stakeholders funders
• Phased implementation scale-up plan

Formulating and disseminating policy
7
GRADE evaluation

• Clear separation
• Recommendation 2 grades
• strong or conditional(optional/weak) for or
  against an intervention
• Balance of benefits and downsides, values and
  preferences, impact, resource use,
• with
• 2) Quality of evidence 4 categories
• ???? (High), ????(Moderate), ????(Low),
  ????(Very low)
• Methodological quality of evidence
• Likelihood of bias
• By outcome and across outcomes

Grades of Recommendation Assessment, Development
and Evaluation
8
Recent WHO laboratory policies (1)

• Automated liquid culture and DST (2007) Use of
  liquid culture systems in the context of a
  comprehensive country plan for strengthening TB
  laboratory capacity in a phased manner starting
  at national/central reference laboratory level
• Rapid speciation (2007) Strip speciation for
  rapid Mycobacterium tuberculosis from
  non-tuberculosis mycobacteria established at
  regional or central reference laboratory level in
  combination with liquid culture
• Line probe assays (2008) Use of line probe
  assays for rapid detection of R resistance within
  the context of country plans for MDR-TB
  management, including development of
  country-specific screening algorithms and timely
  access to quality-assured second-line
  anti-tuberculosis drugs do not eliminate the
  need for conventional culture and DST capability
  should be phased in, starting at national/central
  reference laboratory or those with proven
  molecular capability

9
Recent WHO laboratory policies ()2

• Second-line drug susceptibility testing (2008)
  Reliable and reproducible for injectables and
  fluoroquinolones to be conducted in
  supranational or national/central reference
  laboratories using standardised methodology and
  drug concentrations
• LED microscopy (2009) alternative for
  fluorescence and conventional light microscope
• Selected non-commercial culture and DST methods
  (2010) not alternatives for gold standards, but
  may provide interim solution
• Cepheid Gene Xpert (2010) should be used as the
  initial diagnostic tests in patients suspected of
  MDR TB or TB/HIV
• Commercial serologic tests and IGRAs (2011)
  should not be used in low and middle income
  settings
• Line probe assay for XDR TB (expected in 2012)

10
Stop T B Partnership Working Groups (WG)

• DOTS Expansion WG
• TB/HIV WG
• MDRTB WG
• New Diagnostics WG
• WG on New TB Drugs
• WG on New TB Vaccines
• Global Laboratory Initiative WG (Nov 08)

11
Global Laboratory Initiative

• Platform of coordination and communication,
  providing the required infrastructure, focused on
  TB laboratory strengthening, in the areas of
• Global policy guidance (norms, standards, best
  practices)
• Laboratory capacity development
• Interface with other laboratory networks,
  enabling integration
• Standardised laboratory quality assurance
• Coordination of technical assistance
• Effective knowledge sharing
• Advocacy and resource mobilisation

12
GLI structure governance
Stop TB Partnership
WHO Stop TB Department
GLI Secretariat
GLI Secretariat
GLI Working Group
GLI Core Group
Evaluates, approves, governs projects Advises
GLI Secretariat
GLI Partners Committee
Advises and approves strategic agenda of GLI
Monitors project progress
Technical Working Groups
Supranational Ref Lab Network Sub-WG
Priority projects and activities Time
limited Partner approach
Human resource development strategy
Laboratory strengthening
Laboratory accreditation
Laboratory biosafety
13
GLI strategic priorities

• Accelerating evidence-based policy development on
  diagnostics and laboratory practices
• Promoting a structured framework/roadmap for TB
  laboratory strengthening within the context of
  national laboratory plans at country level
• Developing a comprehensive set of tools, norms
  and standards based on international standards
  and best-practice
• Advancing laboratory strengthening through
  global, regional and local partnerships
• Developing multi-level laboratory human resource
  strategies to address the capacity crisis
• Accelerating new diagnostics into countries

GLI Website http//www.stoptb.org/wg/gli/
14
GenoType MTBDRplus test procedure
3) Hybridization Reverse hybridization of
amplified nucleic acids to specific DNA probes
bound on strips

• DNA
• Extraction
• From NALC/NaOH
• Processed sputum

2) Amplification by PCR
4) Evaluation identification of Mtb complex and
RIF-R and INH-R
15
MTBDRplus Strips
16
Conclusions from Cape TownEvaluation Study

• Overall performance of MTBDRplus assay is
  superior to conventional culture/DST speed,
  accuracy, interpretable results, high throughput
• Cost of testing may be less than culture/DST
• Can substantially reduce the need for culture/
  DST when screening for MDR TB
• May be much easier to establish than new
  culture-capable laboratory
• New sputum processing methods may both increase
  sensitivity and facilitate transport
• Barnard M, et al. AJRCCM 2008177787-792

17
FIND Hain Demonstration Projects
Samara, RF
China
Turkey
Vietnam
India
Brazil
Uganda
South Africa
Thailand
Completed
Underway
18
WHO Policy Statement on LPAs

• Integrated national plan for LPAs together with
  MDR-TB management and lab capacity strengthening
• Use of LPAs on smear-positive sputum and cultures
  (insufficient evidence on smear negatives)
• LPAs do not replace culture DST
• Commercial assays recommended
• Lab infrastructure, procedures and biosafety
• Human resources
• Training, technical support and supplies
• EQA

19
MTBDRsl Line Probe Assay - Background

• June 2008 WHO approves LPAs for MDR TB screening
• Mach 2009 Hain introduces LPA for XDR TB based
  on single published study
• April 2010 FIND enters into agreement with Hain
• FIND to conduct evaluation (accuracy) studies of
  the test
• Hain to provide the test at a favorable price to
  the public sector in developing countries
• FIND supported studies at CDC, UCT and Korea ITRC
• Data from 2 additional studies (SA and Nepal)
  provided to FIND

20
Conclusions from FIND and Other Unpublished
Evaluation Studies

• The studies (excepting UCT) found a high level of
  valid test results when performed on culture
  isolates
• Test sensitivity for FQ-R and injectables met
  FINDs pre-defined performance targets at three
  of the study sites
• Test specificity was over 95 for both drug
  classes at all study sites, except for CDC where
  it was 80-94 depending on the reference standard
• Test performance (sensitivity) for the injectable
  drugs was unacceptably low in the SAMRC study
• Banding problems with rrs probes at 2 SA sites
  presently unexplained but not thought to be
  related to strain differences
• None of the studies evaluated the test on sputum
  specimens
• Operational issues, cost, and patient impact not
  assessed

21
Published Evaluation Studies of MTBDRsl Test

• PNTH, HCMC 62 isolates (41 FQ-R, 21 MDR/FQ-S)
• Pitié-Salpêtrière, Paris 52 isolates (41 MDR, 8
  XDR)
• NRL, Borstel 106 isolates (63 resistant) and 64
  frozen sputa

Drug Sensitivity Specificity Agreement
FQ 83 99 93
AMK 87 100 96
KAN 83 100 97
CAP 86 98 94
EMB 65 97 77
42 AFB, 94 valid results (4 invalid on AFB
specimens)
22
September 2010 WHO EGM on SL LPA

• The EG was considering a conditional
  recommendation for use of the test as an
  alternative to conventional SL DST in screening
  for XDR TB but on culture isolates only
• Based on acceptable performance in evaluation
  studies testing culture isolates
• Additional data needed on
• Direct testing on sputum specimens
• Performance in other settings/populations (Taiwan
  specifically mentioned)
• Availability of genetic sequencing data on
  strains with discrepant LPA and DST results
• EG accepted a suggestion to defer a
  recommendation until these additional data are
  available

23

• MTB / Rif-resistance test
• Essential features of test
• Workflow
• sputum
• simple 1-step external sample prep. procedure
• time-to-result lt 2 h
• throughput gt 16 tests / day / module
• no need for biosafety cabinet
• integrated controls
• true random access
• Performance
• specific for MTB
• sensitivity better than smear, similar to
  culture
• detection of rif-resistance via rpoB gene
• Product and system design
• test cartridges for GeneXpert System

GeneXpert System module
cartridge
Automated Sample Prep, Amplification and
Detection 90 minutes
24
GeneXpert - a Molecular Lab in a
CartridgeFully-Integrated Sample Preparation,
Amplification and Detection

• Universal sample prep
• Closed test system provides
• Nested PCR
• Reflexive test capability
• Same Basic Cartridge works With all Tests and
  GeneXpert Systems

25
(No Transcript)
26
WHO Expert Group Review
Endorsement 6 Dec 10
Evaluation
Development
Demonstration
Feasibility
Collecting evidence for scale-up
Scale-up
Global Consultation
STAG Meeting

• 1. Multi-centre clinical evaluation studies
• 2. Multi-centre demonstration studies
• 3. Single-centre evaluation studies

• 1,730 subjects in five evaluation sites (four
  countries)
• 6,648 subjects in nine evaluation sites (six
  countries)
• 4,575 subjects in 12 studies (nine countries)

27
Evaluation Study Results

• 1730 TB/MDR suspected patients enrolled in
  Azerbaijan, India, Peru, South Africa
• A single, direct Xpert detected 92.2 of all C
  patients.
• Sensitivity in S-C patients was 72.5 and
  increased to 90.2 when three samples were
  tested. Specificity was 99.
• A single, direct Xpert identified a greater
  proportion of culture-positive patients than did
  a single LJ culture.
• Xpert MTB/RIF detected rifampicin resistance with
  99.1 sensitivity and excluded resistance with
  100 specificity.

Boehme C, et al. NEJM 20103631005-14
28
Multicenter Implementation Study Single, direct
Xpert in routine settings Performance similar to
solid culture
Sensitivity All C Sensitivity SC Sensitivity S-C Specificity Non-TB
Lima, Peru 96.6 99.3 88.1 99.6
Baku, Azerbaijan 88.6 97.8 74.7 98.7
Cape Town, SA 86.3 100.0 79.1 99.7
Kampala, Uganda 83.4 97.8 57.7 100.0
Vellore, India 100.0 100.0 100.0 97.7
Manila, Philippines 91.9 96.2 56.3 97.9
TOTAL 90.3 (933/1033) 98.3 (637/648) 76.9 (296/385) 99.0 (2846/2876)

• Routine smear microscopy (2-3 smears per patient)
  had a sensitivity of 61.
• Xpert identified at least as many patients as a
  single LJ culture (89.8 CI 87- 92).
• High positive and negative predictive values in
  all settings
• 2.5 indeterminate rate 0.3 after repetition.
  Culture indet. rate 4.7.

29
Sensitivity and specificity of rifampin
resistance detection
Sensitivity in RIF-resistant Specificity in RIF-sensitive
Total (Correct / total) CI 94.4 (236/250)90.8 - 96.6 98.3 (796/810)97.1 - 99.0

• Rifampin resistance was a good marker for MDR at
  all sites.
• Positive predictive value suboptimal in low
  MDR-prevalence settings
• Confirmation of resistance by culture at present
  recommended for low MDR prevalence settings
• Optimization ongoing

30
WHO recommendation on use of Xpert MTB/RIF
31
A phased roll out
MOH, SA World TB Day 2011
Through Dec 2011, 460 GeneXpert instruments
(2401 modules) have been ordered by 47 countries
45 of the capacity is in South Africa!
32
TB LAMP

• Isothermal
• Rapid
• Closed system
• Visible readout

33
Status TB LAMP
Oct 2011
WHO submission
August 2005
34
Evaluation Study (1063 TB suspects) Sensitivity
and specificity remain below target
Performance of single direct LAMP Reference
standard 2 direct smears 2 LJ and 2 MGIT
cultures per patient
Sensitivity in C Sensitivity in SC Sensitivity in S-C Specificity in S-C-
Peru 88.7 (86/97) 80.8-93.5 98.3 (58/59) 91.0-99.7 73.7 (28/38) 58.0-85.0 95.7 (180/188) 91.8-97.8
South Africa 77.5 (62/80) 67.2-85.3 90.6 (48/53) 79.7-95.9 51.9 (14/27) 34.0-69.3 92.1 (175/190) 87.4-95.2
Vietnam 67.3 (113/168) 59.8-73.9 100 (59/59) 93.9-100 49.5 (54/109) 40.3-58.8 96.1 (123/128) 91.2-98.3
Brazil 85.3 (81/95) 76.8-91.0 98.7 (74/75) 92.8-99.8 35.0 (7/20) 18.1-56.7 95.6 (109/114) 90.1-98.1
Overall 77.7 (342/440) 73.6-81.4 97.2 (239/246) 94.2-98.6 53.1 (103/194) 46.1-60.0 94.7 (587/620) 92.6-96.2
35
TB LAMP performance Improved in demo studies
Lima, Peru Kampala, Uganda Sevegram, India All
Sensitivity in C 94.3 (100/106)88.2 - 97.4 76.1 (140/184)69.4 - 81.7 87.3 (89/102)79.4 - 92.4 83.9 (329/392)80.0 - 87.2
Sensitivity in SC 100.0 (67/67)94.6 - 100.0 95.5 (105/110)89.8 - 98.0 95.9 (70/73)88.6 - 98.6 96.8 (242/250)93.8 - 98.4
Sensitivity in S-C 84.6 (33/39)70.3 - 92.8 47.3 (35/74)36.3 - 58.5 65.5 (19/29)47.3 - 80.1 61.3 (87/142)53.1 - 68.9
Specificity in S-C- 97.8 (364/372)95.8 - 98.9 97.2 (520/535)95.4 - 98.3 93.7 (448/478)91.2 - 95.6 96.2 (1332/1385)95.0 - 97.1

• Sensitivity target in SC (gt95) met
• Point estimates close to meeting S-C target
  (65) and S-C- target (97)

36
Low indeterminate rate for LAMP compared to
culture
Lima, Peru Kampala, Uganda Sevegram, India All
Overall LAMP Indeterminate rate 0.0 (0/480) 0.0 (0/770) 4.4 (29/665) 1.5 (29/1915)
LJ indeterminate rate 0.2 (1/480) 6.5 (50/770) 4.2 (28/662) 4.1 (78/1912)
MGIT indeterminate rate 2.1 (10/480) 13.0 (100/769) 5.5 (37/669) 7.6 (145/1918)

• No serious DNA contamination events reported
• Negative control 1x positive in Uganda 1 x in
  India

37
User appraisal "Easier than smear microscopy"
38
Consolidating LAMP the way forward
Deliverables Key notes
Next steps Validate minor changes and move to demonstration phase II Involve multiple partners for phase II Submit evidence base to WHO in 2012/2013
Platform expansion Malaria HAT Leishmaniasis
39
Acknowledgement

• Catharina Boehme, FIND