New Molecular Tools in Multicenter Evaluations...Source: UNITAID Tuberculosis Diagnostics Technology...
Transcript of New Molecular Tools in Multicenter Evaluations...Source: UNITAID Tuberculosis Diagnostics Technology...
New Molecular Tools in Multicenter Evaluations
Susan E. Dorman, MDJohns Hopkins University School of MedicineTB Clinical Diagnostics Research Consortium
7th FIND Symposium29 October 2014; Barcelona, Spain
Beyond Xpert MTB/RIF: What is needed/wanted?
• More robust platform
• Lower cost
• Performance as good or better
• Choice, competition
Source: UNITAID Tuberculosis Diagnostics Technology and Market Landscape, 2014www.unitaid.eu
Loopamp® MTBC Detection Kit(Eiken Chemical Company, Japan)
Truenat™ MTB (Molbio Diagnostics, India)
Genedrive® MTB ID (Epistem Ltd, UK)
Ustar EasyNAT™ TB (Ustar Biotechnologies, China)
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Truenat™ MTB(Molbio Diagnostics)
• 2‐step process:–DNA extraction from sputum using Trueprep™ AUTO, a magnetic nanoparticle‐based system
–Real time PCR amplification/fluorescent probe‐based detection using Truenat™ MTB chip and Truelab™ Uno instrument
1.Battery powered,
portable, menu‐driven system for sample prep, DNAextraction (20 min)
2.Smart chip with pre‐loaded,
stabilized, lyophilizedreal time PCR reagents for
MTB detection(disposable, single use)
3.Real time quantitative microPCR system.
Battery powered, portable, with network data transfer
(35 min)
Result screens
• Single site, blinded, cross‐sectional, pulm TB suspects• 1 sputum for smear, MGIT culture, Truenat MTB• 226 sputa analyzed
• Truenat SENSITIVITY– Smear POS/Culture POS: 99% (111/112)– Smear NEG/Culture POS: 76% (22/29)
• Truenat SPECIFICITY– Using clinical case definition: 100% (35/35)– Using CX: Not reported but < 100%
Nikam et al, PLoSOne 2013;8:e51121
Genedrive® MTB ID(Epistem Ltd)
• 2‐step process:– Sample prep
• Paper‐based DNA extraction method that is simple, no instrumentation
– PCR amplification/detection using small battery operable instrument• Lyophilized reagents pre‐loaded into Assay Cartridge
• Assymetric PCR and proprietary hybridization probe technology
Genedrive® ‐Workflow 2. Load
Assay Cartridgecontains
pre-loadedlyophilizedreagents; add water
10
1. Sample PrepApply sputum to paper in
Sample Prep Cassette, let dry.Punch 3 discs,
transfer to Assay Cartridge
20 minutes
3. RunPCR reaction
and results readout within battery-operable
device
45 minutes
Result Interpretation
Undetectable, OK Negative for Mtb
Detected, high Positive for Mtb
Detected, Low Positive for Mtb
Retest Mtb not detected, internal control failure
Error System error with failure to generate a result
Results Readouts
1111
• Analytical LOD: 5 genomic DNA copies• Sputum spiked with Mtb:
≥ 1000 CFU/ml, sensitivity 100%≤ 100 CFU/ml, sensitivity 86.4%
• 31 Clinical sputum specimens: – sensitivity 100%
Castan et al, JCM 2014;52:502
‘Evaluation of Non‐Inferiority of Truenat MTB and Genedrive MTB ID for Diagnosis of
Pulmonary TB in Comparison to Xpert MTB/RIF’ • Diagnostic accuracy study
• 3 centers– Infectious Disease Institute in Kampala, Uganda
(L. Nakiyingi, M. Joloba)– Univ. of Cape Town in South Africa (M. Nicol)– Federal Univ. of Espirito Santo in Vitoria, Brazil (R. Dietze)
• Sponsors: FIND & NIH/DMID via TB‐CDRC
• Objectives– Assess sensitivity and specificity of each test, vs. XpertMTB/RIF, using gold standard of culture
– Assess operational feasibility
Screening of TB suspects
Collect sputa
Medical history, CXR, HIV tes ng,
Clinical examina on
INFORMED CONSENT
Does not meet inclusion criteria Meet inclusion criteria
DO NOT ENROLL!
Report conventional results back to Clinic
NALC‐NaOH Decontamina on
LJ MGIT
Sputum 2
≥2.5 ml
Smear
Sputum 1
≥2.5 ml
Homogenize with glass beads
Xpert MTB Rif®
Epistem from pellet
NALC‐NaOH Decontamina on
LJ MGIT
Sputum 3
≥2.5 ml
Day 1 Day 2
Smear
MGIT DST*
MGIT DST*
Store le over pellets
Store le over pellets
Epistem
Split 1.5ml for decontamina on and freeze the rest.
Smear Epistem
Split 1.5ml for decontamina on
Store le over sputa
Add buffer to remainder, use 500uL to perform Molbio tes ng
*MGIT DST Performed only on MTB(+) RIF (R) samples (1/par cipant) White box: Inves ga onal Tests; Purple box: Rou ne Lab Tes ng
Add buffer to remainder, use 500uL to perform Molbio
tes ng
AUG2014
end OCT2014
AUG2015
DEC2014
N=208
Completeinterimanalysisof 300
Hypothesis:investigational tests have sensitivity (for detection of Mtb) that is non‐inferior to that of Xpert MTB/RIF
Sample size: 1200 participants
Enrollmentstart
With thanks to:• FIND
– C. Denkinger, C. Boehme, D. Dollinger, E. Valli, N. Champouillon, M. Perkins• CDRC
– D. Armstrong, D. Alland, J. Ellner, M. Gaeddert, S. Armakovitch, D. Hom, Y. Manabe, A. Nonyane
• Univ. of Cape Town– M. Nicol, W. Zemanay
• Infectious Disease Institute (Uganda)– L. Nakiyingi, M. Joloba, W. Ssengooba
• Federal Univ. of Espirito Santo (Brazil)– R. Dietze, M. Palaci, C. Cheibub
• Molbio Diagnostics for donation of cartridges & instruments• Epistem Ltd for donation of cartridges & instruments
This work is funded in part by contract #HHSN2722000900050C from the National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Department of Health and Human Services, USA.