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)

✔

✔

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.