Point of Care TB testing: Experience with the Gene Xpert …regist2.virology-education.com/4thINTEREST/docs/28_Scott.pdf · 2010-08-23 · Presented at the 4th INTEREST Workshop 25-28

Presented at the 4th INTEREST Workshop25-28 May 2010, Maputo Mozambique

Lesley ScottDepartment of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa

Point of Care TB testing: Experience with the Gene XpertMTB/RIF


Background >9mil new cases of Mycobacterium tuberculosis (TB)

infection and ~1.8mil deaths annually. Smear microscopy continues to be the primary method of

TB diagnosis, but has modest sensitivity (35-80%) and cannot identify drug sensitive/resistant strains.

Culture of M.tb is the current gold standard which is highly sensitive and can differentiate drug sensitivity: Limitations:

▪ time to reportable result 2-6weeks and beyond▪ Requires laboratory level of biosafety▪ Costly

In the era of HIV/TB coinfection, TB diagnosis in resource poor countries remains a challenge.


New diagnostics (WHO, stop TB,FIND partnership)

WHO endorsed: Liquid culture (BACTEC/MGIT) Molecular line probe assays Rapid strip based detection and

speciation Tools in late stage development and

evaluation NAAT Cepheid IGRA Light emitting diode-fluorescent

microscopy Colourimetric redox indicator MODS (microscope observed drug

sensitivity) Nitrate reductase (Thin layer agar) Front loaded smear microscopy

Tools in early stage development Antigen based (LAM) Breathalyser screening NAAT TB-LAMP Phage based Bleach microscopy Sputum filtration Vital fluorescent staining TB patch for latent

infection


The promise of NAAT (nucleic acid amplification technology)

Meta-analysis show existing NAAT’s have high specificity, reduced sensitivity in smear negatives (Ling,D, Plos One 2008;Pai,M, BMC Inf Dis, 2004, Pai,M Lancet Inf Dis 2003)

Diagnostic method for pulmonary TB

(Drugre, IAS 2009)

Limit of detection (colony forming units/ml)

Liquid culture MGIT 960 10-100 cfu/ml

NAAT (LAMP/Xpert) 50-150 cfu/ml

Antigen 150-10,000 cfu/ml

Line Probe Assay 10,000 cfu/ml

Fluorescent microscopy 10, 000cfu/ml


The promise of TB Point of Care testing

Properties: simple, low-tech, perform rapidly, yield accurate results.

Minimum specifications (Pai,M, Clin Chest Med 2009)

Required value

Medical decision Treatment initiation Sensitivity (adult, pulmonary TB) ~95% S+ C+; ~80% S- C+

Specificity (adults) 95% compared with cultureTime to result Max 3 hoursThroughput 20 tests/daySample preparation Minimum 3 stepsReadout; waste disposal; controls; reagents; storage; stability;instrumentation (maintenance etc); power; training; cost (<US$10)


GeneXpert MTB/RIF(Cepheid) Rapid TB diagnosis and MDR (Rif) diagnosis <2hrs TB and DST result 4 samples processed/module/day Hands on: add lysis/inactivator buffer

2 min 15 min


Molecular technology of the Xpert MTB/Rif

Real time PCR using molecular beacons (6 colour, IC, 5 probes)

Ease of assay interpretation Pos: 2 regions amplify; Rif resistant if any probe >3.5cycle diff, or 1 -3 probes not amplify



Raw Sample and BuffersLoaded intoCartridge

Sample is Prefilteredto remove

large inhibitory debris

Target OrganismsAre Isolated,Concentrated and Washed

Cells and Organisms are Lysedto Release their DNA

DisposableMicro-fluidic Cartridge

MixtureDelivered to Integrated

Reaction Tube forAmplification and

fluorescent Detection

DNA Molecules Mixedwith Amplification and Detection Chemicals


Reportable results


International evaluation


Helb, D, et al, JCM 2010 evaluation of the Xpert Spiked sputum showed limit of detection = 131cfu/ml Killing studies showed the buffer decreased M.tb

viability by 8 logs 23 commonly occurring rifampin resistance mutations

showed 100% identification Showed high specificity in presence of non

tuberculosis mycobacteria. Clinical samples (Vietnam) showed 71.7% and 84.6%

detection of smear-negative culture positive. Clinical study in Uganda of re-treatment TB cases

detected 98.4% culture-positive and 100% rifampinresistance.


South African evaluation: Johannesburg

South Africa: ~20% worlds reported HIV-associated TB cases and 2nd largest reported numbers of MDR.

South Africa context ~50mil people (National TB ref lab annual report, 2009): NHLS 2009 (8 provinces):

~3mil smears, ~794,000 cultures, 25% smear negative were

culture positive (Barnard 2007 Cape Town)

TB suspects consecutively enrolled at a primary health care clinic and investigated according to National TB Control program algorithms.

Participants offered HIV testing (67% HIV+)

Data: total n=286, culture n=232,HIV status, Line probe and Xpert n=107

A third sputum specimen was requested.


Comparison to cultureTotal

Group (n=107)

Smear(n=107)

MDRPlus(Hain)

(n=100)*

Gene Xpert(n=107)

Sensitivity 63.6% (47.7; 77.1)

73.1% (56.7;85,2)

86.3% (71.9;94.3)

Specificity 100% (92.8;100)

94.9% (84.9;98.6)

95.2%(85.8;98.7)

•n=2-3 not done, •n=4 indeterminate

HIV positive

Smear(n=71)

MDRPlus(Hain)

(n=65)*

Gene Xpert(n=71)

Sensitivity 59.3%(39.0;76.9)

62.5% (40;80.4)

85.1%(65.4;95.1)

Specificity 100% (89.9;100)

95.1% (82.2;99.1)

93.1%(80.3;98.2)


MDRPlus(Hain)

Gene Xpert

Smear Negative, culture positiven=16 (15%)

n=5 (31.3%) n=10 (62.5%)

Smear positive, culture positiven=27 (25.5%)

n=24 (88.8% n=27 (100%)


Rifampin sensitivity/resistance MDR=resistance against at least rifampicin

(RMP) and isoniazid. The key determinant for treatment failure is

RMP resistance, so detection of RMP resistance is proposed as a proxy for MDR TB diagnosis and epidemiology.

South Africa NHLS 2009 (8 provinces): 9,070 MDR 594 XDR

Culture+DST = 42 days+/-9days (23-99days)


Summary Diagnosing TB in the context of HIV

Xpert is easy to perform and easy to interpret Found a 7% invalid rate (Power failure, no result, cartridge error,

operator error) 4.5% MGIT culture contamination. Hain MDRPlus 3% (indeterminate).

Sensitivity and specificity in HIV- (86%,95%) and HIV+ (85%,93%), 100% detection in S+C+, 65% detection in S-C+

Diagnosing MDR Xpert has improved MDR detection directly from sputum than

existing line probe assays. Complies with most POC criteria: time to result, simple, non-

biohazard, waste disposal, read out, controls.


Clinic evaluation: Witkoppen, Johannesburg, n=23



Acknowledgements University of the

Witwatersrand The National Health

Laboratory Service USAID, PEPFAR, CHRU/Right to Care, RHRU, UNC Esselin Street Clinic Witkoppen Clinic

Wendy Stevens Natasha Gous Kerrigan McCarthy Ian Sanne Francois Venter Adrian Duse Annelies Van Rie Liesl Page-Shipp Matilda Nduna Fred Phakathi Patients and participants

Documents

Point of Care TB testing: Experience with the Gene Xpert …regist2.virology-education.com/4thINTEREST/docs/28_Scott.pdf · 2010-08-23 · Presented at the 4th INTEREST Workshop 25-28