1. Revision of new diagnostics for TB
Churchyard GJ

2. Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM
Diagnostics pipeline
Conclusion

3. Introduction The global burden of TB is declining slowly
The lack of a rapid & accurate diagnostics is compromising progress towards TB elimination
Sputum microscopy remains the mainstay of TB diagnosis in many resource poor countries
Globally <10% of MDR TB patients are diagnosed & treated
HIV associated TB
Is more difficult to diagnose
If undiagnosed, is associated with a high mortality

4. Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM
Diagnostics pipeline
Conclusion

5. Xpert MTB/RIF
Detects M. tuberculosis and common mutations that confer resistance to rifampin
Is a hemi-nested real-time PCR of MTB- specific region of rpoB gene, which is then probed with molecular beacons for mutations
Fully automated
Uses GeneXpert platform (Cepheid, CA)

6. Assay Procedure for the MTB/RIF Test
2 manual steps: addition of bactericidal buffer to sputum then transfer of defined volume to cartridge
Integrated sample processing and PCR; disposable plastic cartridge contains all reagents

7. Evaluation Study of Xpert MTB/RIF
Cross-sectional study of diagnostic test accuracy
Population and Procedures
Peru, Azerbaijan, South Africa x 2, India
Adults with pulmonary TB symptoms
3 sputa obtained (2 spot, 1 morning)
Results
1730 eligible participants
976 with HIV status known; 40.2% HIV-positive
(C. Boehme et al. NEJM 2010;363:1005)

8. Evaluation Study of Xpert MTB/RIF
# Xpert tests per participant
Sensitivity
Specificity
All CX POS
SM POS,
CX POS
SM NEG, CX POS
1 sputum
675/732
92.2%
551/561
98.2%
124/171
72.5%
604/609
99.2%
3 sputa
723/741
97.6%
566/567
99.8%
157/174
90.2%
604/616
98.1%
Among culture positive patients, a single direct Xpert test correctly detected TB in 551/561 amear positive patients (sensitivity of 98.2%), and in 124/171 smear neg patients. A single Xpert test was specific in 604/609 participants (specificity 99.2%).
Two additional Xpert tests slightly increased the overall yield for smear positive patients, but more substantially increased the yield for smear-negative patients (from 72.5% to 90.2%).
(C. Boehme et al. NEJM 2010;363:1005)

9. Evaluation Study of Xpert MTB/RIF
Xpert SENSITIVITY for Rifampin Resistance
# correct/# total %
Xpert SPECIFICITY for Rifampin Resistance
Phenotypic DST
200/205
97.6%
505/515
98.1%
and Discrepant Resolution by Sequencing
209/211
99.1%
506/506
100.0%
(C. Boehme et al. NEJM 2010;363:1005)

10. Xpert MTB/RIF: false positives
WHO estimates Positive Predictive value at
>90% if greater than 15% of isolates are Rif resistant
<70% if less than 5% of isolates are Rif resistant
Further culture based DST to first and second line drugs recommended
Patients should receive MDR TB treatment pending further results
The software & cartridge have been redesigned to address these limitations

11. Xpert MTB/RIF: Operational performance Detection of MTB
Sensitivity
Specificity
Decentralised implementation
90.3%
99%
HIV-associated TB
58%-73%
99.2%-93.9%
Extrapulmonary TB
53%-95%
98.2%-100%
Active case finding
62.6%
99.6%
(Lawn, Nicol. Future Micobiol. 2012; Dorman et al. PLoS ONE. 2012)

12. Xpert MTB/RIF Attributes & Advantages
Simple to perform, minimal training required
Not prone to cross-contamination
Requires minimal biosafety facilities
“Near-care”
Shortcomings & Disadvantages
Complex instrument (calibration, power supply)
Cost for instrument
Cost of cartridges reduced to ~$10
Single supplier

13. Xpert MTB/RIF WHO expert group recommendations:
“Xpert should be used as the initial diagnostic test in individuals suspected of having MDR-TB or HIV- associated TB” (strong recommendation)
“Xpert may be used as a follow-on test to microscopy where MDR and/or HIV is of lesser concern, especially in smear-negative specimens” (conditional recommendation, recognizing resource implications)

14. Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM
Diagnostics pipeline
Conclusion

15. GenoType MTBDRplus (Hain)
Is a molecular line probe assay with probes for
MTB
Almost all of the common rifampicin resistance-conferring mutations
A subset of the mutations conferring resistance to isoniazid

16. GenoType MTBDRplus (Hain)
Ling D. 2008
Sensitivity
Specificity
Rifampicin
98.4%
98.9%
Isoniazid
88.7%
99.2%
Validation study* showed that performance on
Smear positive specimens was good
Smear negative specimens was reasonable
(* Barnard M. Am J Respir Crit Care Med 2008;177: )

17. Genotype MTBDRplus vs MGIT for detection of MTB, by smear status
Missed 15% of RIF resistant and 37% of INH resistant TB
(Dorman S. PLoS One In press)

18. Genotype MTBDRplus 2.0 vs MGIT & clinical TB
Crudu. JCM, 2012
Sensitivity
Specificity
MTB
Overall
87.6%
99.2%
Smear negative
79.8%
Rifampicin resistance
94.3%
96.0%
90.7%
Isoniazid resistance
95.8%
88.9%
93.5%
82.3%

19. GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011
Fluoroquinolone
86.2%
100%
Kiet,V.S. JCM. 2010
75.6%
kanamycin
Ethambutol
64.2%
Hillemann,D. JCM. 2009
88.9%
Amikacin
75.0%
38.5%

20. GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011
Fluoroquinolone
86.2%
100%
Kiet,V.S. JCM. 2010
75.6%
kanamycin
Ethambutol
64.2%
Hillemann,D. JCM. 2009
88.9%
Amikacin
75.0%
38.5%

21. GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011
Fluoroquinolone
86.2%
100%
Kiet,V.S. JCM. 2010
75.6%
kanamycin
Ethambutol
64.2%
Hillemann,D. JCM. 2009
88.9%
Amikacin
75.0%
38.5%

22. GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011
Fluoroquinolone
86.2%
100%
Kiet,V.S. JCM. 2010
75.6%
kanamycin
Ethambutol
64.2%
Hillemann,D. JCM. 2009
88.9%
Amikacin
75.0%
38.5%

23. Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM
Diagnostics pipeline
Conclusion

24. Urine Assays for Mycobacterial Lipoarabinomannan (LAM)
A lipopolysaccharide component of MTB cell wall
Released from metabolically active or degraded MTB
Urine-based test
Urine easy to obtain
Lacks infection control issues of blood, sputum
Inverness: ELISA format
Alere: lateral flow

25. Urine Assays for Mycobacterial Lipoarabinomannan (LAM)
A lipopolysaccharide component of MTB cell wall
Released from metabolically active or degraded MTB
Urine-based test
Urine easy to obtain
Lacks infection control issues of blood, sputum
Inverness: ELISA format
Alere: lateral flow

26. Determine LAM lateral flow assay (Alere)
uses Determine testing platform
No sample processing; results in 25 minutes
Analytical sensitivity reported to be 0.25 ng/ml
Reporting scale: no band (neg), 1+ to 5+ (pos)
Analytical sensitivity reported by Alere to be 0.25 ng/ml
Please note reporting scale of 1+ to 5+ instead of +/- to 4+
sample
application
pad
patient
result
window
control
window

27. Determine TB-LAM Author/ Year N Setting Sensitivity Specificity
Overall
CD4<100
Peter, 2012
335
Inpatients
45%
96%
Lawn, 2012
516
ART clinic
28%
52%
99%
Dorman S,
2012 *
561
Outpatients
80%
90%
(* Dorman S, Interim unpublished data)

28. Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM
Diagnostics pipeline
Conclusion

29. Global TB diagnostics pipeline

30. Conclusion
In many high burden countries sputum microscopy remains the mainstay of TB diagnosis
New diagnostics can substantially reduce the time to diagnosis of TB and drug resistant TB
A point of care test is urgently required

31. Acknowledgements
Susan Dorman