Tuberculosis is a raging problem round the globe. Eradicating TB is a herculean task but is possible is efforts from all corners from the world. The diagnostics have taken a big leap and with effective medications, our dream of TB free world may come true. But unlimited efforts are need to reach our goal.

1. Gene Xpert
& Advances
Dr. Sayan Chakraborty, MD
Senior Resident, DM Infectious diseases
AIIMS, New Delhi
Email: dr.sayan@gmail.com

2. Breaking NEWS
• 10 million new cases in 2017
• India (27%), China (9%),
Indonesia (8%), Pakistan (5%),
Bangladesh (4%)
• 9% in PLHIV (72% in Africa)
• 1.3 million deaths in HIV –
• 300,000 deaths in HIV +

3. Global Drug resistant TB in 2017
• 558,000 developed Rifampicin resistant (RR) TB
• 82% were multidrug-resistant (MDR-TB)
• India (24%), China (13%), Russian Federation
(10%)
• 3.5% of new cases & 18% of previously treated
cases had MDR/RR-TB
• 8.5% (95% confidence interval, 6.2–11%) had
extensively drug-resistant TB (XDR-TB)
Global Tuberculosis report WHO 2018

4. Indian Scenario
Estimates Number Rate (per 100,000)
Incidence (includes
HIV+TB)
2.7 million 204 (140–281)
Incidence (HIV+TB
only)
86,000 6.4 (4.3–9)
Incidence
(MDR/RR-TB)
135,000 10 (5.8–16)
Mortality (excludes
HIV+TB)
410,000 31 (28–33)
Mortality (HIV+TB
only)
11,000 0.79 (0.48–1.2)
Global Tuberculosis report WHO 2018

5. Indian scenario - Treatment
• TB treatment coverage in 2017: 65% (47–96)
Treatment success Rate
New and relapse cases in 2016 69%
Previously treated cases, excluding relapse in
2016
70%
HIV-positive TB cases in 2016 75%
MDR/RR-TB cases started on second-line
treatment in 2015
46%
XDR-TB cases started on second-line treatment
in 2015
28%
Global Tuberculosis report WHO 2018

8. WHO
endors
ed
tests
Gene
xpert
Mtb/Ri
f
Gene
xpert
ultra
Mtb/Ri
f
LPA
(1st
line &
2nd
line)
TB
LAMP
Interfe
ron
gamm
a
release
assays
TB
LAM
Cultur
e
(MGIT,
LJ,
Middle
brook)
Micros
copy
(light &
LED;M
ODS)

9. WHO endorsements in TB diagnostics
Timeline
YEAR TECHNOLOGY TURNAROUND
TIME
Before 2007 ZN stain
Solid culture
2 days
30-60 days
2007 Liquid culture Upto 42 days
2008 Line probe assay
(Genotype MTBDR plus)
24 – 48 hours
2011 LED based fluorescence
microscopy
1 day
2011 Xpert MTB/RIF 112 mins
2013 Line probe assay SL
(Genotype MTBDR SL)
24 – 48 hours
2017 Xpert MTB/RIF Ultra 65 – 87 mins

10. Xpert MTB/RIF

11. Principle of Xpert MTB/RIF
• Semi- quantitative hemi-nested Real time PCR
• Segment Amplified – 192 bp region of
M.tuberculosis rpoB gene
• Set of three primers amplify the target region
• Detection is by five overlapping molecular beacon
probes
Lawn et al. Future Microbiology, 2011, 6(9), 1067–1082

12. RIF RESISTANCE is either NOT DETECTED or DETECTED
There is no high or low detection for RIF RESISTANCE
Interpretation

13. Interpretation
MTB DETECTED
Any Negative Probe (Ct = 0)
or Δ Ctmax > 3.5 between
any two probes
All the 5 probes Positive &
Δ Ctmax ≤ 3.5 between
them
MTB DETECTED
RIF Resistance DETECTED
MTB DETECTED
RIF Resistance NOT DETECTED
∆ Ctmin ≤ 2.0

14. Min = 22.4 Max = 23.9
 Ct <3.5 : 23.9-22.4 = 1.5 : Resistance NOT DETECTED
RIF Resistance NOT DETECTED

15.  Ct
9.6
RIF Resistance DETECTED
Max. =
36.5
Min. =
26.9
 Ct >3.5
Rif Resistance
DETECTED

16. Interpretation
• RIF Resistance INDETERMINATE :
1. If Ct of any probe exceeds the Valid Ctmax ( 39 for
A, B & C & 36 for D & E )
2. Earliest Ct value is greater than (Valid Ctmax of
probe in condition 1) - (delta Ct max cut-off of
3.5 )
• Invalid: Sample Processing Control (SPC) not
detected in a negative test
• Error: Probe check fails

17. Diagnostic value in Pulmonary TB
• Smear +VE/ Culture +VE TB:
Pooled sensitivity 98% (95% CI, 97-99%)
(23 studies, 1952 participants)
• Smear –VE/ Culture +VE TB:
Pooled sensitivity 68% (95% CI, 61-74%)
(23 studies, 7151 participants)
 Pooled specificity of 99% (95% CI, 98-99%)
(23 studies, 7151 participants)
WHO Xpert MTB/RIF implementation manual 2014

18. Diagnostic value in Pulmonary TB
• PLHIV:
Pooled sensitivity 79% (95% CI, 70-86%)
(7 studies, 1789 participants)
• Detecting rifampicin resistance:
Pooled sensitivity 95% (95% CI, 90-97%)
(17 studies, 555/2624 total specimens)
Pooled specificity of 98% (95% CI, 97-99%)
(24 studies, 2414 specimens).
WHO Xpert MTB/RIF implementation manual 2014

19. M.tuberculosis vs NTM
• Xpert MTB/RIF highly accurate in
distinguishing TB from NTM in clinical
specimens
• Among 180 specimens with NTM, Xpert MTB/
RIF had a positive result in only 1 specimen
that grew NTM (14 studies, 2626 participants)
WHO Xpert MTB/RIF implementation manual 2014

21. Sharma SK, et al. PLoS ONE 2015; 10(10): e0141011

22. Sharma SK, et al. PLoS ONE 2015; 10(10): e0141011

25. Diagnose extra-pulmonary TB
in adults & children
Review: 15 published studies, 7 unpublished studies (5922 samples)
WHO Xpert MTB/RIF implementation manual 2014
Pooled sensitivity Studies/Samples
Lymph node aspirate 84.9% 14 studies, 849 samples
Pleural fluid 43.7% 17 studies, 1385
specimen
CSF 79.5% 16 studies,
709 specimens
Gastric fluid 83.8% 12 studies, 1258 samples
Other tissue
specimens
81.2% 12 studies, 699 specimens

26. EPTB in India
Pooled sensitivity Studies/Samples
Lymph node aspirate 83.1% 13 studies, 955 samples
Pleural fluid 46.4% 14 studies, 841
specimen
CSF 80.5% 13 studies,
839 specimens
Index TB guidelines 2017

27. Sample Sensitivity Specificity Samples
Pleural fluid 40% 97% 364
Lymph node 88% 91% 273
CSF 68% 98% 230
Cold abscess 95% 71% 153
Ascitic fluid 18% 99% 102
Endometrial
aspirate
33% 100% 95
Urine 33% 100% 55
Pericardial
fluid
25% 94% 20
Overall 71% 95% 1292
EPTB from AIIMS, New Delhi
SK Sharma et al. Eur Respir J 2014, 44(4), 1090- 1093

28. • 60 patients, 52 (88.3%) had a final diagnosis of PTB and 16 (26.7%)
were culture confirmed.
• Xpert® MTB/RIF: In culture confirmed cases, sensitivity 81%
(54%–96%) and specificity of 73% (56%–85%)
• In culture negative cases, Sensitivity 32% (17%–51%) and
Specificity 100% (54%–100%)
• Culture had a sensitivity of 32% (20%–47%) for the final diagnosis.
Lung India 2018;35:295-300

29. • The sensitivity of stool Xpert was 11.54% and
specificity 98.65% as compared to culture
• Conclusion: Stool sample for Xpert cannot
replace gastric aspirate and induced sputum for
diagnosis, and hence should not be used as a first
line test

30. Need for Xpert MTB/RIF Ultra
• Limitations of Xpert MTB/RIF:
Sputum negative Pulmonary TB:
Pooled sensitivity 68% (95% CI, 61-74%)
CSF: Pooled sensitivity 79.5% (95% CI, 62.0-90.2%
PLHIV:
Pooled sensitivity 79% (95% CI, 70-86%)
WHO Xpert MTB/RIF implementation manual 2014

32. MTB/RIF MTB/RIF Ultra
Diagnosis MTB complex MTB complex
Resistance Detects rifampicin
resistance asa
surrogate for MDR-
TB
Detects rifampicin
resistanceas a
surrogate for MDR-TB
Amplification
forTB
detection
Single target:
rpoB core
region
Multi-copy target:
rpoBcore region
Insertion elements:
IS6110+ IS1081
Resistance
detection
Real-time PCR
5 probes bind to
RpoBgene
Nested PCR, Melting
curve; 4 probes bind to
RpoBgene
Sputum input 2ml 2ml
PCRreaction 25ul 50ul
AssayTAT 112 min 65-87min
Limit of
detection
114 cfu/ml 16 cfu/ml
Short pieces of
DNA, occurs
multiple times
in genome,
conserved in
MTBC

33. Temperature
Fluorescence
Melting curve analysis: If amutation is present, dsDNA(probe +
TBDNA) dissociates sooner thanif ‘normal’ DNApresent
Therange of Tmis known for mutant and normal
Mutant
’Normal’
Mixed population
Tm
mutant TB
Tm
normal TB
Principle ofdetection
WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)

34. Resultsfor Ultra MTBcategories
Category MTB/RIF Xpert Ultra Interpretation
Not
detected
X X No TBdetected
High X X TBdetected
Med X X TBdetected
Low X X TBdetected
Verylow X X TBdetected
Trace X Traceamounts MTB detected
MTBnot detected =neither of multi-copy target probes are positive but
SPC(Samplie Processing Control) is positive (valid)
MTBdetected =one or both probes for multi-copy target are positive and
at least two rpoB probes positive
WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)

35. Newcategory: MTBtrace
• A newresultcategory:
•Trace=Improvedsensitivity =Lowestbacillaryburden detected
•Oneor both probes for multi-copy targetsarepositive withCt
<37 andno more than onerpoBprobes haveaCt<40
Considerationsfor interpretation of “Trace"results:
•In HIV-positives, children andEPTB = TBpositive
•If in apatient with no-risk of HIVor previoushistory ofTB =
repeat test on newspecimen
•if trace detected on repeat =TB positive
WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)

37. Performanceof Ultra vsMTB/RIF
• 1,520 persons with suspected TB enrolled.
• Overall, sensitivity of the Ultra was 5% higher than
that of Xpert MTB/RIF (95%CI +2.7, +7.8)
• Sensitivity increases were highest among smear-
negative culture-positive patients (+17%, 95%CI +10,
+25) and among PLHIV (+12%, 95%CI +4.9, +21).
• But specificity was 3.2% lower (95%CI -2.1, -4.7).
• Specificity-decrease were higher in patients with a
history of TB (-5.4%, 95%CI -9.1, -3.1) than in
patients with no history of TB (-2.4%, 95%CI -4.0, -
1.3)
WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)

38. • In CSF: Sensitivity raised to 95% by Ultra for
detection of TB meningitis
• Respiratory samples from children: Sensitivity
71% for Ultra versus 47% for Xpert MTB/RIF
• Primarily due to the ‘trace call’
• In low TB burden settings: Specificity of Ultra
is very high (99.3%, 95%CI 96-99)
Performanceof Ultra vsMTB/RIF
WHO meeting report of a technical expert consultation 2017 (WHO/HTM/TB/2017.04)

39. Lancet Infect Dis 2018; 18: 76–84

40. Lancet Infect Dis 2018; 18: 68–75

41. Lancet Infect Dis 2018; 18: 68–75

42. • Detection of hetero-resistance: rpoB S531L, L511P
and H526N RRDR in mixtures containing as little as
10% mutant DNA (even 5%)
Chakravorty et al. 2017; mBio 8:e00812-17.

43. GeneXpert Omni
• Single-cartridge, point-of-care platform
• Low power consumption
• Integrated battery (4 hrs) + supplemental
battery (12 hrs)
• Automatic connectivity
Rapid, onsite molecular testing
at primary care clinics in high-burden countries
http://www.cepheid.com/en/genexpert-omni/

44. GeneXpert Xtend
• Identifies resistance to INH, the FQs, amikacin
and kanamycin
• Same testing procedures and platform as the
Xpert,
• Largely restricted to patients who are Xpert +ve &
RIF resistant
• Funding Agency: National Institute of Health
(NIH)
• Project Start: 2014-04-01
• Project End: 2019-03-31
http://grantome.com/grant/NIH/R01-AI111397-01

46. Long pipeline

47. Thank you