This presentation compares the principles of Xpert MTB/RIF with Xpert Ultra.

2. 2010 – CBNAAT – GeneXpert (Cepheid / Sunnyvale LA) rolled out.
Screening tool – Pulmonary Tuberculosis
Technical Aspect – Cartridge - Till date – Four Generations
G1, G2-G3 and G4 G4 was launched in December 2011
WHAT IS XPERT MTB/RIF – A CBNAAT

3. XPERT MTB/RIF
Heminested Real time PCR, Chemistry – Molecular Beacons
Intended Use – Specimens from patients clinically suspected of Tuberculosis and
are not on anti-tubercular drugs
Segment Amplified – 192 bp region of M.tuberculosis rpoB gene
Internal Control – Bacillus globigii.
Set of three primers amplify the target region. The presence of MTB and corresponding Rif.
Resistance is detected by FIVE OVERLAPPING MOLECULAR BEACON PROBES

4. PROBE -GENE XPERT. CODON POSITION Frequency
PROBE A 510, 511 1 %
PROBE B 513, 516 2% ; 10%
PROBE C 522 2%
PROBE D 526 25%
PROBE E 531, 533 56%; 2%
Principle – XPERT MTB/RIF

5. Principle – XPERT MTB/RIF
How does this Molecular Beacon Work -
HYBRIDIZATION PROBE – developed in
1996.
ANATOMY of the probe
Quenching Technology –
CONTACT QUENCHING
Reporter dyes used in Xpert –
1. CF-1
2. FAM
3. Alexa Fluor - 532
4. Texas Red – 647
5. CF 6

6. RESULT INTERPRETATION – XPERT MTB/RIF
If two of the five rpoB-specific molecular beacons -
positive signal – CT - < 38.
If CT ≥ 38.
Δ CT Max is ≤ 3.5 cycles (G1)
MTB DETECTED
MTB NOT DETECTED
MTB DETECTED
RIFAMPICIN RESISTANCE
DETECTED
Rif. Resistance - Difference in Ct between the first (early Ct) and last (later Ct)
M. tuberculosis- specific molecular beacon
ΔCT Max is ≥ 3.5 cycles.(G1)
MTB DETECTED
RIFAMPICIN RESISTANCE
NOT DETECTED
ERROR / INVALID
Δ CT Max is ≤ 4 cycles (G4) ΔCT Max is ≥ 4 cycles.(G4)

7. Information about test
Test Result
Real Time PCR
Curve
Probe Ct Values
RESULT INTERPRETATION – XPERT MTB/RIF

8. RESULT INTERPRETATION – XPERT MTB/RIF

9. RESULT INTERPRETATION – XPERT MTB/RIF
Possible outcomes from Xpert Test -
ERROR - Probe Check Control
(PCC) Fails
Sputum Viscosity / Inappropriate
sample volume / Probe integrity
problem – 5006, 5007, 5008
Pressure exceeds permissible value
- 2008
INVALID PCC Pass; SPC Fails
(MTB DNA
Not determined) PCR Inhibited
Specimen not processed adequetly

10. RESULT INTERPRETATION – XPERT MTB/RIF
Possible outcomes from Xpert Test -
ERROR -
Probe Check Control
(PCC) Fails
Sputum Viscosity / Inappropriate
sample volume / Probe integrity
problem – 5006, 5007, 5008
Pressure exceeds permissible value
- 2008
INVALID
PCC Pass; SPC Fails
(MTB DNA
Not determined) PCR Inhibited
Specimen not processed adequetly

11. RESULT INTERPRETATION – XPERT MTB/RIF

12. RESULT INTERPRETATION – XPERT MTB/RIF

13. RESULT INTERPRETATION – XPERT MTB/RIF
Δ CT Max is ≤ 4 cycles (G4)

14. RESULT INTERPRETATION – XPERT MTB/RIF
Δ CT Max is ≥ 4 cycles (G4)

15. RESULT INTERPRETATION – XPERT MTB/RIF

16. RESULT INTERPRETATION – XPERT MTB/RIF
Last probe gives Ct > 38 and earliest probe gives Ct of 34.5.
According to PCR program, PCR terminates after 38 cycles
Δ Ct of > 4 (or 3.5 in G1) cannot be measured.

17. Summary of error codes in Xpert MTB/RIF
Non-reportable results – Error / Invalid / No Result /
ERROR CODE CATEGORY INFERENCE
1001, 1002, 1004, 2014 A External environment factors
5006, 5007, 2008 B Human error – specimen processing
5011, 2037 C Cartridge malfunction – storage
2017 – Next generation cartridge – launched – XPERT ULTRA
False RIF-R due to improper signals from probe D/E

18. WAS THERE A NEED FOR GENEXPERT ULTRA
Previous MTB/RIF – LIMITATIONS
1. Low sensitivity (28 To 67%) - Smear Negative Sputum Specimen.
2. Sub-optimal Negative Predictive Value.
3. Sub-optimal performance - extra-pulmonary specimen.
4. Limited capacity to detect Rif/R associated mutation – Mixed Sample.
5. Reduced capacity to detect C533G mutation – Rif/R.
6. False positive Rif/R – paucibacillary - delay In Probe D/E signal.
7. False positive Rif/R – Silent mutation.

19. PROCESS INVOLVED IN PCR
Denaturation / melting
Annealing
Elongation / Extension
40 - 45
cycles

20. ANALYSIS OF PCR PRODUCT
MELTING
CURVE
ANALYSIS

21. MELTING CURVE ANALYSIS
DNA Binding Dyes - SYBR Green
- Fluoresces only when bound to dsDNA
Melting Point / Inflection point of DNA –
Temperature at which 50 % of DNA is Single
stranded

22. MELTING CURVE ANALYSIS . . contd .
At end of amplification – target
sequences – double stranded –
Fluorescence is maximum
(Low Temperature)
AS TEMPERATURE INCREASES –
DNA stands separate ( DISSOCATE /
MELT) – release dyes molecules –
fluoresce drop – Dissociation Curve

23. CONCEPT OF SLOPPY MOLECULAR BEACONS (SMB)
2009 – Hajj et al - sloppy molecular beacons
SMB – unusually long probe sequences (40 – 60 nucleotides)
Unlike MB (that bind only to perfectly complementary target sequence), SMB –
tolerate mismatches and bind to wide range of target sequence.

24. COMBINING SMB AND MELTING CURVE ANALYSIS
A homogenous method -
distinguish one amplicon from another-
Single gene amplification assay –
four different SMB with different fluorophores
After completion of amplification – SMB –
Hybridize to amplicons at low temperature
Slowly raise the temperature – determine Tm -
temperature at which probe – target hybrid falls
apart.
Basic Principle – Stability of SMB probe-target hybrid provides a characteristic Tm that
indicates identity of target

25. XPERT ULTRA - CARTRIDGE CONFIGURATION
Xpert Ultra – Modified G4 cartridge, for detection of MTBC
Modifications -
PARAMETER XPERT MTB/RIF (G4) XPERT MTB RIF ULTRA
PCR Heminested Nested
Reaction volume 25 microlitres 50 microlitres
Target rpoB gene rpoB Gene, IS 6110 and IS1081
Chemistry Fluorescence based
Molecular Beacons (5) for
MTB detection and Rif-R
MTB Detection - Multicopy IS6110 (TM ) & IS 1081
(MB)
Four Sloppy Molecular Beacons (SMB) – for RiF-R
(each labelled with different fluorophore) - Melting
Curve Analysis

26. XPERT ULTRA - CARTRIDGE CONFIGURATION .. contd
Modifications -
PARAMETER XPERT MTB/RIF (G4) XPERT MTB RIF ULTRA
Semi
Quantification
•High,
•Medium,
•Low,
•Very low
High, Medium, Low, Very low,
“Trace” – based on Ct value of first positive
rpoB SMB
Assay Turn
Around Time
112 minutes 65 – 87 minutes
Limit Of Detection 131 CFU / ml 16 CFU /ml

27. XPERT ULTRA – PRINCIPLE OF DETECTION
The wild type DNA has fixed Tm
Tm of Normal and mutant DNA is known and
precaliberated
The mutated DNA may have higher Tm or lower Tm
depending on design of SMB and its corresponding
complementarity
In this example – the probe – target
hybrid of mutant DNA dissociates /
melts sooner (at lower temperature) than
that of wild type DNA

28. XPERT ULTRA - PROCEDURE
PROBE CHECK CONTROL (PCC)
FAILED
ERROR
PASS
PROCEED FOR MTB DETECTION –
IS6110 & IS1081 DETECTION
POSITIVE SIGNAL - FAM
rpoB
< 2 rpoB Positive
No melting
curve Analysis
MTB “Trace” DETECTED;
RIF-R INDETERMINATE
≥ 2 rpoB Positive
MTB DETECTED
rpoB Ct based Semi-
Quantification and
Melting Curve Analysis
4 valid peaks, all wild type
RIF-R Not Detected
4 valid peaks, at least 1
MUT peak -
RIF-R Detected
< 4 valid peaks,
RIF-R INDETERMINATE
NO SIGNAL - FAM
SPC
POSITIVE
MTB
NOT DETECTED
NEGATIVE
INVALID

29. XPERT ULTRA – DETERMINATION OF ANALYTICAL SENSITIVITY,
LIMIT OF DETECTION AND COMPARISON WITH XPERT MTB/RIF . .contd
At 95% CI, 100% MTB detection and
RIF susceptibility till 200 CFU / ml.
XPERT MTB / RIF
Sensitivity 85% 50% 10%
CFU / ml 100 50 25
LOD – 112.6 CFU / ml
TB detection LOD – Ultra – 15.6 CFU /
ml
At 2.5 CFU / ml – 48% sensitive.
USING attenuated M.tuberculosis H37Rv

30. XPERT ULTRA – DETERMINATION OF ANALYTICAL SENSITIVITY,
LIMIT OF DETECTION AND COMPARISON WITH XPERT MTB/RIF . .contd
LOD for generating
RIF Susceptible result.
(Rather than Indeterminate Result)
USING attenuated M.tuberculosis H37Rv
XPERT MTB / RIF ULTRA
112.6 CFU/ ml 105.4 CFU / ml

31. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
USING DNA from FIND, DNA from clinical isolates maintained at Rutgers University
Mutation panel in XPERT MTB / RIF
PROBE A 510, 511
PROBE B 513, 516
PROBE C 522
PROBE D 526
PROBE E 531, 533
MUTATION PANEL IN ULTRA
511, 513 rpo 1
510 + 516 rpo 1 + rpo 2
516 + 522 rpo2 + rpo 3
526, 533 + 526 rpo 3
529, 531, 533
530 + 531
rpo 4

32. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
XPERT MTB/RIF – False positive RIF – R – delayed signals from Probe D & Probe E

33. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
Temperature
dF/dT
rpo1 produces Tm lower than wild type in presence of mutation
rpo2 produces Tm lower than wild type in presence of mutation

34. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
rpo4 produces Tm higher
than wild type in presence
of mutation
At cfu ≤ 200 /ml – NO FALSE RESISATNCE BY ULTRA
XPERT MTB/RIF – 2.7% - FALSE POSITIVE RIF RESISTANCE

35. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
RIF-R clinical isolates → RRDR → Sequenced→ Quantify (Nanodrop)
Wild type DNA Χ Mutant DNA (90% to 0.5%)
Isolates with rpoB S531L mutation used.

36. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
Assay failed to detect RIF –R at / below 5% mutant specimen
Other than 531, other mutation were not picked in mixture upto 60&-80%

37. DETERMINATION OF Mutation – Heteroresistance – Inclusivity . .contd
Assay Inclusivity – DNA of 22 clinical isolate, M. bovis, M. bovis BCG, M.tuberculosis H37Rv
- 0 to 18 copies of IS6110
In isolates with 0 copy number of
IS6110, MTB was detected due to
presence of IS1081

38. XPERT ULTRA – Analytical Specificity / Exclusivity
Xpert MTB / RIF (G4) – did not pick NTM (at low bacterial load)
At high bacterial load – Xpert has tendency of Misidentifying NTM as
MTB.

39. XPERT ULTRA – Analytical Specificity / Exclusivity
28 NTM were screened at 107 to 108 CFU/ml
M.abscessus M.gastri M.microgenicum M.thermoresistible
Msmegmatis M.genavense M.perigrinum M.triviale
M.asiaticum M.gordonae M.phlei M.xenopi
M.avium M.goodii M.scrofulaceum M.vaccae
M.celatum M.hemophillum M.shimoidei
M.chelonae M.interjectum M.simiae
M.flaviscens M.intracellularae M.szulgai
M.fortuitum M.malmoense M.terrae
MTB NOT DETECTED
106 CFU/ml of clinically relevant NTM + 50 CFU/ml of M.tuberculosis – MTB DETECTED

40. XPERT ULTRA – Dynamic Range and Semiquantitative measurement
29-40
25-28.9
19-24.9
15-18.9

41. XPERT ULTRA Vs XPERT MTB/RIF
200 CULTURE POSITIVE CASES
Sensitivity Specificity
Ultra 87.5% 98.7%
Xpert 81% 98.7 %
109 SMEAR NEGATIVE – CULTURE POSITIVE SPECIMEN
Sensitivity Specificity
Ultra 78.9 98.7
Xpert 66.1 98.7
187 / 200 – RIF SUCCEPTIBILITY
Sensitivity Specificity
Ultra 92.7 98.0
Xpert 92.7 99.0

42. REFERENCE
Chakravorty S, Simmons AM, Rowneki M, Parmar H, Cao Y, Ryan J, et al. The New Xpert MTB/RIF Ultra:
Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.
mBio. 2017 Aug 29;8(4).