Seminar on methods, advantages, disadvantages, new developments in LPA and Genexpert/CBNAAT/Xpert MTB/Rif

1. Presenter: Dr.Kalaiyarasan
Moderator: Dr.Ritu Singhal
15-Dec-2015

2. Topics
• Introduction – MDR/XDR global & Indian scenario
• Importance of detecting DR-TB
• Why Xpert MTB/RIF & LPA?
• LPA-Method, advantages, disadvantages, evidence
base, newer developments
• Xpert MTB/RIF-Method, advantages, disadvantages,
evidence base , newer developments
• Conclusion

3. Introduction
• Drug-resistant TB poses a major threat to control of TB
worldwide.
• Among notified pulmonary TB patients in 2014, an estimated
300 000 (range: 220 000–370 000) had MDR-TB.
• Globally in 2014, 123 000 patients with MDR -TB
or rifampicin resistant tuberculosis (RR-TB) were
notified.

4. MDR TB
• Globally, 3.3% of new cases (95% CI: 2.2–4.4%) and 20% of
previously treated cases (95%CI: 14–27%) have MDR-TB.
• In India (2014), an estimated 2.2% of new cases (95% CI: 1.9–
2.6%) and 15% of previously treated cases (95%CI: 11–19%)
have MDR-TB.
WHO Global tuberculosis report 2015

5. WHO Global tuberculosis report 2015

6. WHO Global tuberculosis report 2015

7. XDR TB
• Extensively drug-resistant TB (XDR-TB) has been reported by
105 countries.1
• On average, an estimated 9.7% of people with MDR-TB have
XDR-TB (95% CI: 7.4–12%).1
• Sub-national drug resistance surveys (2005-09) showed
4-7% XDR & 21-24% Ofx resistance in MDR isolates.
1. WHO Global tuberculosis report 2015

8. WHO Global tuberculosis report 2015

9. Importance of detecting
drug resistant TB
• Earlier treatment initiation
• Improved patient outcomes
• Reducing transmission
• Reducing the emergence MDR-TB and XDR-TB

11. Drug-susceptibility testing
• Phenotypic methods involve culturing M.tuberculosis
in the presence of anti-TB agents to detect growth or
inhibition of growth.
• Genotypic methods target specific molecular
mutations associated with resistance against
individual anti-TB agents.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

12. Advantages of Culture methods
• Bacterial growth can be identified visually or by automated
detection of its metabolism.
• Provides a definitive diagnosis of TB.
• Significantly increases the number of cases identified (30–
50%) when compared with microscopy.
• Provides the necessary isolates for conventional DST.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

13. Disadvantages of culture methods
• More complex and expensive.
• Takes longer time.
• Needs appropriate lab infrastructure & bio-safety
precautions.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

14. Advantages of molecular
methods
• May be applied directly on clinical specimens.
• Can also be used on specimens not suitable for growth-based
assays.
• Simplification and reduction of bio-safety requirements for both
laboratory testing and specimen transport.
• Large number of specimens can be processed at the same time.

15. Advantages of molecular
methods contd.
• Automation provides standardization & quality assurance of
testing.
• Able to detect mutations associated with lower levels of
phenotypic resistance.
• Detection of specific mutations allows to predict drug resistance
to an entire family of drugs (eg: Aminoglycosides)1.
1. Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to
Amikacin, Kanamycin and Capreomycin: A Systematic Review - Sophia B. Georghiou
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033275

16. Why Genexpert/LPA?
Phenotypic DST
• Solid LJ media (around 84 days)
• Liquid Culture (MGIT) (around 42 days)
Molecular DST
• LPA (around 72 hours)
• CB-NAAT (around 2 hours)
PMDT guidelines May 2012

17. Polymerase chain reaction
(PCR)
• Amplifies a single copy or a few copies of a piece of DNA,
generating thousands to millions of copies of a particular DNA
sequence.
• Denaturation (DNA melting): Two strands of the DNA double helix
are physically separated at a high temperature.
• Annealing step: Temperature is lowered and the two DNA strands
become templates for DNA polymerase to selectively amplify the
target DNA.
• Extension/elongation step
• Final elongation
• Final hold

18. Three steps of PCR: Denaturation,
annealing and extension

19. • Primers are target sequence specific oligonucleotides that
serve as template for the DNA polymerase
• Forward and reverse primers flanking the target sequence
allow both DNA strands to be copied simultaneously in both
directions.
Primers

21. HAINs test: MTB DR plus: Line probe assay
• Based on PCR line probe assay
• Identifies M.tuberculosis and detects
Rif & INH resistance in a day
21

23. Principle of Reverse Hybridization
23

24. Interpretation
24

25. Advantages
• Rapid detection of resistance to rifampicin (in less
than 48 hours, alone or in combination with
resistance to isoniazid).
• Up to 48 specimens to be processed simultaneously.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

26. Disadvantages
• Does not eliminate the need for conventional culture and DST.
• Available LPAs are recommended for use only on smear-positive sputum
specimens and isolates of M. tuberculosis.
• Current LPAs cannot replace phenotypic DST for second-line anti-TB
agents.
• LPAs cannot identify resistance to specific second-line injectable agents;
thus, they cannot be used to guide the choice of second-line agents
included in individualized MDR-TB regimens.1
• The sensitivity of LPAs to detect resistance to INH is lower (approximately
85%) than that of culture methods.
1. The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs: expert group meeting report.
Geneva, World Health Organization, 2013 (WHO/HTM/TB/2013.01) (available at
http://apps.who.int/iris/bitstream/10665/78099/1/WHO_HTM_TB_2013.01.eng.pdf).

27. Line Probe Assay

28. • A systematic review and meta-analysis.
• After the literature searches, 14 comparisons for rifampicin and 15
comparisons for isoniazid were identified in 10 articles that used
GenoType MTBDR assays.

29. Results
• The pooled sensitivity (98.1%, 95% confidence interval (CI) 95.9–
99.1) and specificity (98.7%, 95% CI 97.3–99.4) estimates for
rifampicin resistance were very high and consistent across all
subgroups, assay versions and specimen types.
• The accuracy for isoniazid was variable, with lower sensitivity
(84.3%, 95% CI 76.6–89.8) and more inconsistent than specificity
(99.5%, 95% CI 97.5–99.9).
• While specificity is excellent for isoniazid, sensitivity estimates
were modest and variable.

30. Conclusion
• The LPA test provides an early diagnosis of resistance to
isoniazid and rifampicin and is highly sensitive and specific for
an early diagnosis of MDR-TB. Sensitivity for isoniazid is
suboptimal
• Based on these findings, it is concluded that the LPA test is to
be implemented as first line test for diagnosis of drug
resistant TB among sputum smear positive patients in high TB
burden countries

31. Results:
• Overall concordance of RIF and INH result between Genotype
MTBDR plus and MGIT 960 was found to be 96.6 and 84.7 per
cent, respectively.
• The sensitivity, specificity, positive and negative predictive
values for detection of RIF resistance by Genotype MTBDR
plus was found to be 97.6, 94.4, 97.6 and 94.4 per cent
respectively.

32. Newer LPA
• In 2009, Hain Lifescience introduced a new LPA, the Genotype
MTBDRsl® test
• For the rapid determination of genetic mutations associated with
resistance to fluoroquinolones, aminoglycosides (kanamycin,
amikacin), cyclic peptides (capreomycin), ethambutol and
streptomycin.
• Allows for testing and reporting results within 48 hours.
• Recommended by WHO to be used as a rule-in test for XDR-TB.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

33. • Objectives: To assess the diagnostic performance of the
GenoType MTBDRsl LPA for the rapid detection of mutations
conferring resistance to ofloxacin (OFX), amikacin (AMK), and
ethambutol and to determine the impact of implementation
on the turnaround time in a high-throughput diagnostic
laboratory.
• 657 patients smear-positive specimens resistant to isoniazid,
rifampin, or both according to the GenoType MTBDRplus
assay were consecutively tested, using the GenoType
MTBDRsl LPA.

34. • A total of 516 of 657 patient specimens had valid results for
both tests.
• Implementation of this test significantly reduced the
turnaround time by 93.3% (P < 0.001), calculated from the
date that the specimen was received at the laboratory to
reporting second-line results.
sensitivity (95%CI) specificity (95%CI)
OFX 90.7% (80.1–96.0%) 98.1% (95% CI, 96.3–99.0%)
AMK 100% (91.8–100%) 99.4% (95% CI, 98.2–99.8%)
XDR 92.3% (75.9–97.9%) 99.6% (95% CI, 98.5–99.9%)

35. Newer LPA in research mode
• In 2011, Nipro Corporation completed development of their
LPA-based NTM+MDRTB Detection Kit 2
• Four strips - NTM/MDR-TB, INH, PZA, and FQ strips.
• The NTM/MDR-TB strip was designed to identify four
Mycobacterium species—M. tuberculosis, M. avium, M.
intracellulare, and M. kansasii—and to detect mutations
associated with RIF and INH resistance in M. tuberculosis.
• The INH, PZA, and FQ strips were designed to detect mutations
associated with INH, PZA, and FQ resistance of M. tuberculosis,
respectively.

36. RESULTS
• In the sputum specimens, sensitivity and specificity for detecting RIF-, PZA-, and
LVX-resistant isolates were both 100% & for detecting INH-resistant isolates were
75.0% and 92.9%, respectively.
Sensitivity Specificity
Rifampicin 98.9 97.3
INH 90.6 100
PZA 89.7 96
Levofloxacin 93 100
A Japanese multi-center study
conducted at 6 hospitals assessed this
new line probe assay in 554
isolates (316 from MTB) and 163
sputum specimens.

37. Using microscopy and line-probe assays in conjunction with
drug-susceptibility testing (with solid or liquid media)
to diagnose TB
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

39. Xpert MTB/RIF
• Automated, cartridge-based nucleic acid amplification test
(NAAT) that uses the multidisease GeneXpert platform.
• Performed directly on sputum, processed sputum sediment
and selected extrapulmonary specimens from adults and
children.
• Capacity to have 1, 2, 4, 16, 48 or 80 independently
functioning modules.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

41. Rifampicin Resistant Sample
 At least 2 of 5 rpoB probes positive within 2 cycles and amplification control positive=
M. tuberculosis complex
 Failure of proper hybridization of one or more rpoB probes= rifampin resistance
41

42. Advantages - Xpert MTB/RIF
• Simultaneously detects M. tuberculosis and rifampicin resistance
in less than 2 hours.
• The sensitivity for detecting TB is similar to that of to liquid
culture (sensitivity 88%); the specificity is also high (99%).
• The superior performance of Xpert MTB/RIF in detecting TB over
that of microscopy makes it a particularly useful tool for case-
finding among people living with HIV.
1. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert
MTB/RIF assay for the diagnosis of pulmonary and
extrapulmonary TB in adults and children. Policy update. Geneva, World Health Organization,
2013 (WHO/HTM/TB/2013.16) http://www.who.int/tb/laboratory/xpert_policyupdate/en/

43. Advantages - Xpert MTB/RIF
• As a tool for detecting rifampicin resistance, Xpert MTB/RIF
has a sensitivity of 95% and specificity of 98% when compared
with phenotypic reference standards.
• Biosafety precautions and the training required are minimal.
• For smear-negative culture-positive TB, the pooled sensitivity
of Xpert MTB/RIF has been found to be 68%.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

44. Disadvantages - Xpert MTB/RIF
• A stable uninterruptable electrical supply is needed.
• The ambient operating temperature of the instrument cannot
exceed 30 °C, cartridges must be stored at less than 28 °C.
• The shelf-life of the cartridges must be monitored to prevent
them from expiring before they are used.
• Security measures must be put in place to prevent the theft of
the accompanying laptop or desktop computer.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

45. Disadvantages - Xpert MTB/RIF
• The modules require annual calibration.
• Does not eliminate the need for conventional microscopy,
culture and DST.
• In patients who are not at risk for drug resistance but tested
positive for Rif resistance, a second Xpert MTB/RIF test should
be performed to control for preanalytical and postanalytical
errors, and to improve the clinician’s confidence in the
diagnosis.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

46. • Infrequent occurrence of false-positive results may be linked
to the detection by Xpert MTB/RIF of strains that are truly
resistant to rifampicin but for which resistance is not detected
by phenotypic culture-based DST.
• In cases of discordant results obtained from Xpert MTB/RIF Vs
phenotypic DST or LPA, the culture isolate should be referred
to a reference laboratory for DNA sequencing.
• while awaiting the results, a clinical decision should be made
whether to continue the MDR-TB regimen.
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

47. Research & Development
GeneXpert Omni
• At just 9 inches tall (about 23 cm) and weighing just 2.2 pounds
(about 1kg), the GeneXpert Omni will run the same PCR-based
cartridge tests.
• Additionally, the GeneXpert Omni is
battery-operated, wireless and web-enabled,
able to transmit instrument & test information
in real-time.
• WHO expects to evaluate the platform in 2016.
GLOBAL TUBERCULOSIS REPORT 2015

48. Research & Development
Xpert MTB/RIF Ultra
• The current Xpert MTB/RIF test has a limit of detection (LOD) of
130 cfu/ml.
• New innovations, including a larger DNA reaction chamber in the
cartridge, will bring the LOD down ten-fold, to approximately10
cfu/ml (across all strains) — a level similar to or potentially better
than liquid culture.
• Both GeneXpert Omni & Xpert MTB/RIF Ultra is expected to be
available in the first half of 2016.

49. Summary of WHO’s 2013 policy
recommendations for using
Xpert MTB/RIF
• Should be used rather than conventional microscopy, culture
and DST as the initial diagnostic test in adults and children
suspected of having MDR-TB or HIV-associated TB.
• May be used rather than conventional microscopy and culture
as the initial diagnostic test in all adults and children
suspected of having TB.
• Should be used in preference to conventional microscopy and
culture as the initial diagnostic test for CSF specimens from
patients suspected of having TB meningitis.
Xpert MTB/RIF implementation manual: technical and operational “how-to”; practical considerations. Geneva: World Health Organization; 2014
(WHO/HTM/TB/2014.1). http://apps.who.int/iris/bitstream/10665/112469/1/9789241506700_eng.pdf.

50. • May be used as a follow-on test to microscopy in adults
suspected of having TB who are not at risk of MDR-TB or HIV-
associated TB, especially when further testing of smear-
negative specimens is necessary.
• May be used as a replacement test for usual practice
(including conventional microscopy, culture or histopathology)
for testing specific non respiratory specimens from patients
suspected of having extrapulmonary TB .
Xpert MTB/RIF implementation manual: technical and operational “how-to”; practical considerations. Geneva: World Health Organization; 2014
(WHO/HTM/TB/2014.1). http://apps.who.int/iris/bitstream/10665/112469/1/9789241506700_eng.pdf.

51. Using the Xpert MTB/RIF assay as an initial diagnostic test for TB followed by drug
susceptibility testing for second-line anti-TB agents when necessary
WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

53. • A review commissioned by WHO included all of the evidence
on the use of Xpert MTB/RIF published until the end of
February 2013.
The reviews focused on four specific areas of interest:
• using Xpert MTB/RIF to diagnose pulmonary TB and rifampicin
resistance in adults
• using Xpert MTB/RIF to diagnose extrapulmonary TB in adults
and children
• using Xpert MTB/RIF to diagnose pulmonary TB and rifampicin
resistance in children
• the affordability and cost effectiveness of Xpert MTB/RIF for
diagnosing TB.
WHO Xpert MTB/RIF implementation manual 2014

54. Pooled Sensitivity Pooled Specificity Studies /
Population
Initial diagnostic test
replacing
smear microscopy
88%
(95% credible
interval [CrI], 84-
92%)
99%
(95% CrI, 98-99%)
22 studies, 9008
participants
add-on test following a
negative smear
microscopy result
68%
(95% CrI, 61-74%)
99%
(95% CrI, 98-99%)
23 studies, 7151
participants
cases of smear-positive,
culture-positive TB
98%
(95% CrI, 97-99%)
- 23 studies,
1952 participants
people living with HIV 79%
(95% CrI, 70-86%)
- 7 studies, 1789
participants
for people without HIV
infection
86%
(95% CrI, 76-92%)
- 7 studies, 1470
participants
to detect rifampicin
resistance in adults
95%
(95% CrI, 90-97%)
98%
(95% CrI, 97-99%)
24 studies, 2414
specimens
WHO Xpert MTB/RIF implementation manual 2014
Using Xpert MTB/RIF to diagnose pulmonary TB & Rif resistance in
adults

55. Using Xpert MTB/RIF to diagnose
extrapulmonary TB in adults &
children
• A total of 15 published studies and 7 unpublished studies (5922
samples) were included in the review.
WHO Xpert MTB/RIF implementation manual 2014
Pooled sensitivity Studies/Samples
Lymph
node/aspirate
84.9% (95% confidence
interval [CI], 72.1-92.4%)
14 studies, 849 samples
Pleural fluid 43.7% (95% CI, 24.8-
64.7%)
17 studies, 1385
specimen
CSF 79.5% (95% CI, 62.0-
90.2%)
16 studies,
709 specimens
Gastric fluid 83.8% (95%
CI, 65.9-93.2%)
12 studies, 1258
samples
Other tissue
specimens
81.2% (95%
CI, 67.7-89.9%)
12 studies, 699
specimens

56. Using Xpert MTB/RIF to diagnose
pulmonary TB and rifampicin resistance
in children
• A total of 16 studies (12 published, 4 unpublished) were included
in the review.
• The diagnosis of pulmonary TB was evaluated in 13 studies that
included 2603 participants.
WHO Xpert MTB/RIF implementation manual 2014
Sensitivity Specificity
To detect pulmonary
TB in children
(overall)
Sputum - 66%
(95% CrI, 52-77%
Gastric lavage - 66%
(95% CrI, 51-81%)
98% with
narrow
confidence
interval
against culture
13 studies, 2603
participants
To detect rifampicin
resistance in children
86%
(95%CrI, 53-98%) - -

57. PMDT guidelines May 2012

59. Conclusion
• LPAs are currently limited to detecting resistance to
rifampicin and isoniazid and are suitable only on smear
positive Specimens.
• Conventional DST is needed to detect resistance to anti-
TB agents other than rifampicin and isoniazid & to detect
XDR-TB.

60. Conclusion
• Overall the LPA test is a reliable, rapid and easy to
perform for the simultaneous detection of RMP and INH
resistance in M.tuberculosis
• It is recommend that the LPA should serve as an early
guidance of therapy, which should be followed by a
phenotypic DST confirmation for all suspected MDR-TB
patients.

61. Conclusion
• Although Xpert MTB/RIF is suitable for use at all levels of the
health system & does not require additional laboratory
equipment, it requires care in handling, a stable and
uninterrupted electrical supply, security against theft, adequate
storage space & bio-safety measures.
• Xpert MTB/RIF does not eliminate the need for capacity for
conventional TB microscopy, culture and DST.
• The Xpert MTB/RIF assay is suitable for diagnosing TB and
detecting RR-TB.
• A negative result accurately excludes the possibility of
rifampicin resistance and no further testing is needed to
confirm the negative results. (NPV>99%)

62. • Microscopy or culture, or both, remain essential for
monitoring treatment since molecular tests based on
DNA detection have not been shown to be suitable.
• Liquid culture, molecular LPAs and the Xpert MTB/RIF
assay should be phased in to programmes in a way that
ensures that the existing capacity for solid culture and
DST is maintained.