Division of Infectious Diseases

Division of Infectious Diseases
Xpert® MTB/RIF Ultra: The Next-Generation Assay for Tuberculosis Diagnosis and Rifampin Resistance Detection Diego P. Peralta, M.D., FACP Assistant Professor Division of Infectious Diseases March 22, 2018 Texas Association for Clinical Laboratory Science

Disclosures No financial disclosures to report.

Objectives At the end of the session, the participant will be able to:
Identify the differences between the Xpert® MTB/RIF Ultra assay and its predecessor. Describe the Xpert® MTB/RIF Ultra assay clinical applications. Appraise the evidence to support the use of Xpert® MTB/RIF Ultra assay in tuberculosis diagnosis and Rifampin resistance detection.

Tuberculosis

Epidemiology WHO Estimates 2015 2016 New TB cases reported
10.4 million TB diagnosis 6.1 million (59%) 6.3 million (61%) Deaths attributable to TB 1.8 million 1.3 million Rifampicin resistance 580,000 600,000 Dorman SE et al. Lancet Infect Dis Jan;18(1):76-84 WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Epidemiology WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Epidemiology TB is the ninth leading cause of death worldwide
TB is the leading cause of death from a single infectious agent, ranking above HIV/AIDS WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Reported TB Cases United States, 1982-2016*
CDC. National Tuberculosis Surveillance System 2016 * As of June 21, 2017

TB Case Rates,* United States, 2016
* Cases per 100,000; as of June 21, 2017. DC, District of Columbia; NYC, New York City (excluded from New York state) CDC. National Tuberculosis Surveillance System 2016

TB Morbidity United States, 2011–2016
* Cases per 100,000 population; as of June 21, 2017. CDC. National Tuberculosis Surveillance System 2016

WHO: The End TB Strategy
WHO aims by 2035: 90% reduction in TB incidence 95% reduction in TB mortality WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Diagnosis Gold standard Smear microscopy is insensitive
Culture and drug susceptibility Smear microscopy is insensitive Culture takes too long to be clinically useful For TB diagnosis, highly sensitive, rapid, and accessible diagnostics are needed WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Diagnosis Several assays are endorsed by the WHO: Line Probe Assays
Gen-Probe Mycobacterium Tuberculosis Direct Urine lateral flow lipoarabinomannan (LF-LAM) Loop-mediated isothermal amplification (TB-LAMP) Xpert® MTB/RIF assay WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Xpert® MTB/RIF Assay Automated in vitro test Uses real-time PCR
Qualitative detection of MTB and RIF resistance Primers amplify a portion of rpoB gene containing the 81 base pair core region Probes differentiate the conserved wild-type sequence and mutations in the core region associated with RIF resistance GeneXpert Technology. Cepheid Website

Xpert® MTB/RIF Assay GeneXpert Technology. Cepheid Website

History 2010 WHO endorsed the Xpert® MTB/RIF assay for initial diagnostic testing of individuals suspected of MDR-TB or HIV-associated TB 2013 FDA permitted marketing of the Xpert® MTB/RIF assay 2014 WHO expanded recommendation for use in all patients (including also extra-pulmonary TB) MMWR Morb Mortal Wkly Rep Oct 18;62(41):821-7. WHO. Global Tuberculosis Report 2017.; doi: WHO/HTM/TB/

Going Ultra: Xpert® MTB/RIF Ultra Xpert® MTB/RIF has limitations
March 2017 WHO recommended: Replacement of Xpert® MTB/RIF by Xpert® MTB/RIF Ultra Going Ultra: Xpert® MTB/RIF Ultra WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultra compared to Xpert MTB/RIF. Geneva: World Health Organization; 2017 (WHO/HTM/TB/ ). Licence: CC BY-NCSA 3.0 IGO.

Differences between Xpert and Ultra
Xpert® MTB/RIF Xpert® MTB/RIF Ultra Two multi-copy amplification targets (IS6110 and IS1081) Fully nested nucleic acid amplification More rapid thermal cycling Improved fluidics and enzymes DNA PCR reaction chamber 25 µl 50 µl Limit of detection 114 bacterial CFU/mL 16 bacterial CFU/mL Semi-quantitative categories High, medium, low and very low High, medium, low, very low, and trace (trace call) 1–log improvement in the lower limit of detection RIF resistance detection Real-time PCR Melting temperature-based analysis Improved differentiation of certain silent RIF mutations and detection of RIF resistance in mixed infections and paucibacillary specimens WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultra compared to Xpert MTB/RIF. Geneva: World Health Organization; 2017 (WHO/HTM/TB/ ). Licence: CC BY-NCSA 3.0 IGO.

Xpert® MTB/RIF Ultra Pros: Higher sensitivity Cons: Lower specificity
Increase diagnosis of paucibacillary forms of TB Childhood TB HIV-associated TB Extrapulmonary TB Cons: Lower specificity Previously treated/history of TB High-incidence countries WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultra compared to Xpert MTB/RIF. Geneva: World Health Organization; 2017 (WHO/HTM/TB/ ). Licence: CC BY-NCSA 3.0 IGO. Arch Bronconeumol Dec;53(12): doi: /j.arbres Epub 2017 Jul 10.

Xpert® MTB/RIF Ultra Lower specificity represents Possible explanation
Higher proportion of false positive results Empiric treatment and possibly overtreatment that might undermine clinical effect Possible explanation Detection of DNA from non-viable M. tuberculosis Prolonged excretion of M. tuberculosis DNA form TB lesions after treatment M. tuberculosis DNA from dead bacilli has high stability (DNA can be detected in millennia-old human remains) WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultra compared to Xpert MTB/RIF. Geneva: World Health Organization; 2017 (WHO/HTM/TB/ ). Licence: CC BY-NCSA 3.0 IGO. Arch Bronconeumol Dec;53(12): doi: /j.arbres Epub 2017 Jul 10.

Study Justification Limitations of Xpert MTB/RIF assay
Imperfect sensitivity for paucibacillary disease (HIV, early disease, children, etc) Imperfect sensitivity for RIF-resistance detection in case of heteroresistance Imperfect specificity for RIF-resistance detection due to silent mutation detection Cross-reactivity with some nontuberculous mycobacteria (NTM) Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay CROI Rodwell T. A Multicenter Diagnostic Accuracy Study of the Xpert Ultra for Tuberculosis Diagnosis.

Study Design: Index test: Comparator: Reference standard:
Blinded, multicenter, prospective non-inferiority study. Index test: Xpert® MTB/RIF Ultra Comparator: Xpert ® MTB/RIF Reference standard: Culture, drug susceptibility, sequencing Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Primary Outcomes: MTB detection RIF detection
∆ Sensitivity: among smear-negative/culture-positive patients ∆ Specificity RIF detection Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Secondary Outcomes Estimates of accuracy for MTB detection
Overall/pooled By smear-status By HIV status By TB history and time since treatment completion By site On secondary study samples Root-cause analysis of discordant results between Ultra and culture Analyses reclassifying the ‘trace’ call Analysis of NTMs Analysis of semi-quantitative results Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Sites Ten sites in eight countries
Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Sample Computed sample: Sample: 1143 1520 eligible for analysis

Flow at Enrollment Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Non-inferiority Margins for Comparison between Xpert and Ultra

Main Results

Demographic and Clinical Characteristics of Sample

Sensitivity and Specificity of Xpert and Ultra for MTB Detection

Sensitivity and Specificity of Xpert and Ultra for RIF Detection

Specificity Overall and by Treatment History Status

Non-inferiority Analysis for MTB Detection

Non-inferiority Analysis for MTB Detection
+17% -3.2% Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Non-inferiority Analysis for RIF Detection

Non-inferiority Analysis for RIF Detection
-0.6% +0.3% Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Primary Analysis Findings
MTB detection Ultra shows improved sensitivity but reduced specificity Performance for RIF detection is very similar Non-inferiority met for specificity, but missed by a small margin for sensitivity Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Secondary Analysis Findings
Specificity of both Xpert and Ultra are affected by TB history Stronger for Ultra and for recent history Trade-off of higher sensitivity is lower specificity of Ultra “trace-calls” Reclassification of “trace-calls” as ‘TB-negative’ mitigates specificity losses while maintaining some of the sensitivity gains To be considered for all patients or for patients with TB history Re-testing of “trace-call” results with a second Ultra is another possibility to mitigate specificity losses leading to similar results as reclassification state above Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Other Secondary Analysis Findings
Analyses by site: Expected variability of sensitivity-estimates between sites due to small sample Specificity-estimates broadly consistent Root cause analysis: FPs on sample 1 mainly due to non-viable bacilli prior TB Lower specificity-estimates for samples 2 and 3 versus sample 1 is likely due cross-contamination Cross-contamination was due to procedures that were performed in certain sites (i.e., beads homogenization) Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Conclusions Ultra offers opportunities for increased TB detection
Ultra has increased sensitivity for TB detection including patients with paucibacillary disease Ultra has decreased specificity in patients with prior history of TB Ultra implementation needs to be considered carefully due to reduced specificity Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Additional Data Published with Main Study: CE-mark, Extra-pulmonary TB, Elimination in non-HBDC, and Pediatric Cases

CE-mark Data Data provided by Cepheid MTB detection:
Sensitivity: Smear-positive: 99.5% (95%CI 98.3, 99.9) Smear-negative: 73.4% (95%CI 67.7, 78.4) Specificity: Overall: 95.5% (95%CI 94.2, 96.6) RIF resistance detection: Sensitivity: 96.2% (95%CI ) Specificity: 96.3% (95%CI ) Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Extra-pulmonary TB TB meningitis Sensitivity: Conclusion: Ultra: 95%
Xpert 45% Culture 45% Conclusion: Ultra detected significantly more TB meningitis than either Xpert or culture Adequate CSF testing volume is critical Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Pediatric Study South Africa
Culture and Ultra Sensitivity: 75.7% Specificity: 96.5% HIV-infected Sensitivity: 70.6% Specificity: 98.2% HIV-uninfected Sensitivity: 77.2% Specificity: 96.1% Conclusion: Ultra provides rapid detection of MTB from a single induced sputum in most children with culture confirmed TB Ultra may detect an additional group of children with TB, who are not detected by culture Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

Evaluation of the Ultra Assay in a Pediatric TB Study from Tanzania

Summary of Findings Shows a significant increase of sensitivity for Ultra in TB meningitis Suggests an increase in sensitivity in paucibacillary disease (i.e., children and active case finding in asylum seekers) Sensitivity increase is attributable to Ultra “trace call” Foundation for Innovative New Diagnostics (FIND). Report for WHO: A Multicentre Non-Inferiority Diagnostic Accuracy Study of the Ultra Assay Compared to Xpert MTB/RIF Assay

WHO Position Regarding Xpert® MTB/RIF Ultra
Ultra assay is non-inferior to Xpert assay for the diagnosis of MTB and detection of RIF resistance Ultra can be used as an alternative to Xpert in all settings Ultra can be used as the initial diagnostic test for all adults and children with signs and symptoms of TB Ultra can be used in the testing of selected extra pulmonary specimens (CSF, lymph nodes and tissue specimens) Interpretation of Ultra is similar to Xpert except for “trace call” WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTF/RIF Ultra compared to Xpert MTB/RIF. Geneva: World Health Organization; 2017 (WHO/HTM/TB/ ). Licence: CC BY-NCSA 3.0 IGO.

Recent Published Studies Using the Xpert® MTB/RIF Ultra Assay

Lancet Infect Dis. 2018 Jan;18(1):76-84. doi: 10
Lancet Infect Dis Jan;18(1): doi: /S (17) Epub 2017 Nov 30.

Lancet Infect Dis. 2018 Jan;18(1):68-75. doi: 10
Lancet Infect Dis Jan;18(1): doi: /S (17) Epub 2017 Sep 14.

PLoS Med. 2017 Dec 14;14(12):e1002472. doi: 10. 1371/journal. pmed
PLoS Med Dec 14;14(12):e doi: /journal.pmed eCollection 2017 Dec.

Pediatr Infect Dis J. 2018 Feb 22. doi: 10. 1097/INF. 0000000000001960
Pediatr Infect Dis J Feb 22. doi: /INF [Epub ahead of print]

Division of Infectious Diseases
Xpert® MTB/RIF Ultra: The Next-Generation Assay for Tuberculosis Diagnosis and Rifampin Resistance Detection Diego P. Peralta, M.D., FACP Assistant Professor Division of Infectious Diseases March 22, 2018 Texas Association for Clinical Laboratory Science

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