GeneXpert Ultra

JOURNAL CLUB

By - 
Dr. Rohit Kumar (JR ID)
Dr. Sayan (SR ID)
What do we need ?
Diagnostic
test

Drug
Detection
susceptibility
of Mtb
(DST)

sensitive specific Phenotypic genotypic

MGIT , SOLID CULTURE Xpert, Xpert ultra, LPA

(LJ), MODS
 Gene xpert
Mtb/Rif
Microscopy Gene xpert
(light & ultra
LED;MODS) Mtb/Rif

Culture WHO
(MGIT, LJ,
Middle endorsed LPA (1st line
& 2nd line)
brook)
tests
TB LAM Interferon TB LAMP
gamma
release
assays
(IGRA)
 Gene Xpert Xpert Xpert ultra

Ultra 50 u l
65-87 min
what’s new 25 ul
112 min 16 cfu/ml
Nested (2 phase)
131 cfu/ml PCR
 Design of the cartridge Heminested PCR rpoB 1-4(4);
 PCR parameters rpoB A-E (5)
IS6110, IS1081
 Mutation detection
 Improvements in both Categories Categories
• High ( < 16 • High 15 -18
TB detection and ) .9
• Medium (1 • Medium 19
6– - 24.9
identification of RIF-R. 22) • Low 25 – 2
• Low (22 – 2 8.9
8) • Very low 2
• Very low (> 9 – 40
28) • TRACES

 Can be used on same GeneXpert® equipment

 Same concessional price (USD 9.98) for all eligible
Chakravorty et al. 2017; mBio 8:e00812-17.
  Diagnostic accuracy study (non inferiority)
 Done at 10 reference laboratories in 8 countries
 South Africa, Uganda, Kenya, India, China, Georgia,
Belarus, and Brazil
 P (POPULATION ) – all suspected PTB (smear -, HIV )

Study
 I (Index test) - GeneXpert Ultra
 C (comparator) – GeneXpert

design
 C (reference) - Culture
 O – accuracy estimates (sensitivity, specificity)

 Protocol reviewed & approved by ethics committees at

study sites & supervising organisations
 Written informed consent obtained from all study
participants.
 Study participation did not affect the standard of care.
Diagnostic
accuracy study
 Test evaluated against
clinical reference standard,
or gold standard; results
reported as estimates of
test’s
 Sensitivity & specificity
 Area under the Receiver
Operating Characteristic
(ROC) curve
 PPV & NPV
 Likelihood ratios
 Non inferiority or
equivalence or superiority
PPV – positive predictive value
NPV – negative predictive value
The sensitivity of a single Xpert ultra test for at –7% for sensitivity to
detection of smear-negative TB is non-inferior detect smear “-” TB

to that of a single Xpert,

at –3% for sensitivity
and and specificity to detect
The sensitivity and the specificity of Xpert ultra for rifampicin resistance
rifampicin resistance detection were noninferior to
those of Xpert A margin was not
predefined for
specificity of TB
detection.

HYPOTHESIS
 PRIMARY OBJECTIVE 

To estimate & compare the sensitivity of a single

To estimate & compare Xpert Ultra &
Xpert Ultra test with that of a single Xpert test
Xpert specificities for detection of
of the same raw sputum specimen for detection
rifampicin resistance
of smear “-” TB & rifampicin resistance
 Inclusion criteria
 Adults presenting at primary health-care centres and hospitals
 with PTB symptoms and 
 who were willing to provide upto 4 sputum specimens at study enrolment

 Case detection group

 willingness to attend study follow-up visits 42–70 days after enrolment and 
 no ATT had been taken in the past 6 months. 
  Multidrug resistance risk group
 high risk of drug resistance on basis of 1 or more of following :
 microbiologically confirmed PTB with documented rifampicin resistance & ATT for 31 days or
less; 
 known PTB with suspected treatment failure; and
 history of DR-TB and off ATT for at least 3 months.
 Methodology
Feb 18 – Dec 24 2016 

Demography, medical history,

chest imaging, HIV status

For reference standard testing

Sample reagent added ;

2:1 dilution
2ml to each carridge

Staff doing Xpert

/Xpert ultra were
Rifampicin concn. = 1ug/ml blinded to results
of other tests
Culture positive isolates –
confirmation done using
MPT64/MPB64 Ag

Included in all Included only

analysis in analysis for
rifamipicin
resistance
detection

Phenotypic DST – all culture positive samples

Genetic DST – using pyrosequencing and Sanger sequencing In discordant results between phenotypic DST and Xpert ultra
Next Gen Sequencing/Pyrosequencing of IS6110/IS1081 & rpoB was done if Xpert ultra positive & culture “-” {India & Italy}
 Data NA for 3 pat.
(Mumbai)

Case
detect-
ion
group
RESULTS

Demographic & clinical characteristics, enrolment group, & distribution in diagnostic smear results
categories of the study participants missing for 2
 115 (NOT 155) Culture -- in case
detection group

Comparative accuracy for detection of tuberculosis and rifampicin resistance

Case detection group
Among culture positive – status known = 274 (HIV status = + (115),Neg = 159)

Phenotypic  Rif R Rif S Phenotypic  Rif R Rif S

Xpert Xpert Ultra
Rif R 167 7 174 Rif R 166 6 172
Rif S 8 369 377 Rif S 9 370 379
175 376 551 175 376 551
Test sensitivity & specificity depending on TB history and different approaches to the interpretation of semiquantitative trace-positive
results for Mtb detection by Xpert MTB/RIF Ultra
Specificity estimates of Xpert MTB/RIF and Xpert MTB/RIF Ultra (Xpert Ultra) for TB case detection in
patients with a ATT history and for different approaches to handling an
initial Xpert Ultra trace-positive result
 17%

10% 24%

-3.2% 1.6% -0.7% 1.5%

-0.6%
0.3%
 Critical Appraisal

Patient selection
Was a consecutive or random sample of patients enrolled? Yes

Was a case-control design avoided? Yes

Did the study avoid inappropriate exclusions? Yes

Was the diagnostic test evaluated in a Representative spectrum of patients? Yes

Index Text
Were the index test results interpreted without knowledge of the results of the reference Yes
standard?
If a threshold was used, was it pre-specified? NA
 Critical Appraisal
Reference standard
Is the reference standard likely to correctly classify the target condition? Yes

Were the reference standard results interpreted without knowledge of the results of the Yes
index test?
Flow and Timing
Was there an appropriate interval between index test(s) and reference standard? Yes

Did all patients receive a reference standard? Yes

Did patients receive the same reference standard? Yes

Were all patients included in the analysis? Yes

 Summary
 Sensitivity of Xpert Ultra was superior to Xpert
 TB case detection sputum smear-negative PTB , HIV-infected participants and in all study
participants.

 High sensitivity of Xpert Ultra could facilitate

 Diagnosis of TB at earlier stages of disease and
 Diagnosis of TB in patients with HIV and smear negative TB (population with high mortality)
 Relevant for diagnosis of TB in children and for diagnosis of EPTB (TBM)

 Loss of specificity
 difference in specificity between patients with/without a history of TB treatment
 https://www.finddx.org/dx-pipeline-status/
 Gene xpert vs Ultra

 M. tuberculosis strain H37Rv contains 16 copies of IS6110 and 5 copies of IS1081.

 calculated TB detection LOD for Xpert was 112.6 CFU/ml (95% CI, 91.9 to 158.2).
 correct detection of TB-positive specimens by Ultra was 100% up to 25 CFU/ml.
 calculated TB detection LOD for Ultra was 15.6 CFU/ml (95% CI, 12.2 to 23.1)
 Rates of detection of RIF susceptibility
 LOD by Xpert - 112.6 CFU/ml [95% CI, 91.9 to 158.2 CFU/ml]
 LOD by Ultra - 105.4 CFU/ml [95% CI, 78.5 to 178.8]
 Ultra - substantial LOD improvement for detection of M. bovis BCG In contrast to H37Rv, BCG contains only 1 copy of
IS6110 and 5 copies of IS1081.
 the BCG detection limit for Xpert was 344.1 CFU/ml (95% CI, 297.5 to 434.0)
 BCG detection limit for Ultra of 143.4 CFU/ml (95% CI, 106.2 to 243.7)
 As with the H37Rv M. tuberculosis strain, with BCG,
 LODs of RIF susceptibility detection BCG by Xpert, 338.8 CFU/ml [95% CI, 294.0 to 425.8];
 LOD for RIF susceptibility in BCG by Ultra, 300.3 CFU/ml [95% CI, 244.2 to 408.2])
 Introduction

 Diagnosis and successful treatment of people with TB averts an estimated 54 million over the period 2000–2017 of deaths
each year
 Worldwide in 2017, 6.4 million new cases( increasing since 2013, in which 5.7–5.8) TB were officially notified to national
authorities
 mainly due to increased reporting of detected cases by the private sector in India and, in 2017, an upturn in notifications
in Indonesia.
 The 6.4 million cases reported represented 64% of the estimated 10.0 million new cases that occurred in 2017.
 Ten countries accounted for 80% of the 3.6 million global gap, the top three being India (26%), Indonesia (11%) and
Nigeria (9%).8
 Gaps between estimated number of new cases and number actually reported - due to a mixture of underreporting of
detected cases, and underdiagnosis .
 There were 464 633 reported cases of TB among PLWHA in 2017 (51% of the estimated 920 000 new cases in the same
year), of whom 84% were on antiretroviral therapy.
 To support countries to close gaps in TB detection and treatment, in 2018 WHO, in collaboration with the Stop TB
Partnership and the Global Fund to Fight AIDS Tuberculosis and Malaria, launched an initiative called Find. Treat. All.9
 The initiative includes a target of detecting and treating 40 million people with TB in the period 2018–2022. The
latest treatment outcome data for new cases show a global treatment success rate of 82% in 2016.
 This is a reduction from 86% in 2013 and 83% in 2015; in countries where notifications have increased, reporting of
treatment outcomes has not kept pace.
 Globally, 160 684 MDR/RR-TB cases were detected and notified in 2017 (153 1193 GLOBAL TUBERCULOSIS
REPORT 2016).
 139 114 people (87%) enrolled on 2nd line ATT, up from 129 689 in 2016 but still only 25% of the estimated 558 000
people who developed MDR/RR-TB in 2017.
 China and India alone accounted for 40% of the global gap
 Closing gaps in detection and treatment requires much higher coverage of DST among people diagnosed with TB,
reducing underdiagnosis of TB, models of care that make it easier to access and continue treatment, new diagnostics,
and new medicines and treatment regimens with higher efficacy and better safety.
 Gene xpert

 less sensitive when testing smear-negative sputum.

 For example, when Xpert was performed following negative smear microscopy result , its pooled sensitivity was
67% (2). Xpert was 43% sensitive in one study of HIV-positive patients with smear-negative TB (5), and its
sensitivity was as low as 28% for smear-negative TB patients from a high-resource country with a low TB incidence
(6).
 Clinical impact has also been lower than originally expected, perhaps due to Xpert’s suboptimal negative predictive
value.
 Assay sensitivity limited in some extrapulmonary samples (known to contain lower levels of bacilli than pulmonary
samples)
 limited capacity to detect RIF-R associated mutations in mixed samples (heteroresistance)
 decreased capacity to detect rpoB C533G mutations responsible for some cases of RIF-R .
 false-positive RIF-R - for paucibacillary samples (delays in the real-time signal generated by assay probes D and E )
 FALSE POSITIVE - recognition of nonfunctional rpoB F514F silent mutation as conferring RIF-R
 Bias (diagnostic accuracy study)

Patient/subject Index test Reference test Data analysis

Progression
Mis-
Selection bias
classificatio
bias Information
n bias
bias
Information Data
bias Differential excluded
verification
Partial
Spectrum bias
verification
bias Incorporation
bias
bias
Bias (diagnostic accuracy study)
 Summary

 Specificity increased with increasing time since completion of ATT since the preceding TB
episode up to 7 years.

 Extraneous M tuberculosis from other specimens or the laboratory environment, or false

negative cultures from over-decontamination are possible explanations for a positive NAAT
result with negative cultures for M tuberculosis.

 presence of M tuberculosis DNA or intact bacilli (living or dead, originating from participant’s
lower respiratory system), or both in sputum