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Experts on Xpert: A Laboratorian and a Clinician Discuss Interpretation of Xpert MTB/RIF ResultsDr. Pennan Barry (MD, MPH) and Grace Lin (MS)

September 26, 2018 1

Experts on Xpert:A laboratorian and a clinician discuss

interpretation of Xpert MTB/RIF Results

Pennan Barry, MD, MPHGrace Lin, MS

September 26, 2018

Curry International Tuberculosis Center

Learning Objectives

By the end of the training, participants will be able to:

• describe the principles behind the Xpert MTB/RIF test

• identify circumstances that should trigger additional testing or where false identification of MTB/RIF resistance should be suspected

• identify probes that most frequently detect mutations conferring RIF resistance and silent mutations not conferring RIF resistance

• identify situations where additional Xpert testing is not indicated

Xpert interpretatiom-Curry Center-9/26/18 2



Disclosures

• None


Agenda

• Intro: – Not an introduction to Xpert use

– Not about release from respiratory isolation

• What is Xpert and how does it work?

• Case discussions:– Illustrate nuances of Xpert interpretation

(focus on RIF resistance result)

– When to ask for more info or more testing

– When not to test more

4Xpert interpretatiom-Curry Center-9/26/18



Xpert MTB/RIF assay

• Great tool for TB detection• FDA Approved• Shortens time significantly • Testing time 2.5 hr; Easy to perform• Real-Time PCR

– 5 molecular beacon probes– rpoB gene (core region, codons 507-533)

5

Xpert Clinical Report




“Hidden” Info: Report on Xpert computer


Methodology: Realtime PCR

• Realtime PCR = PCR + probes

• PCR = Polymerase Chain Reaction– Generates copies of specific segments

of DNA

– DNA copies double with each PCR cycle

• Probes report whether specific sequence is present– Probes bind to amplicons; generate

signals with each PCR cycle


PCR cycles



PCR Cycles

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

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10

Molecular Beacon probe(Hairpin structure—head & two arms)

Head (functional part, MTBC sequence, ~20 nt)

Two arms (5-7 nt)

Fluorophore Quencher

At “rest” stage, two arms bind together forming a stem.Fluorophore is quenched. No signals are produced.



11

MB head does not bind to mutant SQ.Arms remain closed.No signals produced.

Mutation Detection with Molecular Beacons (Head containing wildtype SQ)

Mutant Sequence Wildtype Sequence

MB in action!MB resting

MB head binds to wildtype SQ.Arms open. Fluorophore away from quencher. Signals produced.

Ct: Threshold Cycleat which signal crosses threshold Low DNA in the specimen High Ct

0

250

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Flu

ore

sce

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PCR Cycle

Threshold

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Sample 1 (Wildtype)

Ct=18




0

250

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Flu

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sce

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p

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PCR Cycle

Threshold

13

Sample 1 (Wildtype)

Sample 2 (Wildtype)

Ct=18 Ct=25


0

250

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Flu

ore

sce

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p

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y p

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PCR Cycle

Threshold

14

Sample 1 (Wildtype)

Sample 2 (Wildtype)

Sample 3

Ct=18 Ct=25



No signals: 3 possibilities

• Mutation(s) present

• MTBC DNA insufficient or not present

– Smear-negatives (if < ~120 colonies/mL)

• Unable to generate enough DNA copies

– Smear positive but not MTBC

• Inhibitory substance

– Inhibit amplification

– Xpert has internal control (SPC) to detect this

• Signals in SPC indicate no inhibitory substance in specimen


Critical Rules for Interpretation(Set by the Xpert software)

• MTBC detected

– at least 2 probes are positive

• RIF-R detected

– If highest and lowest Ct differs by more than 4 (Δ Ct max > 4)

• See more rules in package insert

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Typical Xpert results

(Examples showing Xpert’s rules work as intended -- No nuance!)

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18

Ct End-pt MTBC: Not detected

• No probe has signal

• SPC has signal. If no signal, test is invalid.



19

MTBC detected• 5 probes up

RIF-SNo mutation• Δ Ct max < 4

(33.9-31.8=2.1)

Ct End-ptCt End-pt

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MTBC detected• 4 probes upRIF-R detected• Probe E no signals

Δ Ct max > 4:24.8-0=24.8

• Most common MDR mutation: S531L, detected by probe E

Ct End-ptCt End-pt



Xpert Probes: Coverage of rpoB

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

Codon#

Most common mutation confers

resistance (531 TTG)

Ask your lab which probe resulted in the RIF-R result

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Can Xpert differentiateM. tuberculosis (MTB) from

M. tuberculosis complex (MTBC)?




M. tuberculosis Complex

• M. tuberculosis

• M. bovis

• M. africanum

• M. caprae

• M. pinnipedii

• M. microti• M. mungi, M. orygis, M.

canettii, M. suricattae(proposed)


All cause tuberculosis

MTB / MTB Complex?

• Species in the MTB complex have same rpoBcore region sequence

– Xpert can only identify MTBC, not MTB species.

• “MTBC” is the correct term.

• Xpert results for M. tuberculosis and M. bovisindistinguishable

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Complications encountered in the real

world TB clinicApply your new knowledge of the

“hidden info” on the Xpert computer


The problems

• Silent mutations – Xpert software does not know they are silent

– Interprets as RIF-R

• Smear-negatives (low MTBC DNA)– May falsely detect RIF-R

– May falsely identify NTM as MTBC [rare]

• Dead bugs– Xpert finds DNA; cannot distinguish between

alive and dead organisms




Case 1

• 70 yo man born in Mexico

• Cough, wt loss, night sweats x 2 months

• CXR shows left upper lobe infiltrate

• Sputum is smear positive

• Xpert is ordered


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MTBC detected• 4 probes up RIF-R detected• Probe B (no signals)Caution:• Most common Silent

mutation in probe B

Ct End-ptCt Ct End-pt



Xpert Probes: Coverage of rpoB

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

Codon#

Most common silent mutation

(514 TTT)

Ask your lab which probe resulted in the RIF-R result

29

Big surprise!

• ~20% of all the mutations detected in the rpoB

core region are Silent!(In California and other low MDR areas)

• Most common is 514TTT

• Mutations detectable by probe B:– 70% is this silent mutation

– Disputed mutation: 516TAC, 516TTC,

– RIF-R mutations: 516GTC, 513AAA, GAA, etc.

• Pennan, how to deal with probe B mutations?




Number and Proportion MDR-TB by Country/Region of Origin, CA 2011–2015

• Countries with >35 cases tested for MDR

Country/Region No. %Former Soviet Republics 5 12.2

Laos 6 5.1

Burma 2 3.4

India 12 3.1

Guatemala 5 3.0

Korea (N&S) 7 2.9

Peru 1 2.6

Ethiopia 1 2.0

Philippines 27 1.7

Vietnam 13 1.4

China (incl Taiwan) 7 1.2

United States 13 0.8

Cambodia 1 0.7

Mexico 11 0.6

31

Number and Proportion MDR-TB by Country/Region of Origin, CA 2011–2015

• Countries with >35 cases tested for MDR

Country/Region No. %

PPV (99% spec)

PPV (98% spec)

Former Soviet Republics 5 12.2 93% 87%

Laos 6 5.1 84% 72%

Burma 2 3.4 77% 63%

India 12 3.1 75% 60%

Guatemala 5 3.0 75% 60%

Korea (N&S) 7 2.9 74% 59%

Peru 1 2.6 72% 56%

Ethiopia 1 2.0 66% 50%

Philippines 27 1.7 62% 45%

Vietnam 13 1.4 57% 40%

China (incl Taiwan) 7 1.2 54% 37%

United States 13 0.8 44% 28%

Cambodia 1 0.7 40% 25%

Mexico 11 0.6 36% 22%

32



MDR-TB Cases by Country/Region of Origin and Years in the US, CA 2011–2015

Country/Region

TotalMDR TB

cases

≤ 2 years in US

No. (%)>2 years in US

No. (%)

All Countries* (excl U.S.)

103 30 (3.7) 71 (1.2)

Vietnam* 13 9 (7.9) 3 (0.4)

China*(incl Taiwan)

7 5 (8.8) 2 (0.4)

Philippines* 27 8 (4.0) 19 (1.4)

* Difference is statistically significant 33

Order Xpert or PSQ for Patients with MDR Risk!

MDR risks:

• Prior TB treatment

• Contact to patient with drug resistant TB

• Non-U.S.-born from country with increased MDR risk (≥2% MDR among TB cases in California)

• Arriver to U.S. within 2 years

• HIV positive

Among 42 smear positive MDR cases with MDR risk, 20 did not get Xpert or PSQ on sputum (California, 2012-2016)

34Lowenthal, NTCA/CTCA poster 2018



Probe B Interpretation

• Interpret in clinical context

– Are there risks for DR TB?

– Is there a pressing need to start treatment immediately?

• In this case: born in Mexico, no prior treatment, not a contact probably silent

• Pyrosequencing: 514TTT (Silent mutation)

35

36

Testing smear-negative samples



Case 2

• 30s yo F cough x 4 weeks

• No prior TB treatment

• Born in country with low MDR prevalence; no travel

• Household contact to pan-S case 1 year prior


MTBC DetectedRIF-R detectedHigh CtsΔCt = 33.8-29.7 = 4.1

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MTBC DetectedRIF-R not detectedHigh CtsΔCt = 31.1-28.2 = 2.9

Case 2

• Treated with RIPE

• Clinically improved after 1 week

• Pyrosequencing: no amplification

• DSTs: PanS




Xpert MTB/RIF Performanceon Smear Negative Sputum Compared with

Culture, U.S. patients

Sensitivity Specificity

1 Xpert 2 Xperts

59.3%(16/27)

71.4%(20/28)

99.2%

Luetkemeyer Clin Infect Dis 2016 41

• Should we always test two samples?

Case 3

• 50 yo man born in Mexico, in U.S. for 10 yrs

• 6 weeks of cough, night sweats

• CXR RUL nodular infiltrate

• No MDR contact, no prior treatment

• All smears are negative

• Xpert pending

– Order testing 2 samples to increase sensitivity!?

42




Sample #1 Sample # 2

Conflicting results! Shall I worry false positivity?Ask lab to test another specimen!?

MTBC not detected

MTBC detectedRif Resistance not detected

44

Sample 3 Sample 4

Sample #3 results were indeterminate. Order another test!!Sample #4 results were RIF-R.Hit the jackpot! 4 tests 4 different results!

MTBC detected Rif resistance indeterminate

MTBC detectedRif resistance detected



Problems with testing smear-negatives

• MTBC DNA – Low, very low or not present

– Xpert’s detection limit is about 120 CFU/mL

• Xpert results less reproducible, because:– Reduced amplification: generate fewer amplicons

• PCR efficacy varies between runs

– Reduced probe binding: generate less signals • Probe’s binding efficacy varies more among probes

• Increased Δ Ct max: Increased false RIF-R detection


Follow-up actions• MTBC not detected

– Does not rule out MTBC. Consider second Xpert– Wait for culture results.

• MTBC detectedRifampin resistance not detected– Most reliable

• MTBC detectedRifampin resistance detected– Need to be confirmed by sequencing.

• MTBC detectedRifampin resistance indeterminate [not discussed today]– Likely happens when testing smear-negatives.– If this is a first test, test 2nd specimen. – If this is a 2nd test. Wait for culture results.




Two probes positive = MTBC?

• This is a dangerous rule!– Especially when the sample is smear-negative

• Two probes pos = 3 or more mutations– Very RARE to have ≥ 3 mutations within 81 bp

– Out of 3754 tested, 2 samples had 3 mutations (MDL data)• None detected by 3 probes!

• One detected by 2 probes (A & B)

• One detected by 1 probe (D)


When should NTM be suspected?

• Smear-negative

– Only two probes are positive. Xpert reports MTBC and RIF-R detected.

• Smear-positive

– Multiple Xperts: MTBC not detected




Can we use Xpert for detection of relapses?


Case 4

• 70 yo man from China

• 5 years ago treated for cavitary TB with RIPEcured

• Presents to ED with 2 days of SOB, no fever, night sweats or weight loss (weight gain)

• CXR with bilateral pleural effusions

• Smear neg, Xpert positive, RIF-R (Probe A)

• No sequence detected by PSQ

• Culture grew a rapid grower

• DNA of the isolate from 5 years ago, showed rpoB 511 CCG (detectable by probe A)

50Kelly et al Am J Resp Crit Care Med 2014



Case 4

• History concerning for MDR

• Clinical presentation atypical for TB, more compatible with CHF

• Treated for 2 weeks then stopped

• All cultures negative, including 12 mos later

• Negative cultures and same rpoB mutation as prior --> dead bugs?

51Kelly et al Am J Resp Crit Care Med 2014

Xpert positivity on treatment

• Insert figure from publication…

Friedrich, Lancet Respir Med 2013; 1: 462–70

Weeks after treatment initiation

27%

52



Don’t get Xpert after treatment start or in previously treated patients

• Xpert cannot determine alive vs dead

• Xpert can remain positive for years after curative treatment

• Package insert recommends against Xpert in anyone treated for >3 days

• If you order it, only helpful if different RIF result from prior (e.g., a previously pan-S case has a probe E RIF-R result)

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Summary Points

• Order Xpert!

• If results surprise you, talk to your lab!– Ask for probe and Ct info

• Interpret in clinical context– How likely is it that this patient has TB and DR-TB?

• MDR risk factors? Prior treatment, contact to MDR case, born in country with high MDR prevalence?

– Can you wait for culture results?

• Get rpoB sequencing for all RIF-R specimens– If smear negative (Ct > 28), wait for culture




Acknowledgments• Ed Desmond

• Phil Lowenthal

• Neha Shah

• MDR Service

• Microbial Disease Lab

55

Pennan Barry

Gayle Schack

(Ret)

Kristen Wendorf

Lisa True

Phil Lowenthal

Jenny Flood

LeslieHenry

Neha Shah

Not pictured: Shereen Katrak

Christy Pak (Ret)

Wendy Cheung

Grace Lin

Lucy Pham

Terry Weber

Stephen Yu

• Zachraias Zachraias

• Kitty Reiher

• Amelia Alonis

Thank You!

• [email protected]

• [email protected]


mailto:[email protected]

mailto:[email protected]



Resources

• California Microbial Diseases Lab PSQ: https://www.cdph.ca.gov/Programs/CID/DCDC/CDPH%20Document%20Library/MDL-Pyrosequencing-for-XDR-TB-Screening.pdf

• Curry/CDPH MDR TB Survival Guide Lab Chapter: http://www.currytbcenter.ucsf.edu/products/drug-resistant-tuberculosis-survival-guide-clinicians-3rd-edition/chapter-3-laboratory

• CDC TB Lab: https://www.cdc.gov/tb/topic/laboratory/default.htm

• NTCA/APHL Consensus Statement on Xpert for release from isolation: http://www.tbcontrollers.org/docs/resources/NTCA_APHL_GeneXpert_Consensus_Statement_Final.pdf

• Khan academy PCR unit: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-

sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

• Xpert MTB/RIF Package Insert: http://www.cepheid.com/administrator/components/com_productcatalog/library-files/f625a0c86cd73ce46ba391c24a3eabc4-3a9ca7ef6b9b88dc736e5a437f41eb3a-Xpert-MTBRIF-ENGLISH-PackageInsert-301-1404-Rev-C.pdf

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rpoB Facts

• Most common mutation conferring RIF-R

– rpoB 531TTG—detectable by Probe E

• 2nd most common mutation conferring RIF-R

– rpoB 526TAC & 526GAC—detectable by probe D

• Most common silent mutation

– rpoB 514TTT—detectable by probe B (>20% of all mutations)

• Disputed mutations are not common, but are detectable

– But you would not know unless sequenced.

• Not common to have 2 mutations, very rare to have 3 mutations. Even more rare to have 3 mutations detected by 3 probes.

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https://www.cdph.ca.gov/Programs/CID/DCDC/CDPH Document Library/MDL-Pyrosequencing-for-XDR-TB-Screening.pdf

http://www.currytbcenter.ucsf.edu/products/drug-resistant-tuberculosis-survival-guide-clinicians-3rd-edition/chapter-3-laboratory

https://www.cdc.gov/tb/topic/laboratory/default.htm

http://www.tbcontrollers.org/docs/resources/NTCA_APHL_GeneXpert_Consensus_Statement_Final.pdf

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

http://www.cepheid.com/administrator/components/com_productcatalog/library-files/f625a0c86cd73ce46ba391c24a3eabc4-3a9ca7ef6b9b88dc736e5a437f41eb3a-Xpert-MTBRIF-ENGLISH-PackageInsert-301-1404-Rev-C.pdf



rpoB codon numbering change

• Historically rpoB codons numbered with E. coliAll other loci numbered according to M. tuberculosis

• Change to M. tuberculosis numbering to align with whole genome sequencing data

• Good news: just subtract 81

– 531 450 526 445 514 433 516 435

• MDL has started using MTBC numbering (As of 8/1/18)

– Codon conversion info provided in comment


Xpert Ultra

• WHO recommends the use of Ultra.

• 2 specific targets for detection of MTBC– IS6110 & IS1081

• Increased sensitivity for MTBC detection– 66% (G4) vs 79% (Ultra)

• Added a step to analyze melting temperature for identification of specific mutations.– 4 “sloppy” MB probes for detecting rpoB mutations.

– F514F Silent mutation is not detected. Great news!

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