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Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
Curry International Tuberculosis Center 1
Molecular Diagnostics for Molecular Diagnostics for Tuberculosis:Tuberculosis:
What are NAATs and What are NAATs and How Do You Use Them?How Do You Use Them?
Gisela Schecter, MD, MPHCalifornia Department of Public Health
January 21, 2015
OutlineOutline• What are NAATs?• What is the added value of NAATs?• Specific NAATs that are commercially
available for detecting tuberculosis (TB)Li it ti f NAAT• Limitations of NAATs
• Performance of NAATs in patients who are AFB smear positive and smear negative
• Use of NAATs in California• Case examples• Introduction to molecular tests for DST
The Treatment of TB is RigorousThe Treatment of TB is Rigorous
• The clinical goal is to treat the disease if present, as early as possible, to avoid death and disability, but not expose patients to the risk of serious side effects of treatment if TB is not present
• The public health goal is to find and treat cases as early as possible in order to interrupt transmission to others
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
Curry International Tuberculosis Center 2
Diagnosing TB before NAATsDiagnosing TB before NAATs• 30 years ago, only 2 tests available
o AFB smear• False Negatives: Missed 40% of culture (+)
active TBF l P iti NTM• False Positives: NTMs
o Culture on solid media• Average time to positive growth, 3-6 weeks• Still needed to do biochemical tests to
determine if TB or NTM, another several weeks
Clearly needed more sensitive, specific and faster tests
What is a NAAT?What is a NAAT?• NAAT: Nucleic Acid Amplification Test• Can be done on the direct clinical specimen;
sputum, CSF, lymph node aspirates, etc.• More sensitive and more specific than the AFB smear• Results available within hours to a few daysy• CDC updated guidelines for NAATs in MMWR,
Jan. 16, 2009: o “CDC recommends that NAA testing be performed on
a least one respiratory specimen from each patient with signs and symptoms of pulmonary TB …”
[if result will impact clinical or public health actions]
• Why aren’t we using NAATs more frequently?
What is the What is the added added value value of a NAAT?of a NAAT?
• For AFB smear (–) patients:o Ability to confirm rapidly the presence of M.tb in
50-80% of AFB smear-negative, culture-positive specimensIf b th th AFB d th NAATo If both the AFB smear and the NAAT are negative, the likelihood of TB becomes very low
• For AFB smear (+) patients: o Greater positive predictive value (>95%) with AFB
smear-positive specimens in settings in which NTM (non tuberculous mycobacteria) are common
o If NAAT negative, in many instances can obviate the need for contact investigation
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Available NAATsAvailable NAATs
• Amplified Mycobacterium tuberculosis Direct Test (MTD, Gen-Probe)
• Xpert MTB/RIF (often called GeneXpert)
P i (PSQ)• Pyrosequencing (PSQ)
• “Home brews”
The GenThe Gen--Probe MTD TestProbe MTD Test• The first FDA approved NAAT for M.tb• Approved in 1995 for AFB smear positive patients• Enhanced MTD (E-MTD) test approved in 1999 for
AFB smear negative specimens• TMA (Transcription-mediated amplification)
T t RNA f M tb• Targets rRNA of M.tb• Sensitivity of E-MTD in culture (+) specimens
o Smear negative = 70%o Smear positive = 97%
• Specificity = 98%• Somewhat labor intensive for the microbiologist• Often not available in hospital labs
Enhanced Amplified Mycobacterium Tuberculosis
Direct Test (“E-MTD”)(Gen-Probe, San Diego, CA)
9
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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XpertXpert MTB/RIFMTB/RIF• FDA approved in 2013 for detection of M.tb in
clinical specimenso Can be used for both AFB smear positive and smear
negative specimenso Using culture as the gold standard, recent meta-
analysis* showed pooled sensitivity estimates of 98%analysis showed pooled sensitivity estimates of 98% for smear (+), and 68% for smear (–) specimens
o Specificity estimates were 99%o Because most studies included in the meta-analysis
were in high TB burden countries, sensitivity may be lower in US
o (Also tests for Rifampin resistance)
* Chang K, et.al. Rapid and effective diagnosis of tuberculosis and rifampin resistance with Xpert MTB/RIF assay: a meta-analysis. J Infect 2012
Xpert MTB/RIF (2)Xpert MTB/RIF (2)• Test performance in a low TB incidence setting:
Montreal, Canada*• 502 consecutive patients in a university hospital
affiliated TB clinico Most with abnormal CXRs, 8% symptomatico 25 had culture (+) TBo 25 had culture ( ) TBo Overall sensitivity of Xpert was 46%; 86% if smear
(+), 28% if smear (-)o Specificity was 100%o Those patients with culture (+)/Xpert (-) took
twice as long for cultures to grow and had minimal disease
*Sohn H, et.al. Xpert MTB/RIF testing in a Low Tb Incidence, High-Resource Setting: Limitations in Accuracy and Clinical Impact. CID, 2014
Xpert MTB/RIF (3)Xpert MTB/RIF (3)
• Automated cartridge-based test
• Uses PCR technology in a self-contained capsule, so contamination is eliminated
• Results available in under 2 hours
• Platform is widely available in many hospitals
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Pyrosequencing (PSQ)Pyrosequencing (PSQ)
• Has been available to us in California since 2012
• Although usually used for detecting mutations associated with drug resistance, is also a NAAT
• Can be used on clinical specimens if at least AFB smear positive 1+
• Result in 1 day (although usually batched twice a week at the CA MDL lab, Grace Lin will run it for you any workday if urgent)
RealReal--Time PCRTime PCR
• 2 componentso PCRo A system to monitor PCR product
• Fluorophore-labeled probes
iCyclerIQ5
• An optical device to detect fluorescence
• No post-PCR manipulationso Fast
• when PCR is done, results are ready for interpretation
o No amplicon contaminations
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Limitations of NAATsLimitations of NAATs• Inhibitors
o Direct clinical specimens can contain inhibitors that prevent amplification
o Results in false negative resultso Xpert MTB/RIF includes a sample processing control
(SPC) to monitor for presence of inhibitorso MTD procedure manual includes instructions on howo MTD procedure manual includes instructions on how
to test for inhibitors o If an AFB smear (+) specimen is NAAT negative, make
sure the lab checked for inhibitors, and repeat the NAAT on another specimen
• Also, none of these tests will tell you if organisms are alive or deado Should NOT be used to follow patients on treatmento Often NAAT will stay positive long after cultures are
negative
Effects of NAAT Use on Effects of NAAT Use on Clinical Care in CaliforniaClinical Care in California
• Since 2010, information about NAAT usage has been collected on the RVCT
• We looked at the utilization of NAAT from 2010-2013
• Overall, 39% of subsequently culture positive patients had a NAAT reported before culture results were reported
• Did use of a NAAT affect clinical care?
Did patients with NAAT start treatment earlier than those without NAAT?
Group NAAT Median time*
No NAAT Median time*
P-value for Wilcoxon rankMedian time*
(N)Median time*
(N)Wilcoxon rank
sums test
All * 3.0 (1960) 14.0 (3101) <0.0001
Smear-positive*
2.0 (1728) 4.0 (1580) <0.0001
Smear-negative*
10.0 (232) 26.0 (1521) <0.0001
*Time (days) between date of earliest specimen collection and treatment initiation
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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How rapidly How rapidly were NAATs were NAATs reported? reported?
• How rapidly were tests reported?• Reported times vs. real world
How rapidly were NAATs reportedHow rapidly were NAATs reported? (2) ? (2)
Among 1878 cases:• Had NAAT• Started treatment after specimen
collection• Started treatment before culture reported
Median TAT = 3 daysMean = 3.77 daysIQR = 2-4 days
*NAAT TAT: working days between NAAT specimen collection and NAAT report date.
Impact of NAATs on Utilization of Impact of NAATs on Utilization of AIIRs and Clinical CareAIIRs and Clinical Care
• CDC sponsored retrospective multisite study* found using NAATS was associated with significant reductions in:o Use of AIIRso Use of diagnostic procedures such as
b h i d CTbronchoscopies and CTso Initiation of CIs for smear positive, culture
negative suspectso Time to diagnose of TB in smear positive caseso Duration of presumptive TB treatment for patients
who did not have TB
*Marks SM, et.al. The health-system benefits and cost-effectiveness of using M.tb direct NAAT to diagnose TB disease in the US. Clin Infect Dis 2013
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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TB or Not TB? Case #1TB or Not TB? Case #1
48 year old man with COPD• 1 month cough weight loss• 1 month cough, weight loss
• Jail inmate
• Rx for TB 15 years ago
TB or Not TB? Case #1TB or Not TB? Case #1
48 year old man with COPD• 1 month cough, weight lossg , g
• Jail inmate
• Rx for TB 15 years ago
Smear positive, is this TB?
• 150 jail contacts, now what?
Laboratory Diagnosis: NAATLaboratory Diagnosis: NAAT
• This CXR could be old TB, current reactivation, or a new process
• If NAAT positive, the diagnosis is confirmed
o (Previous treatment 15 years ago will not give a positive result now)
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Laboratory Diagnosis: Laboratory Diagnosis: NAAT (2)NAAT (2)
• This CXR could be old TB, current reactivation, or a new process
• If NAAT positive, the diagnosis is confirmed
• A NAAT test, if negative, would make it much more likely that this is an NTM, quite common in COPD
Laboratory Diagnosis: Laboratory Diagnosis: NAAT (3)NAAT (3)
• This CXR could be old TB, current reactivation, or a new process
• If NAAT positive, the diagnosis is confirmed• A NAAT test if negative would make it• A NAAT test, if negative, would make it
much more likely that this is an NTM, quite common in COPD
• Could this be a false negative NAAT? o Check for inhibitors
o Repeat the NAAT on another specimen
TB or Not TB? Case #1 TB or Not TB? Case #1
48-year-old, male with COPD• 1 month cough, weight
lossloss
• Jail inmate
• Rx TB 15 years ago
What if he was smear negative instead?
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Laboratory Diagnosis:Laboratory Diagnosis:NAAT in Smear Negative PatientNAAT in Smear Negative Patient
• About 70 % of smear negative specimens that subsequently grow M.tb will be NAAT positive by the E-MTD test or Xpert MTB/RIFpositive by the E MTD test or Xpert MTB/RIF
• If you have a high index of suspicion for TB, a NAAT test should be ordered
Case #2Case #2
• 56 year old white woman from a small town in northern California
• Never traveled out of US, no known TB exposure, no case of TB in her county for e posu e, o case o e cou y opast 5 years
• 40 pack year smoker, cough for several months with recent weight loss
• Abnormal CXR
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Case #2 Case #2 (2)(2)
• Sputa were ordered and were AFB (+) X 3• Health Department consulted and NAAT
suggested• NAAT negative!NAAT negative!
Is this a false negative NAAT or does this woman without any risk factors have TB?
How to answer this question?
Case #2 Case #2 (3)(3)
• Lab was requested to check for inhibitors, none found
• NAAT was repeated on another smear (+) specimen, also negative
• Out of caution, treatment with RIPE begun, but county held off on contact evaluation beyond the immediate family (one person)
• Culture grew in 8 days, identified as M. kansasii, an NTM
• No further public health action needed
Case #3Case #3
• 44 year old man originally from Mexico applying for work as maintenance worker at a hospital
• TST (+) at 28 mm• 20 pack year history of smoking• C/o some cough for several weeks,
occasionally productive, but no fever, sweats of weight loss
• Abnormal CXR
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Case #Case #3 (2)3 (2)
• Sputum X 3 ordered for AFB; all 3 negative• Scheduled for bronchoscopy because of
concern for malignancy• Xpert MTB/RIF ordered; Positive!• Xpert MTB/RIF ordered; Positive!• Bronchoscopy canceled, RIPE begun
• CXR 2 months later:
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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Case #Case #3 (3)3 (3)
• Marked improvement in CXR• Culture positive after 21 days, M.tb
• Patient spared invasive procedure, fewer persons potentially exposed to TB
SummarySummary• NAAT technology for diagnosing TB is
underutilized• NAATs are more sensitive than AFB smears
for detecting TB• Time for treatment initiation is shorter when• Time for treatment initiation is shorter when
NAATs are used• More frequent use of NAATs for TB diagnosis
may affect both clinical care and public health action
• Recent FDA approval of Xpert MTB/RIF will likely increase NAAT use
The Treatment of TB is The Treatment of TB is RigorousRigorousAnd MDR TB And MDR TB Treatment is Even More Treatment is Even More soso
• The clinical goal is to treat the disease if present, as early as possible, to avoid death and disability, but not expose patients to the risk of serious side effects of treatment ifthe risk of serious side effects of treatment if TB is not present
• The public health goal is to find and treat cases as early as possible in order to interrupt transmission to others
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
Curry International Tuberculosis Center 14
Drug Susceptibility Testing (DST)Drug Susceptibility Testing (DST)
• Phenotypic DSTs:o Gold standard is agar proportion method on
solid media: very slow and seldom used nowo MGIT liquid system is now commonly used. Takes
about 1 week after a positive culture for resultsabout 1 week after a positive culture for results
• Molecular DSTs:o Pyrosequencingo Line probe assays (Hain test)o Xpert MTB/RIFo MDDR Service
Pyrosequencing (PSQ)Pyrosequencing (PSQ)• Available at the California state lab since
2012.• Performed on cultures or clinical specimens if
at least 1+ AFB positiveR lt i 1 d ( lth h ll b t h d• Result in 1 day (although usually batched twice a week at the CA MDL lab, Grace Lin will run it for you any workday if urgent)
• Tests for INH (inhA, katG, ahpC) and Rifampin (rpoB) resistance mutations, as well as mutations found in resistance to quinolones (gyrA)and injectable drugs (rrs)
PSQ (2)PSQ (2)
• Sensitivity: Will pick up about 90 % of INH resistant isolates and over 95% of Rifampin resistant isolates
• Specificity is over 95% for both INH and Rif Spec c y s o e 95% o bo a dresistance
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
Curry International Tuberculosis Center 15
HainHain TestTest• Commercially available and used in much
of the world• Not FDA approved in the US• Line Probe Assay detects INH and Rifampin• Line Probe Assay, detects INH and Rifampin
resistance. Second generation test also looks for resistance to quinolones and injectable drugs
XpertXpert MTB/RIFMTB/RIF• Xpert MTB/RIF does not just detect M.tb
DNA; it also tests for rifampin resistance using a molecular beacon technology
• In Chang’s meta-analysis, using cultures and C a g s e a a a ys s, us g cu u es a dphenotypic drug susceptibility tests as the gold standard, the Xpert assay had a pooled sensitivity of 95% and specificity of 98% for detecting rifampin resistance
*Chang K, et.al. Rapid and effective diagnosis of tuberculosis and rifampin resistance with Xpert MTB/RIF assay: a meta-analysis. J Infect 2012
Molecular Diagnostics for TB: January 21, 2015What are NAATS and How Do You Use Them?
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MDDR Service from CDCMDDR Service from CDC• Launched in 2010 by CDC• Molecular Detection of Drug Resistance• Intended primarily for patients with MDR TB who
will be treated with second line drugs• Uses DNA sequencing to find mutations
associated with o INH and Rifampin, o Ethambutol and PZA, o Fluoroquinolones (Levo/Moxi) o Aminoglycosides (Kanamycin/Amikacin) and
Capreomycin• Excellent turn around time of < 48 hours• Drawback: Done on positive cultures, not clinical
specimens
AcknowledgementsAcknowledgements
• Pennan Barry• Amit Chitnis• Ed Desmond• Grace Lin• Lisa Pascopella• Gianna Peralta• TB Branch California Department of Public Health
MOLECULAR DIAGNOSTICS FOR TB: WHAT ARE NAATS AND HOW DO YOU USE THEM? WEBINAR – JANUARY 21, 2015
Curry International Tuberculosis Center, UCSF
300 Frank Ogawa Plaza, Suite 520 Oakland, CA 94612 Phone (510) 238-5100 Email [email protected]
Warmline TB Medical Consultation: 877-390-6682 (toll-free) http://www.currytbcenter.ucsf.edu
REFERENCES Molecular Diagnostics for TB: What Are NAATs and How Do You Use Them? Gisela Schecter, M.D., M.P.H.
Chang K, et.al. Rapid and Effective Diagnosis of Tuberculosis and Rifampin Resistance with Xpert MTB/RIF Assay: A Meta-Analysis. J Infect 2012, doi:10.1016/j.jinf.2012.02.012 http://tbevidence.org/wp-content/uploads/2012/04/Chang-J-Infect-2012.pdf
Marks SM, et.al. The Health-System Benefits and Cost-Effectiveness of Using Mybcobacterium tuberculosis Direct Nucleic Acid Amplification Testing to Diagnose Tuberculosis Disease in the United States. Clin Infect Dis 2013. http://cid.oxfordjournals.org/content/57/4/532.full.pdf+html
Sohn H, et.al. Xpert MTB/RIF Testing in a Low Tuberculosis Incidence, High-Resource Setting: Limitations in Accuracy and Clinical Impact. Clin Infect Dis, 2014;58(7):970-976. http://cid.oxfordjournals.org/content/early/2014/02/13/cid.ciu022