BENEFITS AND LIMITATIONS OF MOLECULAR TESTING

Transcription

1 BENEFITS AND LIMITATIONS OF MOLECULAR TESTING Test performance/ selection in various settings Edward Desmond, Ph.D., D (ABMM) California Dept. of Public Health

2 Problem Pascopella, et al Laboratory reporting of tuberculosis test results and treatment initiation in California. JCM 47:4209 Median time to initiation of therapy: AF smear + patients: 1 day AF smear neg patients: 22 days

3 How increased NAA testing for TB will help NAA testing is more sensitive than acid-fast microscopy NAA will identify some TB patients more quickly (up to 3 weeks sooner)

4 Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis MMWR 58(1): 7-10 January 16, 2009 CDC recommends that NAA testing be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary tuberculosis for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities.

5 Looking more closely at CDC guideline NAA is recommended from each patient with signs and symptoms of pulmonary tuberculosis What about the sputum or bronchoscopy sample that arrives in the laboratory with a request for routine, fungal, and AFB culture? Does that patient really have signs and symptoms of tuberculosis?

6 Close look at CDC guidelines, cont d NAAT is recommended for patients for whom a diagnosis of TB is being considered but has not been established However, some specimens are followup specimens from patients already diagnosed with tuberculosis. These do not need NAA.

7 Close look at CDC guidelines, cont d NAA testing is recommended for TB suspect for whom the test result would alter case management or TB control activities What about an acid-fast smear positive patient who has cavitary lung disease on X-ray and other classic symptoms? NAA would not likely alter case management or TB control activities

8 Implementing the new CDC guidelines Lab not likely to know Whether patient really has signs and symptoms of TB Whether TB diagnosis has already been established Whether test result would alter case management of TB control activities NAA must be ordered by a physician who does know answers to above questions

9 Assuring physician orders appropriate laboratory testing is part of preanalytical quality assurance.

10 How can we, clinicians, laboratorians, and TB control program staff promote increased use of NAA testing?

11 Detection of Drug Resistance Mutations Drugs of interest: For XDR screening INH, RIF, KAN, AMK, CAP, fqs Targeted Genes katg, inha promoter for INH rpob for RIF rrs for KAN, AMK & CAP gyra for Quinolones G Lin PSQ

12 GenoType MTBDRplus 1) DNA Extraction 3) Hybridization 2) Amplification by PCR 4) Evaluation

13 Examples of GenoType MTBDRplus Results

14 FIND Evaluation Study MTBDRplus assay performed on 536 consecutive AFB+ sputum specimens 100 AFB- specimens also tested Specimens cultured on MGIT, DST on 7H11 Primary outcome: performance for INH R, RIF R and MDR TB on AFB+ specimens compared to conventional DST Richard O Brien Foundation for Innovative New Diagnostics

15 Results of MTBDRplus Testing 97% of specimens had interpretable results within 1-2 days Sensitivity, specificity, PPV, and NPV for: RIF-R: 99%, 99%, 98%, 100% INH-R: 94%, 100, 99%, 98% MDR TB: 99%, 100%, 100%, 100% Accurate results for 14/15 AFB-/cult+ sputum specimens

16 Conclusions - MTBDRplus assay Overall performance is superior to conventional culture/dst: speed, accuracy, interpretable results, throughput Cost may be less than culture/dst Can substantially reduce need for culture/ DST when screening for MDR TB May be easier to establish than culture

17 Detection of Mutations with a Molecular Beacon (Loop portion containing wildtype SQ) Mutant Sequence + Wildtype Sequence Amplicon Loop Fluorophore Heat Fluorophore Light Quencher Molecular Beacon (off) Hybrid (Molecular Beacon - On)

18 Detecting INH resistance using molecular beacons 3 years experience with cultures and specimen sediments Molecular beacon result Mutation detected Mutation not detected Beacons test inconclusive Culture INH resistant Culture INH susceptible

19 Detecting rifampin resistance using molecular beacons 3 years experience with cultures and specimen sediments Molecular beacon result Mutation detected Mutation not detected Beacons test inconclusive Culture rifampin resistant Culture rifampin susceptible

20 MB Performance (% Agreement between MB and phenotypic drug results) INH RIF Cultures 98.4% 99% Sediments 93.6% 94.9% Overall 96.1% 97.2%

21 Molecular beacons & realtime PCR advantage Amplification and detection of the amplified product take place inside a sealed tube or microtiter plate No chance that any of the numerous pieces of amplified DNA will contaminate the lab & cause false positive reactions in the future Detection of the amplified product takes place during the amplification saves time and work

22 MB Stories Mtb & M. chelonae mixed culture Unable to isolate; MDRTB identified by MB. Abdominal lesion, HIV+, Smear +, Culture- TB identified by MB, no mutations detected. Use standard regimen to treat. Hmong refugees MDRTB were identified in <1 d of specimen receipt. Proper treatment initiated immediately. A white teenage female in a boot camp MDRTB identified in <1 d of specimen receipt. G Lin UCSD XDR

23 Limitations of molecular beacons o Beacons and line probes detect absence of wild-type sequence: doesn t always mean drug resistance: o Silent mutations o Mutations causing small increases in MIC--?clinical significance? o Molecular beacons good for detecting absence of wild type sequence in a small (about 15 bases) region of a gene with a highly conserved sequence o Fine for rpob, katg, inha promoter region o Not good for detecting mutations associated with resistance if the normal gene sequence is variable (e.g. gyra 95) o Not good for detecting mutations that are spread out over a wide region (e.g. pnca mutations associated with PZA resistance)

24 Pyrosequencing Primer DNA template datp degrades dttp degrades dgtp chemiluminescence G dctp degrades datp chemiluminescence GA Copyright motifolio.com

25 G Lin PSQ

26 Instruments needed for pyrosequencing o Thermocycler for PCR. o Plate shaker to keep streptavidin beads in suspension and allow binding of biotin-dna onto beads. o Vacuum work station to capture beads. o Heating block to heat DNA. When cooling, SQ primer anneals to DNA template. o Pyrosequencer to do sequencing work.

27 Sequencing other genes related to drug resistance o pnca: to detect resistance to pyrazinamide o embb: to detect resistance to ethambutol o More data are needed to show which mutations are or are not associated with drug resistance

28 Comparison of MB & PSQ Interpretation of silent mutations or mutations not conferring R MB Mutation present or not Misinterpret as R PSQ Exact SQ Does not misinterpret Objectivity on interpreting results More subjective More objective Hand-on time 1.5 hr, simple 2.5 hr, more steps Total test time 3.5 hr 5 hr 28

29 Advantages of MB over PSQ Easier to perform, less steps. Less hand-on time. Shorter total testing time. If no mutations present, easy to screen and eliminate susceptible strains. This may yield savings on materials and labor. G Lin PSQ

30 Strategy If affordable to have both MB & PSQ, Use MB to screen INH & RIF % may not have mutations. No further required If mutations detected by MB, PSQ of 7 targets will follow. If affordable for only one instrument, use PSQ. G Lin PSQ

31 Pyrosequencing: what to look forward to Same day detection of patients with extensively drug-resistant tuberculosis (XDRTB) = resistant to INH, rifampin, injectable drug, and fluoroquinolone Greater confidence when a mutation is detected, that it actually confers resistance Ability to test for resistance to other drugs, particularly those that give less reliable results with culture-based testing, e.g. ethambutol and pyrazinamide G Lin PSQ

32 Correlation of specific mutations with level of drug resistance MIC values may differ, even for organisms with the same resistance-conferring mutations Culture-based testing will still be required, or at least desired Molecular and culture-based results can confirm and complement each other Knowing exact mutated sequence can provide some guidance for treatment pending susceptibility testing results

33 What is the future of laboratory-developed tests? CLIA rules for verification Role of FDA in regulating products that are used in laboratory-developed tests Process for FDA clearance of TB diagnostics

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35 Thank you