1 Laboratory: Molecular TestingSean R. Toney Immigrant, Refugee, and Migrant Health Branch Division of Global Migration and Quarantine Centers for Disease Control and Prevention Panel Physician Training Summit March 2017 National Center for Emerging and Zoonotic Infectious Diseases Division of Global Migration and Quarantine

2 Learning Objectives Identify types of molecular laboratory tests.After this session, participants should be able to: Identify types of molecular laboratory tests. Understand the use of molecular laboratory tests for TB and molecular assays for gonorrhea. Understand the limitations of molecular testing for TB and gonorrhea.

3 Types of TB Molecular Laboratory Tests

4 TB Molecular Laboratory TestsTuberculosis is one of the leading infectious diseases in the world Annually: 2 million deaths; ~ 10.4 million new (incident) TB cases worldwide Mycobacterium tuberculosis has a slow growth rate Isolation, identification, and drug susceptibility testing of this organism (and other clinically important mycobacteria) can take weeks Several molecular methods have been developed in the past few years These methods can potentially reduce diagnostic time from several weeks to days. Source: WHO Global TB Report and CDC

5 TB Molecular Laboratory TestsTraditional laboratory procedure for clinical specimens involves: Specimen decontamination and digestion, microscopic examination for the presence of acid-fast bacilli (AFB), isolation of the organism by culture, identification, and drug susceptibility testing of the recovered organism. Nucleic acid amplification (NAA) methods allow for detection of mycobacterial DNA or RNA directly from the specimen before the culture results are available Decontamination and Digestion AFB Microscopic Examination Culture Isolation Identification Drug Susceptibility Testing

6 TB Molecular Laboratory TestsDiagnostic Identification Drug Resistance Source: CDC PHIL

7 Definitions Nucleic acid amplification test (NAAT)Molecular technique designed to amplify sequences that are specific for the organism being detected (e.g., a virus, bacterium, fungi, parasites) Two most common types Polymerase chain reaction (PCR) Transcription-mediated amplification (TMA) Tissue, blood, or other body fluids

8 Definitions Polymerase chain reaction (PCR)Technique which utilizes genus-specific (species-specific) primers to amplify a single copy or multiple copies of a DNA sequence specific to the M. tuberculosis complex IS6110 katG gene 16S ribosomal RNA gene Transcription-mediated amplification (TMA) Technique that utilizes a two enzyme amplification system that drives reaction s of RNA polymerase and reverse transcriptase Can amplify either DNA or RNA Multiple targets can be amplified Isothermal

9 Definitions Clinical Specimen Isolate Direct Detection ProbeMaterial taken directly from the patient (e.g., sputum, CSF, pleural fluid); may be “raw” specimen or may be “processed” specimen (e.g., sediment) Isolate Organism isolated (i.e., grown) from culture of a clinical specimen (e.g., an LJ tube with MTBC growth) Direct Detection Detection of RNA or DNA sequences of interest in organisms present in a clinical specimen; currently requires nucleic acid amplification (NAA) Probe A piece of DNA that hybridizes specifically to a target nucleic acid sequence

10 Types of Molecular Laboratory TestsPCR-based sequencing Technique targets rpoB gene Mutations associated with rifampin (RIF) resistance are concentrated in a very short segment of the gene Line probe assay (e.g., INNO-LiPA® MYCOBACTERIA v2) Simultaneous detection and identification of the genus Mycobacterium and 16 different mycobacterial species Based on the nucleotide differences in the 16S-23S rRNA spacer region Can be performed from either liquid or solid culture < 48 hours Source: Fujirebio

11 Types of Molecular Laboratory TestsDNA Probes Identification of clinically important mycobacterial species, including M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae Tests are based on species-specific DNA probes that hybridize with rRNA released from bacteria ~2 hours DNA Microarrays Technology initially for identification of mycobacterial species Also used for rapid detection of mutations associated with resistance to TB drugs Based on hybridization of labeled PCR amplicons from bacterial colonies to a DNA array containing nucleotide probe ~4 hours

12 Types of Molecular Laboratory TestsAMPLICOR® (Roche Diagnostic Systems, Inc.) Test based on PCR Mycobacterial DNA is amplified with genus-specific primers formulated on the basis of the 16S rRNA gene ~6.5 hours Xpert® (Cepheid) Real-time PCR using molecular beacons Detects MTB complex and RIF resistance All-in-one test cartridge ~2 hours Source: Cepheid

13 Types of Molecular Laboratory TestsGenoType MTBDRplus (Hain Lifescience) Molecular line probe assay (LPA) containing probes specific for MTB complex Also has probes for rifampin (RIF) resistance-conferring mutations and a subset of mutations for isoniazid (INH) resistance ~ 6-7 hours Source: Hain Lifescience

14 Types of Molecular Laboratory TestsDetermined by: Laboratory capacity Resources Personnel Volume / demand Assay cost / price per test Assay throughput Space

15 Use of Molecular Laboratory Tests

16 Use of Molecular Laboratory TestsSputum specimens collected on 3 different days for AFB smear and culture Perform NAAT on the first specimen collected, or the first smear-positive specimen Additional specimens if needed NAAT should be performed according to the manufacturer's instructions or a validated standard operating procedure Interpret NAAT results in correlation with the AFB smear results Source: MMWR :7-10

17 Use of Molecular Laboratory TestsThe positive predictive value of FDA-approved NAATs for TB is >95% in AFB smear-positive cases If the NAAT result is Positive and the AFB smear result is Positive Presume the patient has TB and begin TB treatment while waiting for culture results. If the NAAT result is Positive and the AFB smear result is Negative Use clinical judgment to decide whether to start TB treatment, while waiting for results of culture. Consider testing an additional specimen to confirm the NAAT result. A patient can be presumed to have TB, pending culture results, if two or more specimens are NAAT Positive. Source: MMWR :7-10

18 Use of Molecular Laboratory TestsIf the NAA result is Negative and the AFB smear result is Positive A test for inhibitors should be performed 3% - 7% sputum specimens contain inhibitors that prevent or reduce amplification and cause false-negative NAAT results. An additional specimen should be tested If inhibitors are detected NAAT will be of no diagnostic help for this specimen. Use clinical judgment to decide whether to start TB treatment, while waiting for results of culture. If inhibitors are not detected Use clinical judgment to decide whether to start TB treatment A patient can be presumed to have an infection with a NTM if a second specimen is smear Positive and NAAT Negative, and no inhibitors are detected. Source: MMWR :7-10

19 Use of Molecular Laboratory TestsIf the NAAT result is Negative and the AFB smear result is Negative Use clinical judgment to decide whether to start TB treatment, while waiting for results of culture and/or other diagnostic tests. Currently available NAATs are not sufficiently sensitive (detecting 50%--80% of AFB smear-negative, culture-positive pulmonary TB cases) to exclude the diagnosis of TB in AFB smear-negative patients suspected to have TB. Source: MMWR :7-10

20 Use of Molecular Laboratory TestsNAATs could provide important information to the clinician Although NAATs have been approved for respiratory specimens, they have also been used to test several types of non respiratory specimens Not approved/validated with non respiratory specimens NAATs may be useful for early identification of MTB from all specimen types (except for blood) grown in liquid cultures

21 Use of Molecular Laboratory TestsIncreasingly becoming standard of practice Does not replace clinical judgment for therapy or isolation practices (less than perfect sensitivity and specificity) Performance improves with increased clinical suspicion of TB Potential benefits – impact on therapy, public health measures, respiratory isolation, invasive procedures MMWR :7-10

22 Intended Use of Xpert® Source: WHO “Rapid Implementation Xpert” March 2010

23 Use of Xpert® for diagnosing TBCDC/DGMQ does not consider this technology established enough to replace MTB culture at this time  Xpert is not as sensitive as MTB culture in certain populations and also does not give the full resistance profile with DST Xpert can be a useful tool in making treatment decisions for smear positive cases while cultures are pending Xpert can be used to better inform treatment regimens and to rule out drug resistance if DR/MDR TB is suspected

24 Use of Xpert® for diagnosing TBXpert testing can be performed in addition to MTB culture, but it cannot replace MTB culture to satisfy immigration requirements MTB culture is still considered to be the gold standard for TB diagnosis CDC/DGMQ does not currently plan to change the culture requirement for immigration purposes. If this policy changes, notifications will be made through proper communications. 

25 Limitations of Molecular Testing

26 Limitations of Molecular TestingNAATs Can enhance diagnostic speed, but do not replace AFB smear or culture Should always be performed in conjunction with microscopy and culture Can only detect M. tuberculosis (complex) Cultures still needed for identification of nontuberculous mycobacteria (NTM) and for DST

27 Limitations of Molecular TestingNAATs Do not provide Drug Susceptibility Testing information Able to detect nucleic acids from both living and dead organisms (do not distinguish) Should not be used to monitor therapy Must be validated in-house if used for specimens other than respiratory specimens

28 Limitations of Molecular TestingNAATs Clinicians should interpret NAAT results based on clinical situation Currently available molecular methods aid in the rapid detection of mutations associated with drug resistance, but test results must always be confirmed by phenotypic (conventional) methods Genotypic vs. Phenotypic

29 N. gonorrhea

30 Types of Molecular Laboratory Assays

31 Types of Molecular Laboratory AssaysAbbott RealTime CT/NG Abbott RealTime CT/NG Polymerase chain reaction (PCR) BD ProbeTec™ Qx (Becton Dickinson) Strand displacement amplification (SPA) An isothermal in vitro method of amplifying a target DNA sequence. BD ProbeTec™ Qx

32 Types of Molecular Laboratory AssaysAptima® Combo 2 for CT/NG Aptima® Combo 2 for CT/NG Transcription-mediated amplification (TMA) Can amplify either DNA or RNA, and produces RNA amplicon, in contrast to most other nucleic acid amplification methods that only produce DNA Xpert® CT/NG (Cepheid) Nucleic acid amplification test (NAAT) Cepheid Xpert® CT/NG

33 Use of Molecular Laboratory Tests

34 Use of Molecular Laboratory TestsGonorrhea Technical Instructions (TIs): CDC Immigration Requirements: TIs for Panel Physicians CDC encourages panel physicians to use the least invasive tests for screening The least invasive tests are the urine NAAT (men and women), or a NAAT performed on a self-collected vaginal swab (women)  Combination (CT/NG) kits are acceptable

35 Use of Molecular Laboratory TestsGonorrhea Technical Instructions (TIs): CDC Gonorrhea TIs and CDC STD Treatment Guidelines specifically state NAAT tests and not “PCR” tests.  Some NAAT tests may use PCR, but others do not CDC will allow the screening for gonorrhea to begin no later than October 1, 2016 under the new/updated TIs 2014 MMWR discusses available NAATs NAAT options may be different in your country 

36 Use of Molecular Laboratory TestsPanel physicians should use tests that have regulatory approval in their country Refer to product / package insert Follow product instructions to ensure the correct specimen is used for the laboratory test, as collection methods and specimen types vary Source: eMaze

37 Limitations of Molecular Testing

38 Limitations of Molecular TestingOnly validated with certain specimen types Not performed / validated with all age groups Varying technical / testing skills Assay Interference Blood Mucin Feminine hygiene products Bilirubin Other

39 Limitations of Molecular TestingRefer to product / package insert Intended vs Actual Use Summary and Explanation of Assay Principles of the Procedure Reagent Storage and Handling Requirements Preferred Specimen - Collection, Storage, Stability

40 Limitations of Molecular TestingAssay Interpretation Limitations Sensitivity / Specificity Reproducibility Clinical Trial / Study Information Contact Information

41 Thank you! [email protected] http://www.cdc.gov/panelphysiciansFor more information please contact Centers for Disease Control and Prevention 1600 Clifton Road NE, Atlanta, GA Telephone: CDC-INFO ( )/TTY: Visit: | Contact CDC at: CDC-INFO or The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. National Center for Emerging and Zoonotic Infectious Diseases Division of Global Migration and Quarantine