UPDATE MOLECULAR DIAGNOSTICS IN SEXUAL HEALTH. Dr Arlo Upton Clinical Microbiologist Labtests Auckland

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1 UPDATE MOLECULAR DIAGNOSTICS IN SEXUAL HEALTH Dr Arlo Upton Clinical Microbiologist Labtests Auckland

2 Talk outline Chlamydia trachomatis NAAT What does a positive test mean Interpreting low level positives Confirmatory testing Neisseria gonorrhoeae NAAT Confirmatory testing NAAT role in extra-genital sites NAAT role in forensics

3 Commercially available NAATs Name Company CT NG Method Cobas 4800 Roche Yes Yes PCR Cobas Taqman Roche Yes No PCR ProbeTec ET (Viper) BD Yes Yes SDA Aptima combo 2 (Tigris) Gen-probe Yes Yes TMA (RNA) Abbott real time (m2000) Abbott Yes Yes PCR NAAT advantages Very sensitive Does not require viable organism Can be used on non-invasive specimens Fully automated systems NAAT disadvantages Specificity issues Detects nucleic acid, not disease state Not licensed/validated for all specimen sites False-negative results caused by sequence variation or inhibition Does not provide antimicrobial resistance data

4 How good is a laboratory test? Reproducibility Consistency.could be repeatedly wrong Validity Test measures what it is intended to measure Analytical validity Clinical validity

5 What does a positive CT NAAT mean? 1. Infection 2. DNA only, no viable organisms 3. False positive

6 What tools do we have? CDC guidelines on confirmatory testing 1. Same specimen, same assay 2. Same specimen, different assay (different target) 3. Different specimen, different assay Clinical history, examination. Other laboratory information e.g. degree of positivity

7 Laboratory false positives -contamination

8 Evaluated all three CDC approaches to confirmatory testing Urine & genital specimens Abbott LCx probe, BD ProbeTec, Aptima combo 2, Amplicor PCR 1. >90% of original positives confirmed with additional testing 2. Failure to confirm mostly low-level positives 3. More confirmed by using combination of different NAATs 4. Can t confirm with less sensitive assay (SDA) 5. False positives may occur but confirmatory testing not the way to identify them

9 Low-level positive assays In our lab, approx 9% are low-level positives (>37 cycles) Repeat testing doesn t clarify as some will be positive, some negativedue to small amount of target present & sample variability dilution 1 in 100 Average Ct Detection rate % 1 in 500 Average Ct Detection rate % 1 in 1000 Average Ct Detection rate %

10 Positive test of cure At least five weeks after therapy Standard of care TOC not required unless Absorption Compliance Pregnant Children (?) Non-standard therapy

11 Young women aged 14 17y, primary care clinics 3 monthly interviews, examinations & sample collection Alternating quarters, wkly vaginal swabs & daily diaries Consenting partners investigated & treated Amplicor PCR + Aptima combo ompa genotyping Almost all treated with DOT azithromycin N = % with history of STI at study entry Point prevalence CT during study = %

12 Antibiotic effectiveness 92.1% Repeat episodes 13.7% probable or possible treatment failures

13 CT antibiotic resistance No standardised methods to detect phenotypic resistance In vitro azithromycin/doxycylineheterotypic resistance seen and occurs where there is high bacterial load Clinical implications of this uncertain It is possible that some organisms survive following treatment with azithromycin True resistance with mutation in rrna genes rare Possible fitness cost Re-treatment of possible treatment failures?????? In vitro resistance seen after passaging CT in sub-inhibitory concentrations of quinolones and rifampicin

14 Persistence CT lifecycle

15 Evidence for CT persistence in vitro Induction Observations Antibiotic exposure Enlarged, pleomorphic, aberrant RB Unable to differentiate to EB Depletion essential nutrients IFN-γ exposure Monocyte infection Aberrant RB Altered RB Unable to differentiate to EB Inhibition of inclusion formation Enlarged and aberrant RB Continuous infection Subpopulation of atypical inclusions with aberrant, enlarged RB Atypical inclusions reduction in susceptibly to azithromycin

16 Evidence for CT persistence in vivo EM visualisation in diseased tissue of aberrant RB Synovial macrophages of CT-associated reactive arthritis Macrophages in degenerative aortic stenosis (CP) CT DNA amplified from culture-negative tubal biopsies RNA detected in synovial biopsies Suggests viable organisms but culture negative

17 Evidence for reactivation Active trachoma in people years after leaving endemic trachoma region Intercurrent NG infection reactives CT infection that was previously latent & below level detection (culture)

18 N. gonorrhoeae NAAT Traditionally poor specificity due to cross-reactions with commensal Neisseria species Frequent genetic exchange between the Neisseria species can result in the acquisition of target N. gonorrhoeae sequences by commensal Neisseria species Assay Target Cross-reacts with strains of - Roche amplicor CMT N. cinerea, N. flavescens, N. lactamica, N. sicca, N. subflava BD ProbeTec Abbott CT/NG Pilin-inverting gene Opacity gene No data N. cinerea, N. flavescens, N. lactamica, N. subflava APTIMA combo 2 16SrRNA No cross reactivity found Roche Cobas 4800 DR-9 No cross reactivity found

19 Do positive NG NAAT require confirmatory testing? Prevalence 1% 1% No tested Sensitivity 99% 99% Specificity 99% 99.9% Total positives True positives PPV 50% 91%

20 Do positive NG NAAT require confirmatory testing? CDC confirmatory testing if PPV < 90% 1. >90% of original positives confirmed with additional testing 2. Failure to confirm mostly low-level positives 3. More (>97%) confirmed by using combination of different NAATs on different specimen 4. Can t confirm with less sensitive assay (SDA) 5. False positives may occur but confirmatory testing not the way to identify them

21 Extra-genital sites Ota et al Sex Transm Infect 2009;85:182-6 MSM, 18 years +, men s clinic Toronto Pharyngeal, rectal swabs, urine +/- urethral swab NAAT x 2, culture N = 248 NG culture NG NAAT CT culture CT NAAT Sens Spec 100 > >98

22 N=812 adults, oral sex + Pharyngeal specimens in addition to genital/rectal Amplicor, Aptima, ProbeTec, culture (direct plating) Sensitivity (%) Specificity (%) Culture ProbeTec Aptima Amplicor Prevalence Women 5.3 vs. 9.1% Men 3.5 vs. 7.0%

23 Role of NAAT in forensics CDC guidelines 2006 currently being updated Data are insufficient to adequately assess the utility of nucleic acid amplification tests in the evaluation of children who might havebeen sexually abused, but these tests might be an alternative if confirmation is available and culture systems for C. trachomatisare unavailable. Confirmation tests should consist of a second FDA-cleared nucleic acid amplification test that targets a different sequence from the initial test.

24 Children with suspected abuse, n = 536 Only 51 males Urine, vaginal, anal (urethral) swabs Culture + NAAT (except anal) Aptima + Probetec Probetec positives had additional in-house PCR confirmatory testing Males all negative

25 4.9% had positive result by any test (3.1% CT, 3.3% NG) Excellent specificity & PPV Sensitivity (CT) significantly better with NAAT (85 100%) compared with culture (39%) Sensitivity (NG) no significant difference

26 Study conclusions Prevalence of STIs low (<5%) in children investigated for sexual abuse NAATs on urine + vaginal swabs, with confirmatory testing, adequate Recommend confirmatory testing with different target Recommend caution for using NAAT for NG Alternative target confirmation Culture for susceptibility testing

27 Lymphogranuloma Venereum Labplus (Auckland Hospital) have assay for LGV Real-time PCR assay, which targets the pmpgene which has a unique gap in LGV serovars of CT compared to other serovars, so it is highly specific Usual specimen is rectal swab Few cases of treatment failure reported after 21 days of doxycyline

28 What are NZ labs doing? BD Probetec x 3 Roche Taqman x 3 Roche Amplicor x 1 Abbott m200 x 1 Roche Cobas 4800 x 3 Many accept forensic specimens, not all have chain of evidence NG NAAT only a few labs Confirmation not standardised but themes appear

29 Concluding comments Keep in mind difference between reproducibility and validity NAAT detects nucleic acid Confirmatory testing doesn t exclude all possible false reactive tests Improved technology More specific assays Limit laboratory error Persistence & resistance -research required Increasing acceptance for extra-genital sites & forensic specimens Unexpected result Talk to lab Consider additional testing

30 Acknowledgements Dr Gerardo Herrera NZ labs who provided details of their testing approaches

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