1. Molecular testing for Neisseria gonorrhoeae J.Dave

2. Guidelines for molecular testing for Neisseria gonorrhoeae Initiated and chaired by Dr Anne Eastaway, HPS Members/representatives: Drs Andy Winter and Kirsty Abu-Rajab (Sandyford), Bill Carman (Gartnavel) Alastair Leonard (Monklands), Lesley Wallace (HPS), Helen Palmer, Jayshree Dave and Hugh Young (SBSTIRL)

3. Guidelines for molecular testing for Neisseria gonorrhoeae: AIMS To ensure that national surveillance for gonococcal infection is maintained To determine the role of SNGRL in the introduction of routine molecular detection by nucleic acid amplification tests (NAATs) To ensure as far as possible that the results from molecular tests are reliable

4. Drivers for detection of gonorrhoea by NAATs Use of NAATs for chlamydia (SIGN) Availability of dual CT/NG tests – eases burden on stretched GUM clinics Small or no additional cost of NG testing Independent of GC viability Suitable for areas remote from laboratory – or any area where there is a delay in getting samples to the lab

5. Performance of tests to detect gonococcal infection Diagnostic methodSensitivity Specificity Gram stain urogenital specimens Males -symptomatic 90-95% 95-100% Males - asymptomatic 50-70% 95-100% Females 50-70% 95-100% Culture Urogenital 80-95% 100% Rectal 50-60% 100% Pharyngeal 40-50% 100% NAATs (urogenital) 65-99% 91-100%

6. Recovery of 35 gonococcal isolates on transport swabs at room temperature (high inoculum) Mean inoculum 5.9 x 10 5 cfu/ml

7. Recovery of 35 gonococcal isolates on transport swabs at room temperature (low inoculum) Mean inoculum 5.9 x 10 3 cfu/ml

8. Currently available NAATs for combined detection of chlamydia and gonorrhoea Cobas Amplicor CT/NG (Roche) – Roche Taqman real time PCR (only for CT)  GC under development ProbTec ET SDA CT/NG (Becton Dickinson) Aptima Combo 2 TMA (GenProbe) – Aptima GC (GenProbe) Realtime CT/NG PCR – under evaluation (Abbott)

9. Aptima versus current diagnostic methods Routine samples tested and reported as per laboratory SOPS – Chlamydia TaqMan with repeat testing of positives – Gonorrhoea culture – direct plating on modified NYC medium (10% lysed horse blood and LCAT) Study samples tested by Aptima Combo2 and positives re-tested by mono-specific test Study results not routinely reported to clinic Study results reviewed weekly Notify GUM if Aptima test Positive and routine test Negative – discordant result explained and treatment recommended

10. Gonorrhoea:1521 patient episodes Endocervical swabs ResultCultureAptima Combo Aptima NG Positive6119* Negative151515061512 Equiv40 Prevalence0.4%0.7%0.6% *1 contact GC Correlation 99.8% (1518/1521)

11. Gonorrhoea: Sensitivity of Culture and Aptima in detecting infected women PatternNumber Culture Positive & Aptima Positive6 Culture Positive & Aptima Negative0 Culture Negative & Aptima Positive3 Prevalence: 0.6% (9/1521) or 0.5% (7/1521) Culture Sensitivity: 66.7% (6/9) or 85.7% (6/7) Aptima Sensitivity: 100% (9/9) or (7/7) Specificity 100% or 99.9% (1512/1514)

12. Gonorrhoea:1158 male episodes urethral culture versus urine ResultCultureAptima Combo Aptima NG Positive212421 Negative113711241137 Equiv100 Prevalence1.8%2.1%1.8% Correlation 100% (1158/1158)

13. Gonorrhoea: 224 male episodes rectal culture versus Aptima ResultCultureAptima Combo Aptima NG Positive82221* Negative216199203 Equiv30 Prevalence3.6%9.8%9.4% Correlation 94.2% (211/224) all culture pos were Aptima pos *10 had additional evidence of gonococcal infection

14. Gonorrhoea: 242 male episodes throat culture versus Aptima ResultCultureAptima Combo Aptima NG Positive82423* Negative236216218 Equiv21 Prevalence3.3%9.9%9.5% Correlation 93.8% (226/241) all culture pos were Aptima pos *12 had supportive evidence of gonococcal infection

15. Gonorrhoea: Sensitivity of Culture and Aptima in detecting infected men PatternNumber Culture Positive & Aptima Positive30 Culture Positive & Aptima Negative0 Culture Negative & Aptima Positive12* Prevalence: 3.5% (42/1205) or 3.2% (38/1205) Culture Sensitivity: 71.4% (30/42) or 78.9% (30/38) Aptima Sensitivity: 100% (42/42) or (38/38) Aptima Specificity: 100% (1163/1163) or 99.7% (1163/1167) * 4 no other evidence of gonorrhoea

16. Gonorrhoea: Infected sites from 1205 male episodes Prevalence by Culture 2.5% (31/1205) Prevalence by Aptima 3.5% (42 /1205) or 3.2% (38/1205)

17. Conclusions on Aptima for GC Aptima was more sensitive than culture in detecting cervical gonorrhoea (100% vs 66.7% or 85.7%) Aptima was equivalent to culture in detecting urethral gonorrhoea in men (100% correlation) Aptima was more sensitive than culture in detecting rectal gonorrhoea in men (100% versus 38% to 44%) Aptima was more sensitive than culture in detecting throat gonorrhoea in men (100% versus 35% to 40%) Overall Aptima detected 23% (7/31) to 35% (11/31) more gonococcal infections A strategy is required to ensure antibiotic resistance surveillance when molecular diagnosis is used

18. Published data on sensitivity of NAATs for gonorrhoea Aptima Combo 2 Cobas Amplicor Probe Tec CT/NG Real Time CT/NG Male urine 97.1%94.1% (sym) 73.1% (asym) 98.1%None New test Prelim data suggests similar to Aptima Combo Female Urine 91.3%64.8% (not FDA approved) 84.9% Cervical swab 99.2%92.4%96.6% Vulvo- vaginal swab 96.2% (Clin) 98.7% (PT) 92.9% (Clin) 90.9% (PT) 100% but 25% inhibited

19. Published data on specificity of NAATs for gonorrhoea Aptima Combo 2 Cobas Amplicor Probe Tec CT/NG Real Time CT/NG Male urine 99.2%99.9%96.8 - 98.7%None New test Prelim data suggests similar to Aptima Combo Female Urine 99.3%99.8%98.8 - 99.8% Cervical swab 98.7%99.5%98.9 - 99.8% Vulvo- vaginal swab 99.4% (Clin) 99.6% (PT) 99.7% (Clin) 99.9% (PT) 99.8% (Clinician) 99.7% all sites 99.9%

20. Which NAAT ? Analytical Specificity - results from testing a panel of relevant organisms TestCross Reacting organism Cobas AmplicorN. flavescens, N. lactamica, N.sicca N.subflava N.cinerea BD ProbetecN. flavescens N. lactamica N. subflava N cinerea Aptima Combo 2none to date Aptima GCnone to date Some of these tests are more likely than others to generate false positives and decrease the Positive Predictive Value of a positive screening test

21. Guidelines for the introduction of NAATs Test platform – May vary depending on local circumstances Specimen – Male urine – Cervical or vulvo-vaginal swab (can be self-taken)  if urine from female is tested add comment “urine is sub- optimal for detecting gonorrhoea in women and will miss up to 1 in 4 infected women” Supplemental testing is essential Discussed with local lead clinician for GUM services Local agreement between laboratory and GUM in terms of reporting initial test result Non-GUM samples should always have a supplemental test before reporting – Patient with positive NAAT should be referred (and/or treated) to GUM for partner notification work

22. Supplemental NAAT testing Scenario 1 – Diagnostic lab performs two separate NAATs each targeting a different region of DNA or RNA  Multiplex reaction  Two separate tests (ideally on a different platform) – Aliquot of original sample from all positives (or equivocals) sent to SBSTIRL for ST Scenario 2 – Diagnostic lab performs a single NAAT  All positive and equivocal results sent to SBSTIRL for a supplemental NAAT targeting a different region of DNA or RNA

23. Culture & Introduction of NAATs In addition to NAAT – Cultures should be taken in GUM patients as follows:  Symptomatic for gonorrhoea  Positive on Gram-stained smear  Known contact of gonorrhoea  Anyone treated on epidemiological grounds  Treatment failure  All return patients who are NAAT positive but not included above – All isolates sent to SBSTIRL  When NAAT is positive local laboratory identification of isolates can be restricted to oxidase positive GNDC  SNGRL will perform susceptibility testing and sequence typing  SNGRL will hold NAAT sample for up to 2 weeks prior to sequence typing to link with cultures

24. SNGRL and the introduction of NAATs Advise on commercial systems, strengths & drawbacks Provide supplemental testing during the period of introduction of NAATs until an acceptable level of concordance between laboratory results is established Provide supplemental testing for labs that detect only one target Perform sensitivity testing on all isolates received to advise on individual treatment and support national surveillance programme Sequence type all strains either from NAAT or culture to support local contact tracing and National surveillance

25. Primary result Supplemental resultsInterpretation/Report Aptima Combo Aptima GC Pos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test. Pos NegN. gonorrhoeae nucleic acid present. This result confirms the original NAAT test PosNegPosN. gonorrhoeae nucleic acid present. This result confirms the original NAAT test Pos*Neg This result does not confirm the original NAAT test. Consider repeat if clinically indicated EquivocalPos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test. EquivocalPosNegN. gonorrhoeae nucleic acid present. This result confirms the original NAAT test. EquivocalNegPosN. gonorrhoeae nucleic acid present. This result confirms the original NAAT test. Equivocal*Neg This result does not confirm the original NAAT test. Consider repeat if clinically indicated. Neg/invalidNot routinely tested. Please discuss * This pattern reflects either a false positive first test or a low level of nucleic acid

26. Acknowledgements Hugh Young Helen Palmer GUM