Gonorrhoea: time for a culture shift

1. Gonorrhoea: time for a culture shift? Thanks to Rory maclean and sashi muthulasssy : who basically did all the work Thanks to Rory maclean and sashi muthulasssy : who basically did all the work

2. Contents Introduction and Background Attendees Total Numbers Male attendees Female attendees Summary and implications Background : reasons for cult neg B1 and a brief run through of the naats tests for b1Background : reasons for cult neg B1 and a brief run through of the naats tests for b1

3. Background 1 Poorly performing EIA for chlamydia leads to introduction of new testing method, which also detects gonorrhoea Decision to run this new GC test (Gen-Probe Aptima Combo 2) alongside existing test (GC culture) for trial period Sensitivity of existing test ~ 95% Dual NAAT Testing : cost neutral AND THAT�S SOMETHING Men urethral microscopy and culture Females : cervical microscopy, and urethral and cervical culture Selective Antibiotic Medium : Vanc/Nystatin/Trimethoprim/Colistin Rectal swabs not performed unless anal sex. Not helpful unless obtained via protcoscopy. 0/115 rectal culture sole diagnostic site in 2001 BAASH GUIDELINES one set of culture 95% sensitive at excluding B1Dual NAAT Testing : cost neutral AND THAT�S SOMETHING Men urethral microscopy and culture Females : cervical microscopy, and urethral and cervical culture Selective Antibiotic Medium : Vanc/Nystatin/Trimethoprim/Colistin Rectal swabs not performed unless anal sex. Not helpful unless obtained via protcoscopy. 0/115 rectal culture sole diagnostic site in 2001 BAASH GUIDELINES one set of culture 95% sensitive at excluding B1

4. Brief overview of B1 NAATS

5. The Tests (B1 NAATs) Roche Cobas Amplicor PCR assay : limited specificty : Cytosine DNA Methyltransferance gene LCx probe system (abbot) OPA gene BD Probe tec : Target gene? TMA Combo 2 assay : target 16S rRNA : Roche Assay This gene present in sub flava and lactobacilli .THIS HAS GIVEN B1 NAATS A BAD NAME Van Doornum et al 2001, Journal Clin Micro , females, population prevelance of 1.2%, PPV 35% for roche PCR when a second confirmatiory 16s rRNA assay used or the LcX assay used Same study 5 positives of 500 for LCX all cult postive . Assay now discontinued? Comercial not performance reasons 2nd study : 5 case reports of +ve roche pcr naat in hawwai (done private lab in low risk monognomous realtionships, mostly with BV), all 5 tma �ve and cult neg. Little if any published data on in vitro data on ability of these tests to detect non NG species. Companies must have the data but don�t publish it as it does not suit themRoche Assay This gene present in sub flava and lactobacilli .THIS HAS GIVEN B1 NAATS A BAD NAME Van Doornum et al 2001, Journal Clin Micro , females, population prevelance of 1.2%, PPV 35% for roche PCR when a second confirmatiory 16s rRNA assay used or the LcX assay used Same study 5 positives of 500 for LCX all cult postive . Assay now discontinued? Comercial not performance reasons 2nd study : 5 case reports of +ve roche pcr naat in hawwai (done private lab in low risk monognomous realtionships, mostly with BV), all 5 tma �ve and cult neg. Little if any published data on in vitro data on ability of these tests to detect non NG species. Companies must have the data but don�t publish it as it does not suit them

6. BD Probe Tec BD Probe Tec : in the UK Urethral swabs in men : 25/152 GC +ve men 5 cases cult neg, 2 microscopy positive Urethral/cervical swabs in men/women Slater et al Rectal swabs in men : high concordance Urines difficult for Lab : centrifugation needed Skinner et al 2005 published in International Journal Ceri watson at st thomas Poster 10 Samarawickrama et al rectal naat compared with rectal culture, Urine need an experienced lab to prevent contmaination etcSkinner et al 2005 published in International Journal Ceri watson at st thomas Poster 10 Samarawickrama et al rectal naat compared with rectal culture, Urine need an experienced lab to prevent contmaination etc

7. Slater et al BAASH poster 2005 BD probe Tec introduced without local evaluation Culture was not performed unless high risk or microscopy positive Men : 49 +ve urethral culture : NAAT detected 48, one missed was also micro +ve also Sensitivity = 97.9% Females : 29 +ve cervical : NAAT detected all Females microscopy was only done for GC if high risk for GC e.g B1 contact , b1 in last 3y, severe PID, frank pus at cervix Men :urethral discharge or B1 contactFemales microscopy was only done for GC if high risk for GC e.g B1 contact , b1 in last 3y, severe PID, frank pus at cervix Men :urethral discharge or B1 contact

8. Slater et al cont BAASH poster 2005 Specificity Men 48/50 NAAT +ve also cult positive 2 NAAT +ve cult negative Both cultures several days post GC tx and one symptomatic GC contact PPV = 96%

9. Slater et al cont BAASH poster 2005 Specificity Females 43/55 NAAT positives had cultures done 14 NAAT +ve cervical cult Neg 3 +ve other sites 6 Abx prior to cult (include one B1 contact) 4/14 possible false Positives PPV 74% 4/14 time elapsed between naat and swab was 25-63 days 32/43 (presuming the Abx ones were false postives) So sensitivity good and PPV acceptable in practice for that clinic4/14 time elapsed between naat and swab was 25-63 days 32/43 (presuming the Abx ones were false postives) So sensitivity good and PPV acceptable in practice for that clinic

10. TMA Transcription Mediated Amplification Target ribosomal RNA Urine sample do not require a centrifugation step

11. Aptima Combo 2 American Multicentre Study : female endocervical and urines GC Prevalence about 8% �Gold standard� cult positive or LCx positive (two sites) 126/1479 : Combo 2 positive Sens 99.2% Spec 98.7 % PPV 90% : No data on PPV as culture �gold� standard Gaydos et al J Clin Micro 2002 : FDA approval for detection using urine and cervical swabs. But of 17 false +ve naats in this study, 15 were confirmed by another TMA NAAT targeting another sequence one was TMA NEG BUT A TRUE postive , no data on whether it was a cult pos or an LCR post data on cross referencing , nice to see USA suffers from same problems as in UK on this issue. Note for the chlamydia part of study specificity was 97.4% and PPV 88%. Confirmatory tests roche PCR and LCx.Gaydos et al J Clin Micro 2002 : FDA approval for detection using urine and cervical swabs. But of 17 false +ve naats in this study, 15 were confirmed by another TMA NAAT targeting another sequence one was TMA NEG BUT A TRUE postive , no data on whether it was a cult pos or an LCR post data on cross referencing , nice to see USA suffers from same problems as in UK on this issue. Note for the chlamydia part of study specificity was 97.4% and PPV 88%. Confirmatory tests roche PCR and LCx.

12. First published UK study of TMA NAAT Identify GC prevalence amongst chlamydia screening population To monitor culture and partner findings in those GC NAAT positive 47 (1%) of 4680 females = F/up 38 8 (1.7%) of 473 men = F/up 5 STIi June 2006 Lavelle et al Liverpool : those postive referred to GUM for findings F/up information quite incredible : agreed f/up I.e go to gum (note previous london FP data of f/up of patient referred to gum form fp) and screened pre treatment 9 of the 47 women no confirmatory testing, 4 accepted antibiotic but refused screen 3 of the 8 men no follow up, one aware but choose no f/up, 2 refused further testing but accepted AbxSTIi June 2006 Lavelle et al Liverpool : those postive referred to GUM for findings F/up information quite incredible : agreed f/up I.e go to gum (note previous london FP data of f/up of patient referred to gum form fp) and screened pre treatment 9 of the 47 women no confirmatory testing, 4 accepted antibiotic but refused screen 3 of the 8 men no follow up, one aware but choose no f/up, 2 refused further testing but accepted Abx

13. TMA study cont Supportive evidence Females 37/38 : 34 culture +ve 3 B1 contacts Males 5/5 : Cult +ve Low prevalence Population (1%) excellent PPV Only 31 were cervical postive U or C 68%, C only 23%, U only 6% culture stats . One female site was pharanyx only postive. Note if microscopy postive, non vanc containing medium was used (lincomycin, containing). Also various in house growth supplements added One female attended three months after test to Gum and partner not tested NPV not known Interesting point is that only 55% of the women post for GC (on TMA) were c4a postive (data on those with f/up only), been used as argument to screen only chlamydia postives for b1Only 31 were cervical postive U or C 68%, C only 23%, U only 6% culture stats . One female site was pharanyx only postive. Note if microscopy postive, non vanc containing medium was used (lincomycin, containing). Also various in house growth supplements added One female attended three months after test to Gum and partner not tested NPV not known Interesting point is that only 55% of the women post for GC (on TMA) were c4a postive (data on those with f/up only), been used as argument to screen only chlamydia postives for b1

14. Reasons for culture Negative GC Recent Antibiotic (dental etc) Selective medium Failure to sustain or clearance (immunity) Organism perishes on culture plate Non Neisseria Species Presuming no human error in labeliing or sampling Cult live organism , DNA dead organism Clearance evidence from cloumbian study in 2004 , where many women cleared chlamydia spontaneously (50% at one year) , hPV study where retrospectively chlamydia samples where examined for chlamydia dnaPresuming no human error in labeliing or sampling Cult live organism , DNA dead organism Clearance evidence from cloumbian study in 2004 , where many women cleared chlamydia spontaneously (50% at one year) , hPV study where retrospectively chlamydia samples where examined for chlamydia dna

15. Background (2) Simultaneous B1 NAAT testing commences 6th June 2005 Endocervical swab for women Urethral swab then First Catch Urine for men Expectation that NAAT will have higher sensitivity (detect more cases) than culture, but are these true or false positives? Decision to perform in house comparison of NAAT vs Culture Wanted to make sure that NAAT did not miss cases in men or women Wanted to try and determine if the NAAT pos cult �ve were true postives or false negative culture? Wanted to make sure that NAAT did not miss cases in men or women Wanted to try and determine if the NAAT pos cult �ve were true postives or false negative culture?

16. Methods of Audit Positive gonorrhoea results from H.A�s list (from lab) G.C proforma for a period of 10 months (June 05 to March 06) supposed to be completed at B1 Tx Case notes were reviewed and data collected using excel. Proforma : additional info recorded e,g b1 contact or recent abx not rotuiinely askedProforma : additional info recorded e,g b1 contact or recent abx not rotuiinely asked

17. Total Cases in Audit Exclude throat and rectal B1 more interested in sensitivity but maybe of use for assessing specificity Exclude throat and rectal B1 more interested in sensitivity but maybe of use for assessing specificity

18. Male Positive Results Males first 6 ,microscopy only positive!!!! 20 equivocal on naat Make 86 in totalMales first 6 ,microscopy only positive!!!! 20 equivocal on naat Make 86 in total

19. Sensitivity and Specificity Sensitivity � the proportion of patients with a disease who have a positive test (ie how well does it identify cases) Specificity � the proportion of patients without the disease who have a negative test (ie how well does it identify healthy subjects)

20. Positive and Negative Predictive Values Positive Predictive Value � the proportion of people with a positive test who actually have the disease (i.e if they�re NAAT +ve, have they got it?) Negative Predictive Value � the proportion of people with a negative test who really don�t have the disease (i.e. if they�re NAAT �ve, do they really not have it?) Depends on PrevelanceDepends on Prevelance

21. Male