Molecular testing for Neisseria gonorrhoeae

J.Dave

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)

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

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

Performance of tests to detect gonococcal infection

Diagnostic method Sensitivity Specificity

Gram stain urogenital specimensMales -symptomatic 90-95% 95-100%Males - asymptomatic 50-70% 95-100%Females 50-70% 95-100%

CultureUrogenital 80-95% 100%Rectal 50-60% 100%Pharyngeal 40-50% 100%

NAATs (urogenital) 65-99% 91-100%

Recovery of 35 gonococcal isolates on transport swabs at room temperature (high inoculum)

0102030405060708090

100

Per

cent

0 6 Hr 12 Hr 24 Hr 48HR

Copan Med Wire charcoal

Mean inoculum 5.9 x 10 5 cfu/ml

Recovery of 35 gonococcal isolates on transport swabs at room temperature (low inoculum)

0102030405060708090

100

Per

cent

0 6 Hr 12 Hr 24 Hr 48HR

Copan Med Wire charcoal

Mean inoculum 5.9 x 10 3 cfu/ml

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)

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 testStudy results not routinely reported to clinicStudy results reviewed weeklyNotify GUM if Aptima test Positive and routine

test Negative– discordant result explained and treatment

recommended

Gonorrhoea:1521 patient episodes Endocervical swabs

Result Culture Aptima

Combo

Aptima

NG

Positive 6 11 9*

Negative 1515 1506 1512

Equiv 4 0

Prevalence 0.4% 0.7% 0.6%

*1 contact GCCorrelation 99.8% (1518/1521)

Gonorrhoea: Sensitivity of Culture and Aptima in detecting infected women

Pattern Number

Culture Positive & Aptima Positive 6

Culture Positive & Aptima Negative 0

Culture Negative & Aptima Positive 3

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)

Gonorrhoea:1158 male episodes urethral culture versus urine

Result Culture Aptima

Combo

Aptima

NG

Positive 21 24 21

Negative 1137 1124 1137

Equiv 10 0

Prevalence 1.8% 2.1% 1.8%

Correlation 100% (1158/1158)

Gonorrhoea: 224 male episodes rectal culture versus Aptima

Result Culture Aptima

Combo

Aptima

NG

Positive 8 22 21*

Negative 216 199 203

Equiv 3 0

Prevalence 3.6% 9.8% 9.4%

Correlation 94.2% (211/224) all culture pos were Aptima pos*10 had additional evidence of gonococcal infection

Gonorrhoea: 242 male episodes throat culture versus Aptima

Result Culture Aptima

Combo

Aptima

NG

Positive 8 24 23*

Negative 236 216 218

Equiv 2 1

Prevalence 3.3% 9.9% 9.5%

Correlation 93.8% (226/241) all culture pos were Aptima pos

*12 had supportive evidence of gonococcal infection

Gonorrhoea: Sensitivity of Culture and Aptima in detecting infected men

Pattern Number

Culture Positive & Aptima Positive 30

Culture Positive & Aptima Negative 0

Culture Negative & Aptima Positive 12*

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

Gonorrhoea: Infected sites from 1205 male episodes

22

8 8

22 22 23

0

5

10

15

20

25

Pos

itiv

e

Culture Aptima (n=137)

Urethra Rectal Throat

Prevalence by Culture 2.5% (31/1205)Prevalence by Aptima 3.5% (42 /1205) or 3.2% (38/1205)

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

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

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%

Which NAAT ? Analytical Specificity- results from testing a panel of relevant organisms

Test Cross Reacting organism

Cobas Amplicor N. flavescens, N. lactamica, N.sicca N.subflava N.cinerea

BD Probetec N. flavescens N. lactamica N. subflava N cinerea

Aptima Combo 2 none to dateAptima GC none 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

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 essentialDiscussed with local lead clinician for GUM servicesLocal agreement between laboratory and GUM in terms

of reporting initial test resultNon-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

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

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

SNGRL and the introduction of NAATs

Advise on commercial systems, strengths & drawbacksProvide 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

Primary result

Supplemental results Interpretation/Report

Aptima Combo

Aptima GC

Pos Pos Pos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test.

Pos Pos Neg N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test

Pos Neg Pos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test

Pos* Neg Neg This result does not confirm the original NAAT test. Consider repeat if clinically indicated

Equivocal Pos Pos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test.

Equivocal Pos Neg N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test.

Equivocal Neg Pos N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test.

Equivocal* Neg Neg This result does not confirm the original NAAT test. Consider repeat if clinically indicated.

Neg/invalid Not routinely tested. Please discuss

* This pattern reflects either a false positive first test or a low level of nucleic acid

Acknowledgements

• Hugh Young

• Helen Palmer

• GUM