molecular testing for neisseria gonorrhoeae j.dave
TRANSCRIPT
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