Background: Use of nucleic acid amplification tests (NAATs), such as strand displacement assay (SDA), for the detection of gonococcal infection in low prevalence populations is controversial because of the likelihood of false positive results. Use of supplementary NAATs with alternative target sites has been recommended for confirmation of primary NAAT results.
Aim: To evaluate if SDA reactive specimens for Neisseria gonorrhoeae, which were either culture positive or negative, can be confirmed by alternative target NAATs such as transcription-mediated assays (TMA).
Methods: SDA reactive specimens were retested by TMA using APTIMA Combo 2 (AC2) and APTIMA GC (AGC) assays. Two different methods of specimen preparation were used to test the specimens. In method A, residual extract after SDA was retested and in method B, the original clinical specimen was re-extracted in TMA medium and then retested. Cervical or urethral swabs were requested to confirm the SDA results by culture.
Results: By method A, 26/49 (53.1%) of SDA positive specimens were positive by AC2 and/or AGC; 14/27 (51.8%) culture confirmed SDA positive tests were positive by AC2 and/or AGC. By method B, 38/39 (97.3%) SDA positive results were confirmed by both AC2 and AGC. All the 25 culture confirmed SDA positive tests were confirmed by both AC2 and AGC; 5/6 SDA positive tests that were culture negative were confirmed by both AC2 and/AGC.
Conclusion: Alternative target site NAATs, such as AC2 and AGC, can be used to confirm SDA positive results using the same clinical specimen. There is high concordance between the three NAATs.
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Neisseria gonorrhoeae is the second most common bacterial sexually transmitted infection (STI) in the UK with 19 007 infections diagnosed in STI clinics in 2006. The highest rates are found in London and urban areas. Lewisham is an inner city south London borough1 and for the past 4 years community sexual and reproductive health clinics and a few general practice clinics have been participating in the National Chlamydia Screening Programme (NCSP) for detecting asymptomatic Chlamydia trachomatis infections in sexually active subjects under 25 years of age.2 In the light of the fact that STI clinics in South London report a high rate of diagnoses of gonococcal infection, we initiated screening for N gonorrhoeae in addition to C trachomatis using a nucleic acid amplification test (NAAT) based on strand displacement assay (SDA) (ProbeTec CT/GC, Becton Dickenson, Maryland, USA)
SDA has been found to be highly specific and sensitive in detecting gonococcal infection in high-risk subjects attending STI clinics but there are only a few studies that report on the use of SDA in a community screening programme.3 In a recent paper describing our experience of screening in the community using SDA, we reported a high prevalence (3.8%) of gonococcal infection in the Lewisham community. At the time of reporting the results, we were concerned whether some of the SDA positive results were “false positive” as only 67% of these results could be confirmed by culture or the cultures were false negative. In an accompanying commentary, Barlow observes that SDA may be prone to false positive results and the actual prevalence may be lower.4 5 Although isolation is the preferred method to confirm the presence of N gonorrhoeae, as it provides information about antibiotic sensitivities and molecular epidemiology, it is being increasingly recognised that culture may not always be possible either because of difficulties in obtaining a suitable specimen or limitations in transport and laboratory support. In instances where it is not possible to confirm using culture, Ison recommends that all positive results should be repeated with a supplementary test that is of equal sensitivity and specificity to the original test. The supplementary NAAT should have a different target.6
In a simulated experiment, Scragg et al were able to confirm the chlamydia results using APTIMA-Combo 2 (AC2) (Gen-Probe, San Diego, California, USA) in specimens previously tested by SDA.7 It is, therefore, conceivable that specimens previously tested for gonococcus infection can be retested using AC2 and APTIMA GC (AGC), which have different target sites for amplification. Furthermore, AC2 and AGC have been found to be highly specific and sensitive even when screening populations where prevalence of gonococcal infection is low.8 AC2 NAAT also identified additional subjects who had partners with gonococcal infection but who themselves were negative by microscopy and culture. Thus, these subjects may have been previously labelled as false positive using culture as a gold standard.9
The aim of this study was to evaluate if SDA reactive specimens for N gonorrhoeae, which were either culture positive or negative, can be confirmed by alternative target NAATs such as AC2 and AGC.
Subjects attending community sexual and reproductive health clinics and general practices in the borough of Lewisham in South London were screened for C trachomatis and gonococcal infection.
“First pass urines” in male and self-taken vulvovaginal, high vaginal or cervical swabs in female subjects were tested by SDA (ProbeTec CT/GC, Becton Dickenson, Maryland, USA).
All positive specimens were re-tested once. If both tests were positive, the test result was reported as “positive”. If only one of the two tests was positive, then the test result was reported as “equivocal” and repeat specimen requested.
Subjects with a positive SDA result for N gonorrhoeae were recalled to the clinics as soon as possible. Cervical swabs (Transport swab, Amies medium with charcoal, Sterilin, Caerphilly, UK) and/or urethral swabs (Transwab, Amies medium with charcoal, Medical Wire and Equipment, Wiltshire, UK) were taken for culture and antibiotic sensitivity tests prior to commencement of treatment.
Confirmation with alternative target NAATs
AC2 and AGC assays, which amplify different target sites, were used to confirm the SDA reactive specimens. These tests were performed at the Clinical Microbiology Laboratory, University Hospital Aintree, Liverpool, UK, using the manufacturer’s instructions.
Two different methods of testing residual specimens were evaluated in separate time periods. In the first time period (January 2007) method A was used and in the second time period (February to December 2007) method B was used.
Method A (using residual extract)
Specimens received at the Clinical Microbiology Laboratory at University Hospital Lewisham were extracted into 2 ml of ProbeTec extraction buffer as per manufacturer’s instructions. The specimens were then tested using ProbeTec CT/GC NAAT. SDA extracts reactive for N gonorrhoeae were retested using some of the remaining extract. After retesting, the residual extract (approximately 800 μl) was transferred to a transcription-mediated assay (TMA) transport tube and transported by postal services to the Clinical Microbiology and Health Protection Agency Collaborating Laboratory, Aintree Hospital, Liverpool, UK, for testing with AC2 and AGC NAATs.
Method B (re-extraction of residual clinical specimen)
The residual clinical specimen was re-extracted. The female swabs were swirled in the TMA tubes for approximately 15 seconds. Some of the remaining original urine was transferred into a TMA tube. The tubes were then sent to the Clinical Microbiology and Health Protection Agency Collaborating Laboratory for confirmation by AC2 and AGC NAATs.
Comparison of SDA with AC2 and AGC and culture using residual extract (method A)
Overall, only 26/49 (53.1%) of SDA positive specimens were positive by AC2 and/or AGC; 17/27 (62.9%) SDA and culture positive specimens were negative by AC2 and/or AGC (table 1).
Comparison of SDA with AC2 and AGC and culture using residual clinical specimen (method B)
Overall, 38/39 (97.3%) SDA positive results were confirmed by both AC2 and AGC. All of the 25 culture confirmed SDA positive tests were confirmed by both AC2 and AGC (table 2)
Use of NAATs for the diagnosis of gonococcal infection, especially as part of a screening programme, remains controversial and has still not gained wide acceptance. The principal concern is that certain NAATs such as SDA may have a high false positive rate and may have serious clinical and medico-legal consequences when screening low prevalence populations.10
This study was undertaken to evaluate if SDA (ProbeTec) reactive specimens for N gonorrhoeae, which are either culture positive or negative can be confirmed by alternative target site NAATs such as AC2 and AGC.
As no laboratory methods have been previously described to carry out such confirmation in clinical specimens, we evaluated two methods with a view to describe the optimal method for confirmation of a primary SDA NAAT reactive result with a supplemental NAAT such as AC2 and AGC.
We found method A (using residual extract) to be unsatisfactory for testing by AC2 and AGC. Over 50% of the SDA positive tests were not confirmed by AC2 and AGC even though 75% of the SDA positive results were confirmed by culture.
Conversely, method B (retesting of the original sample) with AC2 and AGC gave very reliable results (97.3% concordance). All but one of the SDA positive results were confirmed by AC2 and AGC. While all culture positive (25/25) SDA positive results were confirmed by AC2 and AGC, all but one of the culture negative (5/6) SDA positive results were also confirmed by AC2 and AGC.
None of the SDA equivocal test results were confirmed by either culture or AC2 and AGC. The number of equivocal results were too few to draw firm conclusions.11 We confirmed positive SDA results by culture in 52/67 (77.6%) of the subjects in whom a culture was obtainable. This shows that culture confirmation is feasible in a majority of the subjects in a community screening programme. Finally, the confirmation of SDA results by AC2 and AGC suggests that the prevalence reported by us in our earlier report is likely to be a true reflection of the prevalence of gonococcal infection in the Lewisham community.4
We conclude that that alternative target site NAATs, such as AC2 and AGC, can be used to confirm SDA positive results from the same urogenital specimens. There is also high concordance between the three NAATs. Laboratories using SDA should consider sending re-extracted clinical specimens in TMA medium for further testing to another laboratory with facilities for TMA assays. Furthermore, it is feasible to confirm by culture and test antibiotic sensitivity in a majority of the cases with SDA positive results in a community screening programme.
Alternative target site nucleic acid amplification tests (NAATs), such as transcription-mediated assays (TMA), can be used to confirm strand displacement assay (SDA) positive results from the same urogenital specimens.
Laboratories using SDA should consider sending re-extracted clinical specimens for further testing to another laboratory with facilities for TMA assays.
It is feasible to confirm by culture and test antibiotic sensitivity in a majority of the cases with SDA positive results in a community screening programme.
We thank the Lewisham Chlamydia and Gonococcus Screening Programme for supporting this study.
Competing interests: None.
Contributors: RH was the principal investigator of the study. RH, GR and HM designed, analysed and contributed to the write up of the study.
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