Objectives Recently marketed nucleic acid amplification tests (NAAT) for the detection of Neisseria gonorrhoeae (NG) have improved specificity over previous generation assays. A study to assess the necessity for confirmation of Roche cobas 4800 NG positive samples was undertaken by the Public Health Wales Microbiology Molecular Diagnostic Unit in Cardiff.
Methods Classical NG culture identification was compared to cobas 4800 (DR-9), opacity (opa) gene and porA pseudogene (pap) results. Confirmatory NAATs (opa/pap) were performed prospectively for 120 cobas 4800 NG positive urogenital and extragenital samples. Retrospective supplementary NAAT and sequence analysis of additional cobas 4800 NG positive extragenital samples was also carried out.
Results Of the 188 classically identified clinical NG isolates, 184 were identified as NG in all 3 molecular targets. Two isolates were only detected by 2 molecular targets. A further 2 isolates were culture false-positives. Combining the results from prospective and retrospective testing, the sensitivity and negative predictive value for cobas 4800 NG detection for urogenital, rectal and oropharyngeal samples was 100%. Specificity for all sample types was greater than 99.7%. Positive predictive value was 96.0% and 96.4% for urogenital and rectal specimens, respectively, and 88.6% for oropharyngeal samples.
Conclusions Molecular tests could be used for culture confirmation where available. Roche cobas 4800 Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG)CT/NG gonorrhoea diagnosis is superior to culture with urogenital and rectal positives not requiring confirmation. Roche cobas 4800 oropharyngeal NG detection findings warrant further prospective study of routine confirmatory testing accounting for its cost and clinical usefulness.
- Neisseria Gonorrhoea
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Microbiological diagnosis of Neisseria gonorrhoeae (NG) infection has historically been performed using microscopy and culture. Although these methods are often adequate for diagnosing patients with overt signs and symptoms of urogenital infection, sensitivity in asymptomatic disease and for non-genital specimens is poor. Additionally, despite the excellent specificity of culture, false positivity may arise.1 ,2 Nucleic acid amplification tests (NAAT) have increased sensitivity of detection compared with microscopy and culture for most sample types.3–9 However, the detection of NG using NAATs has been compromised historically by unacceptably high rates of non-specific reactivity with non-gonococcal Neisseria species, leading to recommendations for routine repeat testing of all NAAT NG positives.10
Recent commercial tests, such as the Roche cobas 4800 CT/NG test, demonstrate improved specificity over earlier generation assays although possible false-positive results have been reported.11 Furthermore, the first account of a clinical NG false-positive result from a pharyngeal specimen using the cobas 4800 Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) test has recently been reported in the literature.12 Nevertheless, due to the significantly superior sensitivity of NAATs for the detection of NG especially in the rectum and oropharynx, they have been recommended as the method of choice for extragenital NG diagnosis in high-risk patients, such as men who have sex with men without the need for confirmation.4 ,13–15 In low-prevalence populations, routine confirmatory testing is still advocated, for extragenital NG positive samples.9 ,10
The cobas 4800 CT/NG test targets a novel and highly conserved direct repeat region, which is repeated three times within the NG genome, called DR-9. The assay incorporates two different sets of NG DR-9 primers in order to detect known combinations of target variations.16
Roche cobas 4800 CT/NG testing was introduced in Wales in 2011. Analysis of culture-positive NG isolates was undertaken to compare the cobas 4800 CT/NG test NG results with classical technique identification. Prospective confirmatory testing of cobas 4800 NG positive samples sought to demonstrate the sensitivity and specificity of the Roche cobas 4800 CT/NG test for the detection of NG in all specimens from patients attending sexual health clinics in Wales where NAAT testing was offered. Further work was undertaken to investigate the performance specifications of the cobas 4800 CT/NG assay for extragenital specimens. The results of this study are presented and the rationale for confirmatory testing strategies is discussed.
All conventional and molecular methods used in this study were appropriately controlled using known positive and negative controls. All controls gave the expected results unless otherwise stated.
N gonorrhoeae culture
One hundred and eighty-eight stored NG patient isolates (on cryopreservation beads at −80°C) were available for testing (isolates from 98 urethral swabs, 50 cervical swabs, 30 rectal swabs, 9 oropharyngeal swabs, and 1 isolate from a swab from an intrauterine contraceptive device (IUCD)). Culture results were also obtained retrospectively for 80 samples in the initial prospective cobas 4800 (Roche Molecular Systems, UK) CT/NG test cohort, and for 71 samples in the extragenital site study. Culture was undertaken according to published national standard method guidance using the API NH test strip (bioMérieux) and the Phadebact monoclonal gonococcal test (Bactus AB).2
cobas 4800 CT/NG testing
Three regional sites in Wales were established to perform the cobas 4800 CT/NG test (Cardiff, Rhyl and Swansea). Specimens were collected in cobas PCR media from patients attending integrated sexual health clinics between August and December 2011 and tested according to the manufacturer's instructions.17 The first 120 NG positive samples (39 urine, 31 oropharyngeal swab, 23 rectal swab, 13 endocervical swab, 11 vaginal swab, 2 urethral swab and 1 eye swab) were sent for prospective confirmatory testing at the Molecular Diagnostic Unit in Cardiff. A total of 97 patients were tested prospectively with 82 samples from 65 male patients (age range 16–61 years) and 38 samples from 32 females (age range 16–47 years except for 1 neonatal eye sample). Seventy-eight samples were from patients where only one anatomical site was tested. Fifteen patients had 2 samples tested (11 male, 4 female) with 5 from duplicate anatomical sites (2 rectal, 2 oropharyngeal, 1 urine/cervical) and 4 patients had 3 samples tested (3 male, 1 female) with 2 tested from urine, oropharyngeal and rectal samples; 1 from cervical, oropharyngeal and rectal samples; and 1 from urethral, oropharyngeal and rectal samples. Sexual orientation data for patients was unavailable.
The extragenital phase of the study was undertaken retrospectively, using 110 stored (4–8°C) NG positive residual samples in cobas buffer previously tested using the cobas 4800 CT/NG test in Cardiff between March and August 2012. Samples included 62 oropharyngeal specimens, 39 rectal specimens and 9 urogenital specimens from 90 patients (68 male and 22 female). One hundred and thirty-two cobas 4800 NG negative samples (57 urogenital, 52 oral and 23 rectal) were also selected for confirmatory testing. Data analysis was undertaken in September 2012. Patient demographics (other than gender) and clinical information was unavailable for all samples tested retrospectively.
Confirmatory opa/pap PCR testing
Four hundred microlitres of sample in cobas PCR media was extracted on an EZ1 Advanced XL using the EZ1 DSP virus kit (Qiagen, UK) according to the manufacturer's instructions,18 with a 60 μL elution volume.
Confirmatory PCR testing was undertaken using a duplex assay based on previously published porA pseudogene (pap) and modified opacity gene (opa) assays.19 ,20 The papTM primers and probe were used and the opa reverse primer (5′-TTTCGGCTCCTTATTCGGTTTG(A)A 3′) and probe (5′VIC-CCGATATAATCCGC(T)CCTTCAACATCAG-TAMRA 3′) sequences were modified (base modifications are shown in bold type with original bases in parentheses with no locked nucleic acid (LNA) bases used in the opa probe). The duplex assay was optimised with an amplification reaction consisting of 5 μL of DNA extract and 15 μL of master mix including: 10 μL TaqMan Fast Universal PCR Master Mix (Life Technologies, UK), final concentrations of 0.3 μM of each primer (Life Technologies, UK) and 0.5 μM of opa (Life Technologies, UK) and pap probe (Eurogentec, Belgium). Amplification and detection were performed on an ABI 7500 fast real-time PCR system (Life Technologies, UK) in fast mode using the following parameters: initial denaturation at 95°C for 20 s followed by 50 cycles of denaturation at 95°C for 3 s and annealing and extension at 60°C for 30 s.
Three different sequencing assays were used for the study. Pan-bacterial 16S sequencing (500 bp fragment) was used to confirm the identity of two stored clinical isolates not detected using the cobas 4800 CT/NG test and opa/pap confirmatory assay. A proprietary sequencing assay (Roche Molecular Diagnostics) based on NG-specific primers within the 16S rRNA gene (353 bp fragment) was employed to further investigate cobas 4800 CT/NG test NG detected clinical samples where the opa and/or pap gene could not be detected. These samples (13 oropharyngeal and 3 rectal) were also analysed using a proprietary DR-9 sequencing assay (Roche Molecular Diagnostics) that employed primers flanking the DR-9 primers (431 bp fragment) used in the cobas 4800 CT/NG test.
Pan-bacterial 16S sequencing identification
Isolates were cultured according to national standard method guidance,2 after which a 1 μL loopful of harvested bacterial colonies was inoculated into 100 μL of a 5% Chelex solution (Bio-Rad, UK). The suspension was then incubated at 100°C for 12 min after which the extracts were centrifuged at 13 000×g for 10 min. Forty microlitres of the supernatant was removed and stored at 4°C. PCR was undertaken using the primers (pA and pH) and parameters described by Hutson et al21 generating a 1500 bp fragment of the 16S gene. The sequencing primer 5′-TTACCGCGGCTGCTGGCACGT-3′ allowed for sequencing of a 500 bp fragment of the PCR product. This fragment was compared by BLAST analysis with publicly available sequence data (National Center for Biotechnology Information (NCBI)).
NG-specific 16S and DR-9 sequencing
These assays were nested PCR assays that used NG-specific 16S and DR-9 primers to generate 353 bp and 431 bp PCR products respectively for sequencing. Sequence homology of these products was then compared with those seen in cobas 4800 CT/NG test confirmed positives.
Samples were re-extracted on the cobas 4800 with 50 μL used in the downstream PCRs. For both assays, primary PCR was performed, using NG-specific 16S and DR-9 primers respectively, in a 100 μL reaction with 50 μL master mixes containing a final concentration of 0.5 μM upstream and downstream primers, PCR buffer, 0.25 mM MgCl2, 5% glycerol, 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 200 μM dUTP, 0.05 U Amplitaq (Life Technologies, USA), and water to volume.
For the NG-specific 16S reaction, initial denaturation was at 95°C for 5 min. DNA amplification was performed at 95°C for 10 s, 60°C for 20 s and 72°C for 40 s for 30 cycles, followed by 72°C for 5 min. This was followed by a nested amplification with 5 μL of amplicon from the primary PCR reaction, 45 μL of water and 50 μL master mix. Initial denaturation was at 95°C for 5 min. PCR amplification was performed at 95°C for 10 s, 60°C for 20 s, 72°C for 40 s for 30 cycles, followed by 72°C for 5 min. This was completed with an incubation step at 72°C for 10 min.
For the DR-9 sequencing assay, the initial denaturation was at 95°C for 5 min. DNA amplification was performed at 95°C for 20 s, 50°C for 40 s and 72°C for 60 s for 5 cycles, then 95°C for 20 s, 60°C for 40 s and 72°C for 60 s for 35 cycles, followed by 72°C for 10 min. A nested amplification was then performed with 5 μL of amplicon from the initial PCR reaction, 45 μL of water and 50 μL master mix. Initial denaturation was at 95°C for 5 min. PCR amplification was performed at 95°C for 20 s, 50°C for 40 s, 72°C for 60 s for 5 cycles, then 95°C for 20 s, 60°C for 40 s and 72°C for 60 s for 20 cycles, followed by 72°C for 10 min.
Reactions for both assays were performed in a GeneAmp PCR System 9600 (Life Technologies, USA).
Amplified products were visualised on a 2% Egel (Life Technologies, USA). The resulting amplicons were purified with ExoSAP-IT (Affymetrix, USA), on another 2% Egel prior to sequencing. Sequencing was performed with the Applied Biosystems BigDye Terminator V.3.1 Cycle Sequencing Kit on an Applied Biosystems 3730 DNA Analyser (Life Technologies, USA).
Sequences were analysed with CLC Bio Genomic Workbench software V.5.5.1., and compared with NCBI reference strains.
Assay performance was determined using cobas 4800 NG detected and undetected cases as defined in table 4. Roche cobas 4800 NG positive and negative patients (from the prospective and retrospective test periods) from the South East Wales area (samples tested in Cardiff) included: 1965 urogenital samples from 1832 patients; 3531 oropharyngeal samples from 3242 patients; and 1062 rectal samples from 961 patients. Of the urogenital, oropharyngeal and rectal specimens tested, 56%, 43% and 75% were from female patients, respectively with the remainder originating from male patients.
Eight rectal specimens reported as ‘invalid’, 1 rectal specimen reported as ‘not tested’, and 11 oropharyngeal and 9 rectal cobas 4800 NG detected specimens discarded prior to retrospective confirmatory testing were excluded from analysis.
Sensitivity, specificity, positive and negative predictive values were calculated for urogenital, extragenital, rectal and oropharyngeal specimens in January 2014 using 2×2 contingency tables. Wilson CIs were also calculated for each specimen type.
Stored positive NG culture testing
One hundred and eighty-four clinical isolates were confirmed by all targets. Four isolates gave discordant results (table 1).
GenBank BLAST analysis of a 500 bp portion of the 16S rRNA gene identified the unconfirmed oropharyngeal swab isolate as Kingella denitrificans (GenBank accession number L06166.1), and the unconfirmed urethral swab isolate as either a Paracrauococcus species or Roseomonas genomospecies 5 (GenBank accession numbers JQ259701.1 and AY150049.1) with 99% sequence homology.
Prospective confirmatory testing
Of the 120 prospectively tested clinical samples, nine were not confirmed with the opa gene and 10 samples were not confirmed with the pap gene of the duplex confirmatory assay. Six samples (three oropharyngeal swabs, two urines and one vaginal swab) from five patients could not be confirmed with either pap or opa targets (table 2).
Culture information was available for 80 of the 120 samples tested, of which 55 (69%) were NG culture positive. Genital and extragenital culture confirmations were successful in 90% and 39% of samples, respectively.
All the 132 NG negative samples (57 urogenital, 52 oral and 23 rectal) evaluated on the cobas 4800 CT/NG Test were not detected using the opa/pap confirmatory assay.
Retrospective extragenital sample confirmatory testing
Of the 39 rectal swabs, 37 were confirmed in either the opa or pap targets, as were 53 of the 62 retrospectively tested oropharyngeal swabs. Culture confirmed 19 (59%) of 32 rectal positives, and 13 (33%) of 39 oropharyngeal positives.
Confirmatory NG-specific 16S and DR-9 sequencing testing
Sequencing data consistent with the presence of NG (>99% homology across the target region) was obtained for three of the 13 oropharyngeal samples using the NG-specific 16S sequencing assay and one sample using the DR-9 sequencing assay. The opa gene was detected in all three of these samples although the pap gene was not detected in any. Sequencing data to confirm the presence of NG (>99% homology across the target region) was generated from one of the three rectal swab samples tested in which the pap gene was also detected. Neither NG-specific 16S or DR-9 sequence could be obtained in one oropharyngeal sample where the cobas 4800 NG test result was confirmed using the opa assay (table 3).
The performance characteristics of the cobas 4800 CT/NG test for different specimen types using the Cardiff patient data from prospective and retrospective test phases is summarised in table 4.
The culture confirmation results from this study highlight the potential for false-positive NG reporting from genital and extragenital specimens using conventional methodologies.1 ,2 ,22 The isolates incorrectly identified as NG in this study demonstrated miniaturised biochemical test profiles identical to those for NG and pseudoagglutination with the coagglutination test used. The potential for false-positive culture confirmation should, therefore, be readily appreciated by laboratory and clinical staff alike. The small number of false-negative pap (n=1) and opa (n=1) results only serves to further illustrate the challenging nature of confirming NG infection by either conventional or molecular means.23–25 Latest generation NAATs employing novel targets, such as the cobas 4800 CT/NG test should be considered as a replacement for conventional confirmation assays.
According to UK guidance confirmation of NG positive samples is recommended where the positive predictive value (PPV) of the test falls below 90%.10 Initial prospective testing resulted in PPV for the cobas 4800 CT/NG test of greater than 90% for all sample types. After completion of the extragenital phase of the study, the PPV of the cobas 4800 CT/NG test for urogenital and rectal samples remained above 90%, which supports the data recently published by Bromhead et al.9
The low-level nature of the cobas 4800 CT/NG test results from the majority of unconfirmed oropharyngeal samples (table 3—CT>37 with clinical samples rarely if ever seen above CT>40), may reflect the typically scant organism load at this anatomical site26 or may be due to less than 100% primer and/or probe target sequence homology .12 The inability to obtain NG-specific 16S or DR-9 sequence data from these samples could also reflect either possibility. The lack of evidence that the unconfirmed oropharyngeal cobas 4800 CT/NG test NG positive samples are NG suggests that these could be false-positive results. Given the promiscuous nature of NG with respect to DNA uptake and loss, and with over 700 bacterial species or phylotypes with which to exchange parts of its genome within the oropharyngeal cavity,27 recombination events could lead to false positivity.
This study demonstrates the presence of unconfirmed NG positive results with the cobas 4800 CT/NG test from oropharyngeal samples, but did not succeed in providing a better understanding of their nature. Unfortunately, the false-positives described by Upton et al,12 were only cultured after the patient had been treated for NG infection on two occasions, and originated from an isolate identified as several Neisseria species using different identification techniques. The true clinical and analytical nature of these unconfirmed positive results, therefore, may only be possible by conducting a prospective study on appropriate samples, although definitive characterisation may prove elusive.
Supplementary testing for positive NG NAATs is advocated by many as it presumptively results in more accurate patient treatment and case reporting, and helps to avoid unnecessary patient and clinician anxiety.12 In light of the growing threat of resistant strain transmission28 and the consequences of inadequate or untreated oropharyngeal infection/colonisation, the true cost and clinical impact of supplementary testing merits additional investigation. For the cobas 4800 CT/NG test oropharyngeal NG positive samples from sexual health settings, clinicians and laboratory staff should be aware that no test is perfect, and that where a positive or negative result arouses suspicion, confirmatory testing may be performed.29 However, the results may not yield meaningful information, and the patient should be managed with careful attention to clinical presentation and sexual history.
Another potential challenge associated with rapid uptake of these easy-to-use molecular screening assays is the possibility of users employing tests without local validation or appreciation of the limitations of molecular diagnostics. Awareness of the performance of assays (frontline and confirmatory) for specific sample types and patient groups is essential in informing discussions regarding the interpretation of unexpected results in a given clinical setting.30 Local surveillance of cultured strains, where available, should also be promoted to ensure the continued efficacy of assay targets. Companies marketing such assays have a responsibility to actively support surveillance and investigation of the use of their assays in unlicensed sample types and different patient groups.
It is recognised that one of the weakness of this study is the lack of information pertaining to patient sexual orientation and clinical history available for the samples included. Further work examining the performance of the cobas 4800 CT/NG test in known patient groups would be beneficial.
In summary, false-positive culture results were demonstrated from retrospective molecular analysis of stored clinical NG isolates. The study confirmed that evaluation of urogential and extragenital samples with the cobas 4800 CT/NG test results in the detection of more true cases of gonorrhoea compared with culture. It also indicated that, according to UK guidelines, routine confirmatory testing is not necessary for cobas 4800 NG positive urogenital and rectal samples. Further prospective work should be conducted to elucidate the reason for unconfirmed oropharyngeal positivity. The cost and clinical usefulness of routine confirmatory testing should also be investigated.
The Roche cobas 4800 CT/NG test is suitable for the detection of Neisseria gonorrhoeae (NG) from urogenital and rectal specimens without confirmatory testing.
A prospective study is required to clarify the usefulness of confirmatory testing for oropharyngeal Roche cobas 4800 CT/NG test positives.
Molecular methods should be recommended for identification of presumptive NG culture isolates where available.
Public Health Wales colleagues for undertaking primary and confirmatory testing during the prospective phase of the study and primary testing during the retrospective period. Dr P Lewis White for advice on statistical analysis and manuscript review. Dr Sarah Copsey for undertaking pan-bacterial 16S sequence identification. Roche Diagnostics for funding the culture and retrospective confirmatory work and undertaking the proprietary sequencing work.
Handling editor Jackie A Cassell
Collaborators Catherine Moore P Lewis White Sarah Copsey Roche Diagnostics.
Contributors MDP undertook the culture and retrospective confirmatory work and some of the prospective confirmatory testing as well as analysing the data and preparing the manuscript. RNJ was involved in designing the study. SAC undertook some of the prospective confirmatory testing and was involved in study design and manuscript review.
Competing interests MDP has received travel grants and honoraria from Roche. SAC has received travel grants from Roche. Public Health Wales received funding from Roche to undertake the culture and retrospective confirmatory testing presented in this study.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement All data is published herein.