Validation of a Laboratory Developed Real-Time PCR Protocol for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine

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1 Validation of a Laboratory Developed Real-Time PCR Protocol for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine Mark J Hopkins, Lynne J Ashton, Fath Alloba, Anura Alawattegama, Ian J Hart To cite this version: Mark J Hopkins, Lynne J Ashton, Fath Alloba, Anura Alawattegama, Ian J Hart. Validation of a Laboratory Developed Real-Time PCR Protocol for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine. Sexually Transmitted Infections, BMJ Publishing Group, 2010, 86 (3), pp.207. < /sti >. <hal > HAL Id: hal Submitted on 19 Jan 2011 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 1 2 3 Validation of a Laboratory Developed Real-Time PCR Protocol for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine 4 5 M J Hopkins, L J Ashton, F Alloba, A Alawattegama, and I J Hart Authors affiliations M J Hopkins, L J Ashton, I J Hart, Liverpool Specialist Virology Centre, Royal Liverpool University Hospital, Liverpool, UK F Alloba, A Alawattegama, Department of Genitourinary Medicine, Royal Liverpool University Hospital, Liverpool, UK Correspondence to: M J Hopkins, Liverpool Specialist Virology Centre, Royal Liverpool University Hospital, Liverpool, L7 8XP, UK; m.hopkins@liv.ac.uk Running title: Chlamydia and Gonorrhoea Quadruplex Assay Keywords: Chlamydia trachomatis, Neisseria gonorrhoeae, real-time PCR, laboratory diagnosis, urine 20 1

3 21 Abbreviations: EQA, external quality assurance; LDA, laboratory developed assay; LDQA, laboratory developed quadruplex assay; NAAT, nucleic acid amplification test The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Sexually Transmitted Infections and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence ( 30 2

4 31 Abstract Objective: To evaluate a sensitive and specific, real-time PCR assay with internal control for Chlamydia trachomatis and Neisseria gonorrhoeae DNA detection in urine specimens. Methods: Diagnostic performance of a laboratory developed quadruplex assay (LDQA) targeting the cryptic plasmid and MOMP genes of C. trachomatis, the pora pseudogene of N. gonorrhoeae, and a synthetic internal control was assessed using 1028 urine specimens. The LDQA was compared to the Roche COBAS Taqman CT test and the COBAS Amplicor NG assay with supplemental confirmation tests. Subsequent performance of the LDQA to detect N. gonorrhoeae was monitored in comparison to bacterial culture from swabs. Results: A total of 88 (8.6%) urines were determined as C. trachomatis positive in the diagnostic evaluation. LDQA sensitivity and specificity were calculated to be 100% and 99.9%, respectively for C. trachomatis. The LDQA showed high specificity with isolates of other Neisseria species and gave complete concordance with resolved data for N. gonorrhoeae detection. However, the incidence of N. gonorrhoeae infection was low with 17 (1.7%) positive patients. A post-implementation audit of 14,316 patients gave the LDQA N. gonorrhoeae urine PCR protocol (pora, OPA, 16s rdna) a sensitivity of 96.9% and specificity of 99.8% in comparison to bacterial culture from swabs. Conclusions: The LDQA was found to be an effective method for detection of C. trachomatis and N. gonorrhoeae DNA in urine samples and the PCR protocol has replaced bacterial culture for screening of N. gonorrhoeae in asymptomatic men and women in our laboratory. 55 3

5 56 INTRODUCTION Nucleic acid amplification test (NAAT) methodologies are currently recommended as the standard of care for diagnosis of genital chlamydia infection in England. 1 The gold standard method for diagnosis of N. gonorrhoeae infection is bacterial culture, although NAATs are also advocated to increase sensitivity of detection in non-invasive sample types. 2 A number of NAATs for detection of C. trachomatis employ, or are pursuing, multiplex formats to reduce the risk of under-reporting infections, and companies are increasingly offering combined tests capable of detecting both C. trachomatis and N. gonorrhoeae in the same analysis. 3 4 Previous reports have identified problems with the sensitivity of N. gonorrhoeae detection in female urine by PCR and also highlighted the absence of 67 antimicrobial sensitivity data from these tests. 5 However, the relatively small increase in laboratory reagent and labour costs associated with the multiplex format and the ease of non-invasive sexual specimen collection makes this an attractive option for clinics. Different genetic targets are often preferred for screening and confirmation assays to increase the overall specificity of NAAT diagnostic procedures. 1 6 Laboratory developed assays (LDA) can offer useful alternatives to the range of commercial tests, particularly when existing equipment can be utilised and the new protocols aligned to established workflows. However, the laboratory must undertake significant validation of LDA to ensure the assay and method of sample preparation are suitable for diagnostic use. A small number of laboratories in the UK employ LDA for routine screening of C. trachomatis but only limited data is available in the literature for real-time LDAs for the multiplex detection of C. trachomatis and N. gonorrhoeae. 7-9 Here we describe a real-time multiplex LDA 4

6 81 82 protocol for the detection of C. trachomatis and N. gonorrhoeae and validate its use with first void urine samples METHODS Bacterial strains and EQA samples Two external quality assurance (EQA) panels for C. trachomatis and N. gonorrhoeae were supplied by the Quality Control for Molecular Diagnostics (QCMD, Glasgow, UK). PCR specificity for N. gonorrhoeae was also assessed using isolates of N. cinerea (n=16), N. flavescens (n=2), N. gonorrhoeae (n=8), N. lactamica (n=7), N. meningitidis (n=4), N. mucosa (n=1), N. perflava (n=1), N. polysaccharea (n=1), N. sicca (n=3), N. subflava (n=3), and Neisseria spp. (n=7). Patients and specimens One thousand and twenty eight urine specimens were obtained from 1,015 patients attending the Genitourinary Medicine Clinic at the Royal Liverpool University Hospital during the period August to October Median age of the patients was 25 years (range 15 to 73 years) with 556 being male (55%), and 459 female (45%). Samples were collected as part of routine testing for sexually transmitted infections with patient consent obtained to test for both chlamydia and gonorrhoea infection. Patients continued to be swabbed for routine microscopy and N. gonorrhoeae culture for comparison with the N. gonorrhoeae PCR results. Results from the LDQA N. gonorrhoeae PCR protocol were audited in comparison to swabs for bacterial culture over the period February 2008 to February A total of 14,319 patients were screened during this time. Of 5

7 these, 3,393 were asymptomatic females, and the remaining 10,926 were symptomatic males and females. DNA isolation For the LDQA, and the N. gonorrhoeae confirmation assays (NsppID and NG- OPA/16s rdna), genomic DNA was extracted from 500µl of urine or bacterial suspension and eluted in 100µl buffer using the Magnapure Classic automated extraction system with the DNA Isolation Large Volume Kit (Roche Diagnostics, Burgess Hill, UK) according to the manufacturer s instructions. Urine samples (1 ml) were prepared for the C. trachomatis confirmation test (Artus CT Plus assay, Qiagen, Crawley, UK) by centrifugation for 5min at xg and resuspension of the cell pellet in 400 µl water. The entire volume was then processed using the Qiagen M48 Biorobot and Magattract Virus Mini M48 Kits (Qiagen), eluting in 50 µl buffer. If necessary, extracts were stored at +4 o C for up to 24h or longer term at -70 o C until required. Laboratory developed quadruplex assay Neisseria gonorrhoeae assay components were introduced to an established C. trachomatis assay with the best performing quadruplex taken forward for the 121 LDQA Details of the oligonucleotides used in the final assay are given in table The assay used LC480 probe master mix (Roche Diagnostics) in accordance with the manufacturer s instructions to a final volume of 25µl, with each reaction containing 10µl purified DNA. Amplification and detection was carried out using a LightCycler 480 PCR machine (Roche Diagnostics) with the following cycling parameters; 5 min at 95 o C, followed by 50 cycles of 10s at 95 o C, 45s at 60 o C, 1s at 72 o C, and a final step of 30s at 40 o C. Amplification signal for the N. gonorrhoeae pora pseudogene fragment or either of the two C. trachomatis PCR 6

8 targets was taken as a positive result. The internal control was required to amplify in order to call a negative result. COBAS Amplicor/Taqman assays Samples (500µl) were processed with 50µl of the resultant lysate being used in the Roche COBAS Amplicor NG or Taqman CT assays according to the manufacturer s instructions (Roche Diagnostics). Results from either assay were deemed inhibitory if the internal control failed to amplify in otherwise negative samples. Confirmation PCR assays All urine samples identified as positive or inhibitory for C. trachomatis by the COBAS Taqman CT assay or LDQA were repeat tested with the Artus CT Plus PCR kit (Qiagen Ltd) on a LightCycler 1.2 PCR machine (Roche Diagnostics) according to the manufacturer s instructions. Published PCRs directed toward the neisseria 16S rrna gene (NSppID), and the opacity protein gene (NG-OPA) were used to confirm the presence of N. gonorrhoeae DNA in any sample identified as positive, inhibitory or grey zone by the COBAS Amplicor NG test or LDQA. The NSppID assay was run as described previously but was later replaced with the NG-OPA PCR multiplexed with a 16s rdna PCR for post-implementation confirmation of the LDQA (Table 1). The NG-OPA/16s rdna duplex PCR was run on the LC480 under the same conditions as the LDQA but the extension time at 72 o C was increased to 10s. Bacterial culture Neisseria gonorrhoeae was isolated using GC agar base with VCAT selective supplement (Oxoid, Basingstoke, UK). Suspect colonies were identified to species 7

9 level on the basis of Gram stain, oxidase test, Phadebact GC kit (Launch Diagnostics, Longfield, UK) and API NH test (BioMerieux, Basingstoke, UK). Statistical analysis Kappa analysis was used to calculate the level of agreement between LDQA for detection of C. trachomatis and/or N. gonorrhoeae in comparison to resolved data from both screening assays and all confirmation tests. Level of agreement was also calculated for confirmed N. gonorrhoeae PCR results against bacterial culture RESULTS Assay sensitivity and specificity The LDQA scored full marks on the C. trachomatis and N. gonorrhoeae EQA panels, correctly identifying all positive samples and giving no false positive results. The COBAS tests achieved a score of 10/10 for the C. trachomatis EQA panel but falsely identified a sample containing N. lactamica as N. gonorrhoeae positive in the N. gonorrhoeae panel. However, the NsppID confirmation assay differentiated this as a non-gonococcal neisseria positive sample on the basis of melt temperature (66 o C). Neither the COBAS NG Amplicor test nor the N. gonorrhoeae pora component of the LDQA amplified any non-gonococcal isolates from the laboratory panel of 53 Neisseria species but correctly identified the eight isolates of N. gonorrhoeae. Diagnostic evaluation Positive urines were identified on the basis of at least two positive results - either two PCRs or PCR with microscopy or culture. Two samples were excluded from 8

10 the C. trachomatis kappa analysis due to repeatedly inhibitory results with both the COBAS and Artus PCR tests. Table 2 shows resolved data identified 88/1026 (8.6%) C. trachomatis positive samples and gave good concordance with LDQA results (kappa value, 0.99). Both the COBAS CT Taqman and the LDQA screen each identified one false positive C. trachomatis sample. The LDQA correctly detected C. trachomatis DNA in two samples that gave negative results from the COBAS Taqman CT screen (Table 3). Overall, the sensitivity and specificity of the LDQA C. trachomatis screen were calculated to be 88/88 (100%) and 937/938 (99.9%), respectively. The LDQA identified seventeen (1.7%) of the 1028 urine samples as pora pseudogene PCR positive which gave 100% concordance with resolved N. gonorrhoeae data (Table 2). All 17 samples were confirmed as NG-OPA PCR positive but four could not be amplified using the NsppID 16s rdna assay (Table 4). The COBAS Amplicor NG PCR identified a larger number of urine samples that required N. gonorrhoeae confirmation tests with a total of 45/1028 giving a positive (n=19), grey zone (n=6), or inhibitory (n=20) result with this screening assay. Seventeen were the resolved N. gonorrhoeae positives but twenty four could not be confirmed by any of the other tests employed (Table 4). The remaining four were identified as non-gonoccocal Neisseria spp. on the basis of NsppID PCR melt curve analysis. Sensitivity and specificity of the LDQA N. gonorrhoeae screen were calculated as 17/17 (100%) and as 1011/1011 (100%), respectively using this data set. Three (0.3%) of the 1028 samples were confirmed positive for dual infection of C. trachomatis and N. gonorrhoeae. 9

11 201 Neisseria gonorrhoeae follow-up audit The LDQA was introduced into routine service in addition to the existing bacterial culture and results from the two techniques were reviewed after 12 months. Kappa analysis of the N. gonorrhoeae urine PCR protocol results showed a strong correlation with bacterial culture (kappa value, 0.99) identifying 210 patients positive by all three PCR targets (pora, OPA, 16s rdna). Twenty five of these were negative by bacterial culture whilst there were an additional six falsenegative PCR results (Table 5). Sensitivity and specificity of the N. gonorrhoeae urine PCR protocol were calculated as 96.9% and 99.8%, respectively using this data set. Gonorrhoea infection was diagnosed in 11 of 3,393 asymptomatic females. One case was culture positive/pcr negative but two cases which were initially negative by bacterial culture from cervical swabs were PCR positive. The latter two cases were culture positive on patient recall DISCUSSION The use of NAATs for detection of C. trachomatis is well established and is increasingly utilised for detection of N. gonorrhoeae, particularly as emphasis shifts towards the use of non-invasive specimen types Several manufacturers now supply NAAT platforms that can screen urine samples for both C. trachomatis and N. gonorrhoeae in the same reaction. Results presented here show the LDQA is a sensitive and specific test for detection of C. trachomatis and N. gonorrhoeae in first void urine samples. Dean et. al. (2008) reported the COBAS Amplicor CT test to have a sensitivity and specificity of 96.9% and 98.2% respectively, but there are no published data available comparing the COBAS TaqMan CT test (used in our evaluation) to the Aptima Combo 2 which was cited 10

12 as the most sensitive assay in that study. 16 Both Roche and Abbott have included secondary chlamydial targets in their assays to facilitate detection of new variant 228 strains. 8 The LDQA detects the cryptic plasmid and MOMP PCR products in different channels of the real-time PCR machine which facilitates local monitoring of this issue. Numerous genetic targets have been assessed for the detection of N. gonorrhoeae although bacterial culture is often considered the diagnostic standard due to genetic heterogeneity in this organism If requested, the LDQA allows flexible detection of C. trachomatis and/or N. gonorrhoeae through addition of a pora pseudogene target to the assay. This PCR was chosen because of the high sensitivity and specificity previously demonstrated with the pseudogene target. The pora PCR was found to be more sensitive and specific than the COBAS Amplicor NG test which has previously been shown to generate a considerable number of inhibitory or reactive results that could not be confirmed, and our findings support this (Table 4). The NSppID assay has been reported as a useful confirmation test to improve the specificity of the COBAS NG assay. 12 However, NSppID assay sensitivity was limited in comparison to the NG- OPA confirmation PCR and an NG-OPA/16s rdna duplex assay was subsequently adopted as the confirmation assay to complement the LDQA (pora) screen. Insufficient data for N. gonorrhoeae infection was generated from the initial diagnostic evaluation due to the low prevalence of this organism in our patient population. The LDQA screen was implemented alongside the existing bacterial culture service and a 12 month review of data showed the urine PCR protocol was comparable to N. gonorrhoeae culture from swabs. Although culture was taken as the standard for comparison, the authors feel the 25 culture negative/urine PCR 11

13 positives (Table 5) could equally be true positives since amplification of all three N. gonorrhoeae targets in the screen and confirmation PCRs (pora, OPA, 16s rdna) was required for a N. gonorrhoeae PCR positive result to be issued. This assumption would improve the performance characteristics of the N. gonorrhoeae urine PCR protocol, although further comparison with additional testing protocols would be needed to establish this. No obvious problems were observed identifying gonorrhoea infection in the 3,393 asymptomatic females tested over this time period. This may be related in part to the use of a nucleic acid purification technique in this protocol to concentrate DNA and efficiently remove inhibitors, unlike the original COBAS method which relied on a simple lysis procedure for sample preparation. Review of this data led to a change of policy in our clinic, with the urine PCR protocol becoming the favoured screen over bacterial culture from swabs for asymptomatic patients. However, antibiotic resistance profiling remains an important consideration and N. gonorrhoeae culture is maintained as part of the confirmation protocol. The authors do not advocate LDA above validated assays from commercial companies which supply support and a degree of accountability, but firmly believe they warrant consideration by specialist molecular diagnostic laboratories when IVD approved tests are not appropriate for existing workflows. Data presented here may be useful to inform laboratory decisions on screening or confirmation protocols, or clinical choice of sample. Key messages This study has validated the C. trachomatis assay previously described by Jalal et al., (2007) for use with first void urine specimens. 7 12

14 LDQA followed by NG-OPA/16s rdna PCR confirmation allowed for the specific and sensitive multiplex detection of gonorrhoea infection from a non-invasive sample. Laboratories may find this NAAT data useful when reviewing their existing C. trachomatis and N. gonorrhoeae workflows ACKNOWLEDGEMENTS The authors are grateful to Dr Helen Palmer, Scottish Bacterial Sexually Transmitted Infections Reference Laboratory, Royal Infirmary of Edinburgh, UK for provision of Neisseria strains, and to Dr Steven Lane, Centre for Medical Statistics and Health Evaluation, University of Liverpool, for statistical advice throughout this study. The authors thank Mrs Jean Howell in the Department of Genitourinary Medicine, Royal Liverpool University Hospital for assistance compiling the N. gonorrhoeae data CONTRIBUTORS IJH and MJH conceived and designed the evaluation. FA and AA coordinated specimen collection and NG audit. MJH and LJA conducted the PCRs and analysed the data. MJH wrote the manuscript in collaboration with all co-authors Competing interest: None declared Word count (abstract 244, body 2484); Text pages = 20; Tables =

15 Table 1. Oligonucleotide primers and probes used in this evaluation. Assay Oligonucleotide Sequence (5 to 3 ) Concentration (µm) Target gene Citation LDQA HJ-Cp-F AACCAAGGTCGATGTGATAG 0.25 C. trachomatis HJ-Cp-R TCAGATAATTGGCGATTCTT 0.25 cryptic plasmid HJ-Cp-FAM (FAM) CGAACTCATCGGCGATAAGG (BHQ1) 0.10 HJ-Momp-F GACTTTGTTTTCGACCGTGTT 0.25 C. trachomatis HJ-Momp-R ACARAATACATCAAARCGATCCCA 0.25 MOMP HJ-Momp-VIC (VIC) ATGTTTACVAAYGCYGCTT (MGB-NFQ) 0.10 Pap-Tm-F CAGCATTCAATTTGTTCCGAGTC 0.25 N. gonorrhoeae Pap-Tm-R GAACTGGTTTCATCTGATTACTTTCCA 0.25 pora Pap-Tm-LC610 (LC610) CGCCTATACGCCTGCTACTTTCACGC (BBQ) 0.10 HJ-Ic-F GTGCTCACACCAGTTGCCGC 0.10 Synthetic HJ-Ic-R GCTTGGCAGCTCGCATCTCG 0.10 internal control HJ-Ic-Cy5 (Cy5) ATTGTGTGGGTGTGGTGTGGGTGTGTGC (BHQ3) NsppID NG767-F AAAGCGTGGGTAGCAA 1.0 Neisseria spp. NG946-R TTCTTCGCGTTGCATC S rdna NG839-FL CAACCTGATTGCTTAGTAGCGTAGCTAACG (FL) 0.2 NG (LC640) GTGAAATTGACCGCCTGGGGAGTACGG (PH) NG-OPA OPA-F TTGAAACACCGCCCGGAA 0.16 N. gonorrhoeae / 16s OPA-R TTTCGGCTCCTTATTCGGTTTAA 0.16 opa Duplex OPA-Cyan500 (Cyan500) CCGATATAATC+CGTC+CTTCAA+CATCAG (BBQ) s-19-F TCGGACGGCAGCACAGGGAA 0.32 N. gonorrhoeae this 16s-432-R GGCCGCCGATATTGGCAACG S rdna study 16s-88-FAM (FAM) TACCGGGTAGCGGG (MGB-NFQ) denotes position of locked nucleic acid bases

16 Table 2. LDQA results in comparison to resolved data from 1028 urines for Chlamydia trachomatis and Neisseria gonorrhoeae. C. trachomatis Results N. gonorrhoeae Results LDQA CT Result LDQA NG Result Positive Negative Positive Negative Resolved CT Result Positive Positive Resolved NG Result Negative Negative a a, Two samples were excluded due to repeatedly inhibitory COBAS and Artus results. LDQA C. trachomatis sensitivity 100%, specificity 99.9%, kappa value 0.99, PPV 98.9%, NPV 100%. LDQA N. gonorrhoeae sensitivity 100%, specificity 100%, kappa value 1.00, PPV 100%, NPV 100%. 15

17 Table 3. Details of C. trachomatis test results for the 13 discordant specimens. Results for all other specimens were concordant. Resolved results were used in LDQA kappa analysis. LDQA screen COBAS screen Additional tests Number of LDQA CT: LDQA CT: COBAS CT Taqman: Artus CT Plus: Resolved Specimens cryptic plasmid MOMP LDQA CT result cryptic plasmid cryptic plasmid & MOMP CT Result (# female) (CP range) (CP range) (CP range) 1 (0) >45 - CT DNA detected - Inhibitory - 2 (1) CT DNA detected CT Positive 1 (0) CT DNA detected Inhibitory - 1 (1) 43 - CT DNA detected CT DNA detected - CT Positive 1 (1) 41 - CT DNA detected Inhibitory 37 CT Positive 1 (0) CT DNA detected CT DNA detected - CT Positive 4 (1) CT DNA detected CT DNA detected CT Positive 2 (0) Inhibitory Inhibitory Unclear a Total 13 (4) -, Negative; CP, real-time PCR crossing point; CT, C. trachomatis. a Two samples were excluded from the CT kappa analysis due to repeatedly inhibitory results. Results for the remaining 79 CT resolved positives were concordant. 16

18 Table 4. Details of N. gonorrhoeae test results for the 34 discordant specimens. Results for all other specimens were concordant. Resolved results were used in LDQA kappa analysis. LDQA screen COBAS screen Additional tests Number of LDQA NG: COBAS NG Amplicor b : NSppID: NG-OPA: NG culture Resolved Specimens pora LDQA NG result methyltransferase 16s rdna opa NG Result (# female) (CP range) (Melt temp) c (CP range) 3 (3) - - Grey zone (3) - - Grey zone 56 o C, 65 o C (9) - - Inhibitory (0) 22 NG DNA detected Inhibitory d NG Positive 1 (0) 25 NG DNA detected Inhibitory 60 o C 23 - d NG Positive 1 (0) 23 NG DNA detected Inhibitory 60 o C 23 NG isolated NG Positive 3 (3) NG DNA detected NG DNA detected NG isolated NG Positive 3 (2) - - NG DNA detected (1) - - NG DNA detected 65 o C Total 34 (21) -, Negative; CP, real-time PCR crossing point; NG, N. gonorrhoeae. b COBAS NG Amplicor defined grey zone results as OD between to and positive results if OD COBAS CT Taqman does not give grey zone results. c N. gonorrhoeae was defined by a melt temperature of 60 o C. Melt temperatures other than 60 o C were indicative of other Neisseria spp. d N. gonorrhoeae not isolated but Gram-negative diplococci were observed when cells were stained directly from the specimen swab. Results for the remaining 11 NG resolved positives were concordant. 17

19 Table 5. Neisseria gonorrhoeae urine PCR protocol validation results in comparison to bacterial culture data from14,319 sexual health screens. N. gonorrhoeae Results Urine PCR Protocol Result Positive Negative Bacterial Culture Result Positive Negative 25 14,100 14, ,106 14,316 N. gonorrhoeae urine PCR protocol sensitivity 96.7%, specificity 99.8%, kappa value 0.99, PPV 88.1%, NPV 100%. 18

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