Mark J Hopkins
email: m.hopkins@liv.ac.uk

Lynne J Ashton

Fath Alloba

Anura Alawattegama

Ian J Hart

J Hart

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

Keywords: Chlamydia trachomatis, Neisseria gonorrhoeae, real-time PCR, laboratory diagnosis, urine

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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 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][8][9] Here we describe a real-time multiplex LDA 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

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 LDQA. 9-11 Details of the oligonucleotides used in the final assay are given in table 1. 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 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 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

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 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.

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. 1 14 15 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 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 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. 9 17-20 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. 10 21 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 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

• 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. a Two samples were excluded from the CT kappa analysis due to repeatedly inhibitory results.

------------------- ----------------------------------------------------------------------------------- ----------- ---------------- NG-
Results for the remaining 79 CT resolved positives were concordant. 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.

  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 2007. 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 2009. A total of 14,319 patients were screened during this time. Of 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 12 000xg 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.

  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 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. 12 13

  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.

Table 1 .

 1 Oligonucleotide primers and probes used in this evaluation.

	Assay	Oligonucleotide	Sequence (5' to 3')

Table 2 .

 2 LDQA results in comparison to resolved data from 1028 urines for Chlamydia trachomatis and Neisseria gonorrhoeae.

	C. trachomatis Results

Table 3 .

 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)	34 -42	36 -39	CT DNA detected	-	33 -36	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)	39	40	CT DNA detected	CT DNA detected	-	CT Positive
	4	(1)	36 -40	-	CT DNA detected	CT DNA detected	34 -37	CT Positive
	2 (0)	-	-	-	Inhibitory	Inhibitory	Unclear a
	Total 13 (4)
	-, Negative; CP, real-time PCR crossing point; CT, C. trachomatis.

Table 4 .

 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. COBAS NG Amplicor defined grey zone results as OD between 0.300 to 0.999 and positive results if OD ≥1.000. 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.

				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	(3)		-	-	Grey zone	56 o C, 65 o C	-	-	-
	18 (9)		-	-	Inhibitory	-	-	-	-
	1	(0)		22	NG DNA detected	Inhibitory	-	22	-	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)		28 -40	NG DNA detected	NG DNA detected	-	28 -38	NG isolated	NG Positive
	3	(2)		-	-	NG DNA detected	-	-	-	-
	1	(1)		-	-	NG DNA detected	65 o C	-	-	-
	Total 34 (21)

-, Negative; CP, real-time PCR crossing point; NG, N. gonorrhoeae.

b

Table 5 .

 5 Neisseria gonorrhoeae urine PCR protocol validation results in comparison to bacterial culture data from14,319 sexual health screens.N. gonorrhoeae urine PCR protocol sensitivity 96.7%, specificity 99.8%, kappa value 0.99, PPV 88.1%, NPV 100%.

		N. gonorrhoeae Results
		Urine PCR Protocol Result
		Positive	Negative
	Positive	185	6	191
	Bacterial
	Culture
	Result
	Negative	25	14,100	14,125
		210	14,106	14,316

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


Merlijn Kramer
email: merlijn.kramer@rivm.nl

Liesbeth Mollema

Gaby Smits

Hein Boot

Hester De Melker

Fiona Van Der Klis

Age-specific HPV seroprevalence among young females in The Netherlands

Keywords: HPV, seroprevalence, women, epidemiology, sexual behaviour

Objectives: To obtain insight into the age-specific seroprevalence for human papillomavirus (HPV) 6, 11, 16, and 18 among females before introduction of HPV vaccination in the Netherlands.

Methods:

In a population-based study in The Netherlands, 637 sera of 11-26-year-old females were tested for HPV6/11/16/18 antibodies. Sera were tested using a competitive Luminex assay with neutralizing monoclonal antibodies specific for each serotype.

Associations between HPV seropositivity, demographics, and sexual behaviour were studied with logistic regression. Seroprevalences were standardized for age and urbanization degree to the general female population in the Netherlands.

Results: Overall prevalence of antibodies against HPV6/11/16/18 was 7.9%. 4.3% had antibodies against HPV types 6/11, and 4.4% had antibodies against HPV types 16/18. HPV seropositivity significantly increased with age (OR 1.2; 95%CI 1.1-1.4), starting at the age of 16 years (median age of sexual debut in The Netherlands). A former diagnosis with sexually transmitted infections was also significantly associated with HPV seropositivity (OR 6.3; 95%CI 2.2-17.9).

Conclusions:

In addition to 12-year-old girls who are targeted for routine HPV vaccination, also girls up to 16 years are likely to benefit substantially from HPV16/18 vaccination.

Testing for the presence of HPV antibodies in females after introduction of vaccination makes it possible to monitor the impact of immunisation at the population level.

INTRODUCTION

Persistent infection by certain high risk human papillomavirus (hrHPV) types is known to be responsible for cervical cancer. However, only a small percentage of women who become infected will develop precancerous lesions which can progress to cervical cancer. [START_REF] Ho | Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia[END_REF] The majority of genital HPV infections are without symptoms and transient. More than 90% of detected infections are cleared within two years. [START_REF] Moscicki | Chapter 5: Updating the natural history of HPV and anogenital cancer[END_REF] A quadrivalent (HPV6, 11, 16 and 18) and a bivalent (HPV16 and 18) prophylactic HPV vaccine have been licensed in Europe recently.

Both vaccines protect against the hrHPV types 16 and 18, which are responsible for an estimated 73% of cervical cancer cases in Europe. [START_REF] Smith | Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update[END_REF] In phase III trials, both vaccines have been shown to prevent more than 90% of precancerous lesions associated with types 16 or 18 among HPV naïve women aged 15 to 26 years. [START_REF] Schiller | An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results[END_REF][START_REF] Paavonen | Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomized study in young women[END_REF] The primary target group to consider for routine vaccination is girls at the age just before the onset of sexual activity, because HPV infections are common and frequently acquired soon after initiation of sexual activity. [START_REF] Jr | Age considerations when vaccinating against HPV[END_REF] Vaccination does not affect the course of an existing vaccine-type infection and the effectiveness of immunization for subsequent infections (of that type) is uncertain. [START_REF] Schiller | An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results[END_REF] In 2008, the Minister of Health in The Netherlands decided to include HPV vaccination in the National Immunization Program (NIP) for girls aged 12-years and a catch-up program for girls aged 13 to 16 years, based on reported age of sexual debut and sexual activity. [START_REF] De Graaf | Seks onder je 25e: Seksuele gezondheid van jongeren in Nederland anno 2005[END_REF] The catch-up program started in 2009 and HPV vaccination through the NIP will start in 2010, both using the bivalent HPV vaccine.

Pre-vaccination data on the occurrence of HPV infection in the population, in particular in women, are essential to monitor the impact of vaccination. While a lot of data are available from Dutch women 30 years of age onwards as cervical cancer screening starts, data from younger women/girls are scarce. [START_REF] Smith | Age-specific prevalence of infection with human papillomavirus in females: a global review[END_REF] In The Netherlands, an estimated HPV16 and -18 prevalence of respectively 2.8% and 1.4% relies on a HPV DNA study among a convenience sample of unscreened women aged 18-29 years. [START_REF] Lenselink | Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in The Netherlands[END_REF] Other reported HPV16 and -18 DNA prevalences in the age group 18-24 years, respectively 7.5% and 2.3%, are based on data of opportunistic cervical screening. [START_REF] Coupe | Age-dependent prevalence of 14 high-risk HPV types in The Netherlands: implications for prophylactic vaccination and screening[END_REF] These data are based on detection of HPV DNA in genital mucosa, where HPV DNA identifies current infections. Although not all infections result in seroconversion, HPV antibodies may persist for many years and can serve as markers of both ongoing and previous infections. [11,[START_REF] Carter | Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection[END_REF] Several studies have shown that HPV (sero)prevalence increases with age soon after the start of sexual activity. [START_REF] Coupe | Age-dependent prevalence of 14 high-risk HPV types in The Netherlands: implications for prophylactic vaccination and screening[END_REF][START_REF] Jit | Prevalence of human papillomavirus antibodies in young female subjects in England[END_REF][START_REF] Dunne | Prevalence of HPV infection among females in the United States[END_REF][START_REF] Burchell | Chapter 6: Epidemiology and transmission dynamics of genital HPV infection[END_REF][START_REF] Moscicki | Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females[END_REF][START_REF] Newall | Population seroprevalence of human papillomavirus types 6, 11, 16, and 18 in men, women, and children in Australia[END_REF][START_REF] Markowitz | Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: National Health and Nutrition Examination Survey 2003-2004[END_REF] Maximum seroconversion rate of IgG after incident HPV16 infection was 56.7% by 8 months in a study of Ho et Despite incomplete seroconversion and waning of antibodies, population-based seroepidemiological studies performed before and at regular intervals after introduction of vaccination is one of the tools to monitor the impact of mass vaccination against HPV on the frequency of HPV infection. This paper describes the first age-specific seroprevalence for HPV6, -11, -16, and -18 among girls and women aged 11-26 years in The Netherlands. Sera and questionnaire data were collected in a cross-sectional population-based study in The Netherlands. [START_REF] Van Der Klis | Second national serum bank for population-based seroprevalence studies in The Netherlands[END_REF]

METHODS

Study population

A cross-sectional population-based study, the PIENTER 2 project, was performed from 2006 to 2007. Dutch inhabitants aged 0-79 years from 40 municipalities throughout the country were invited to participate. Of all invitees, 33% took part in the study. This sample of the general population in The Netherlands will be used to evaluate the present existing NIP and to determine age-specific seroprevalences against future NIP vaccine candidates, such as HPV.

A more detailed description of the study design and objectives of the PIENTER 2 project has been reported previously. [START_REF] Van Der Klis | Second national serum bank for population-based seroprevalence studies in The Netherlands[END_REF] The study proposal was approved by the Medical Ethics Testing Committee of the foundation of therapeutic evaluation of medicines (METC-STEG) in Almere, the Netherlands.

The objective of this study was to provide age-specific data on HPV infection in girls/women prior to the national HPV-immunization program. Therefore, all females aged 11-26 years were selected from the nationwide sample of the PIENTER 2 project (n=637).

Serological testing

Serum samples were tested for specific neutralizing antibodies to HPV6, -11, -16 and -18 using a competitive Luminex assay. [START_REF] Dias | Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18[END_REF][START_REF] Opalka | Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay[END_REF] Antigen HPV specific virus-like particles (VLPs) [START_REF] Cook | Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein LI from Saccharomyces cerivisiae[END_REF] were coupled to distinct fluorescent Luminex microspheres. Sera were incubated with the coupled beads in the presence of HPV type-specific neutralizing monoclonals which compete for the binding sites on the VLPs. The monoclonals are HPV type-specific, bind neutralizing epitopes on the VLPs and show minimal cross-reactivity. [START_REF] Dias | Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18[END_REF][START_REF] Yeager | Neutralization of human papillomavirus (HPV) pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies[END_REF] Sera were called seropositive above the cutoffs as determined in [START_REF] Dias | Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18[END_REF]: 20, 16, 20 and 24 milli-Merck Units per milliliter (mMU/ml) for HPV6, -11, -16, and -18 respectively. The serological method used in this study is well validated and bridging to former neutralising assays has been performed.

Data analyses

Information on age, ethnic origin, educational level, and urbanization degree was available for the total study population. Ethnic origin was classified as indigenous Dutch, first generation and second generation of migrants according to the country of birth of the participant and the country of birth of the participant's parents. Educational level of the participant or the mother (for participants <15 years, only the educational level of the mother was available) was classified as low (primary school), medium (lower vocational school, secondary school), or high (higher vocational school, university). Urbanization degree was divided into two classes based on the number of addresses per square meter, namely high or middle/low urbanization degree.

[24] Information on marital status and sexual behaviour was only available from participants aged 15-26 years. Information on the sexual behaviour included data on age of sexual debut, steady and casual partnerships, number of partners in the last 6 months, and sexually transmitted infections (STI) including HIV diagnosed ever.

Seroprevalence and geometric mean titres (GMT) were estimated for HPV6, -11, -16, and -18. For GMT, only titres of seropositive samples (i.e. samples with titers above specified cutoff values) were included in the analyses. Associations between HPV6/11/16/18 seropositivity and demographic variables or sexual behaviour were studied with univariate logistic regression. For women aged 15-26 years, variables significantly (p<0.2) related to HPV infection in univariate analyses were entered into multivariate logistic regression analysis. Multivariate analyses were not studied separately by HPV type due to a low number of seropositives. To account for non-response, each person was assigned a sampling weight that incorporated the probability of selection and included adjustment for age and urbanization degree. [START_REF] Van Der Klis | Second national serum bank for population-based seroprevalence studies in The Netherlands[END_REF] Moreover, we also adjusted for the two stage cluster sampling by taking into account the strata (regions) and clusters (municipalities). In this way results were representative for the general female population in The Netherlands on January 2007.

RESULTS

General characteristics

The median age of our study population (n=637) was 19 years (Interquartile range 14-23 years). The majority of the total sample population (84%) was of indigenous Dutch origin, 10% was second generation migrant and 6% was first generation migrant. Of those 15 years and older (n=473), 68% had a medium education level (including those who may go on to complete higher vocational school or university) and 73% of the females were not married or living together. Of the 15-year-old females, 6% reported to be sexually active compared to 57% of the 17-year-old females (Figure 1). A quarter of the females older than 15 years reported having had no sexual contact yet.

HPV seroprevalence and risk factors

The overall prevalence of antibodies against at least one of the HPV types 6/11/16/18 amounted to 7.9% among 11-26-year-old females. 4.3% had antibodies against low-risk HPV (lrHPV) types 6 and/or -11 and 4.4% had antibodies against hrHPV types 16 and/or 18 (Table 1). HPV types 6 and 16 were more prevalent (each 3.9%) than HPV11 (1.2%) and -18 (1.1%).

Evidence for multiple infections with the different HPV types tested for, was found in the sera of 10 females (1.6%). The majority (8 of those 10 women) had antibodies against HPV16 and any of the other three HPV types. Women with multiple infections were older than 18 years, sexually active for more than two years and reported at least one sex partner in the last 6 months.

None of the girls between 11 and 16 years of age had detectable antibodies against hrHPV16 or -18 (Table 1). After this age the HPV seroprevalence increased with age showing a similar increase with age for HPV6/11 and HPV16/18. Between the ages 21-26 years the overall HPV6/11/16/18 seroprevalence remained at a percentage of about 16% with the highest prevalence of 18% among 23-24-year-old girls (Figure 2). CI, confidence interval a For 41 females not sufficient material was available for HPV6 test.

b For 139 females not sufficient material was available for HPV11 test.

c For 4 females not sufficient material was available for HPV18 test.

As described above, increasing age was associated with HPV6, -11, -16, or -18 seropositivity (Table 2). For females in the age of 15-26 years, we also found that those who reported to have ever been diagnosed with an STI were more often HPV seropositive (Table 2). No association was found for ethnicity, educational level, marital status, and number of partners in the last 6 months. Age at sexual debut and the number of years being sexually active were associated univariately with being HPV seropositive for types 6, 11, 16 and/or 18, but were not significant after adjustment in multivariate analysis (Table 2). Table 2 also shows that four women who reported not to be sexually active yet had antibodies against HPV. Three of them had antibodies against HPV6 or -11, while the other one had antibodies against HPV16.

Geometric mean titres

A large proportion of the HPV seronegative samples had titres below the detection limit (i.e.

the lower limit of quantitation), respectively 88%, 96%, 95%, and 95%. The GMT was highest for HPV16 (82 mMU/ml (95%CI 57-118)) and lowest for HPV11 (30 mMU/ml (95%CI 14-65)). We found a GMT of 76 mMU/ml (95%CI 45-129) for HPV18 antibodies and of 67 mMU/ml (95%CI 45-102) for HPV6 antibodies. In line with our study, large-scale surveys of HPV seroprevalences among children younger than 13 years of age found low seroprevalences. [START_REF] Dunne | Seroprevalence of human papillomavirus type 16 in children[END_REF] Various studies also have shown increasing HPV seroprevalences with age, starting around the age of sexual debut. [START_REF] Jit | Prevalence of human papillomavirus antibodies in young female subjects in England[END_REF][START_REF] Newall | Population seroprevalence of human papillomavirus types 6, 11, 16, and 18 in men, women, and children in Australia[END_REF][START_REF] Markowitz | Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: National Health and Nutrition Examination Survey 2003-2004[END_REF] Comparison of HPV serological data should be done with caution due to differences in type of assay used and associated seropositivity cut-off values. Despite they used identical assays and cut-off values as in our study, studies in the United States, England, Norway, Germany, and Australia found higher seroprevalences for HPV in females aged 10-29 years. [START_REF] Jit | Prevalence of human papillomavirus antibodies in young female subjects in England[END_REF][START_REF] Newall | Population seroprevalence of human papillomavirus types 6, 11, 16, and 18 in men, women, and children in Australia[END_REF][START_REF] Markowitz | Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: National Health and Nutrition Examination Survey 2003-2004[END_REF][START_REF] Skjeldestad | Seroprevalence and genital DNA prevalence of HPV types 6,11,16 and 18 in a cohort of young Norwegian women: study design and cohort characteristics[END_REF][START_REF] Barthell | Baseline characteristics and prevalence of HPV 6, 11, 16, 18 in young German women participating in phase III clinical trials of a quadrivalent HPV (6/11/16/18) vaccine[END_REF] Compared to our study, females in the population-based American study reported more often to have had their sexual debut before their 16 th birthday. In the Norwegian and German study, regular partners and new partners in the past 6 months were more often reported compared to our study. As both the age of sexual debut and the number of partners were found to be associated with a higher HPV prevalence, this could explain the differences in seroprevalence. [START_REF] Burchell | Chapter 6: Epidemiology and transmission dynamics of genital HPV infection[END_REF] However consistent to these studies, we found the highest seroprevalences for HPV6 and -16. Moreover in line with our study, all females younger than 17 years in the German study were seronegative for HPV16 and 18. [START_REF] Barthell | Baseline characteristics and prevalence of HPV 6, 11, 16, 18 in young German women participating in phase III clinical trials of a quadrivalent HPV (6/11/16/18) vaccine[END_REF] For our risk factor analysis we have to acknowledge that the number of seropositives were low and we were unable to perform multivariate analysis separately by HPV type. Our study was population-based and we have no indications that our results are influenced to a considerable extent by demographic characteristics (age, marital status, educational level).

The distribution of these characteristics in our study population is similar to that in the general female population in this age group [24], except that our sample population consisting of more persons of indigenous Dutch ethnic origin compared to the general population (84% vs. 76%). However, no association was found between seropositivity for HPV and ethnicity.

The reported median age of sexual debut in our study was similar to those reported by earlier studies on sexual behaviour in The Netherlands (16 vs. 16.7 years). [START_REF] De Graaf | Seks onder je 25e: Seksuele gezondheid van jongeren in Nederland anno 2005[END_REF][START_REF] Bakker | Seksuele gezondheid in Nederland 2009[END_REF] Other sexual behavioural characteristics of our study population such as diagnosed with an STI ever, steady partnership and condom use with this partner were comparable to behavioural factors described in earlier studies. [START_REF] De Graaf | Seks onder je 25e: Seksuele gezondheid van jongeren in Nederland anno 2005[END_REF][START_REF] Bakker | Seksuele gezondheid in Nederland 2009[END_REF] However, underestimation of the HPV seroprevalence is conceivable as in our study population condom use with casual partners was more frequent and a smaller percentage of females aged 15-17 years reported to be already sexually active (27% vs. 42%). [START_REF] De Graaf | Seks onder je 25e: Seksuele gezondheid van jongeren in Nederland anno 2005[END_REF] Sexual behaviour was self-reported which could result in underreporting of sexual activity.

This underreporting may potentially have been somewhat larger among young females.

Underreporting of sexual behaviour might explain seropositivity among persons who reported no sex partners. Females who reported having had no sexual contact yet were mainly seropositive for HPV6 or -11. LrHPV types 6 and 11 cause the majority of genital warts.

Although, in particular lrHPV can be transmitted through skin-to-skin contact as well, without having sexual intercourse. In the present study, HPV6 and/or -11 infections were found from the age of 13 years onwards and increased with age. Genital warts were the most frequently diagnosed viral STI reported in all Dutch STI centres in 2008. [START_REF] Koedijk | Sexually transmitted infections, including HIV, in The Netherlands in 2008[END_REF] In women with genital warts, 59% was younger than 25 years and for 11% a history of gonorrhoea, infectious syphilis or Chlamydia infection was reported. [START_REF] Koedijk | Sexually transmitted infections, including HIV, in The Netherlands in 2008[END_REF] Similar to our study, earlier studies have shown an independent association between diagnosed ever with an STI and HPV infection. [START_REF] Moscicki | Risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females[END_REF][START_REF] Nielsen | Type-Specific HPV Infection and Multiple HPV Types: Prevalence and Risk Factor Profile in Nearly 12,000 Younger and Older Danish Women[END_REF] To be diagnosed with an STI ever could serve as a marker for sexual exposure to HPV among women. Or STIs might increase women's susceptibility to HPV infection. [START_REF] Burchell | Chapter 6: Epidemiology and transmission dynamics of genital HPV infection[END_REF] Although the number of years of sexual activity was associated with HPV seroprevalence in univariate analyses, in multivariate analyses age was found to be a stronger predictor for HPV6/11/16/18 seropositivity than age of first sexual contact.

Taking into account that half of the individuals show an antibody response after infection would imply that the percentage of females who have previously been infected with HPV6, -11, -16, or -18 is higher. [START_REF] Carter | Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection[END_REF] Although, in the oldest age groups (21-26 years) no further increase in seroprevalence was observed; HPV seroprevalences were about similar, which might suggest waning antibody levels over time. [START_REF] Carter | Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection[END_REF] Alternatively this could result from a small sample size or reflect a cohort effect.

This study gives information on the baseline HPV6/11/16/18 seroprevalence in 11-26-yearold girls/women in The Netherlands before introduction of vaccination. No antibodies against HPV16/18 were found among women younger than 17 years. As the median age of sexual debut is 16 years, the majority of the girls aged 13-16 years is not sexually active yet. Taking this and the serological results of our study into consideration, girls aged 13-16 years are likely to benefit substantially from HPV16/18 vaccination in addition to 12-year-old girls.

This supports the decision to implement a catch-up HPV vaccination for this age group.

Despite the lower sensitivity compared to HPV DNA testing, serological surveillance is an important additional tool to study the impact of HPV vaccination. In contrast to HPV DNA testing where vaginal swabs or cervical smears are needed, serum is easier to collect in younger age groups and less prone to sampling errors. After implementation of routine HPV16/18 vaccination and the catch-up campaign, comparable studies among vaccinated and unvaccinated women will be repeated to study the impact of HPV-vaccination. Testing for the presence of HPV antibodies and DNA in females in a broader age range will make it possible to study the vaccine impact at the population level, i.e. including the effects on those not directly targeted by the HPV-vaccination program.

Figure 1 Figure 2

 12 Figure 1 Reported age of first sexual intercourse (bars) and percentage of ever had sexual intercourse by age (line) for female participants (15-26 years) in the PIENTER 2 project, 2006-2007, The Netherlands

  al. among young female students (mean age 20 years).[11] A study ofCarter et al. observed seropositivity 18 months after incident HPV6, -16, or -18 infection in about 60% of the women.[START_REF] Carter | Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection[END_REF]

Table 1

 1 Weighted age-specific seroprevalences for HPV6, -11, -16, and -18 among 11-26-year-old females, The Netherlands, 2007

	Proportion seropositive, % (95%CI)

Table 2

 2 General characteristics of the total sample population and the sample population of 15 years and older

		Females	HPV seroprevalence		Logistic regression
						Univariate		Multivariate
		N (%)	N (%)*	OR (95%CI)	p-value	OR (95%CI)	p-value
	Total population	637 (100)	47 (8)
	Age						<0.0001	Not applicable
	11-14	164	(26)	1	(1)	1.0
	15-18	133	(21)	3	(2)	2.8 (0.4-21.2)
	19-22	174	(27)	17	(11)	14.6 (2.4-88.8)
	23-26	166	(26)	26	(17)	23.4 (3.9-139.1)
	Ethnicity						0.1007
	Indigenous Dutch	537	(84)	38	(7)	1.0
	Non-indigenous Dutch	100	(16)	9	(9)	1.8 (0.8-3.6)
	Population ≥15 years	473		46
	Age						<0.0001	0.0007
	15-18	133	(28)	3	(2)	1.0		1.0
	19-22	174	(37)	17	(11)	5.2 (1.6-16.5)		5.4 (1.6-17.4)
	23-26	166	(35)	26	(17)	8.3 (2.7-25.6)		9.2 (2.9-29.1)

  Our findings show that the percentage of females aged 11-26 years in the Netherlands with antibodies against HPV6/11 and HPV16/18 amounted both to about 4%. Seroprevalence increased steeply with age from 17 years onwards following the increasing percentage of women who started being sexually active. Below the age of 17, all girls were seronegative for HPV16/18.

					0.0258
				1.1 (0.1-8.8)
			(14)	4.3 (1.6-11.5)
			(18)	5.6 (1.5-21.2)
			(9)	2.5 (0.5-11.7)
	STI diagnosed ever	19 (4)	7 (38)	6.6 (2.4-17.9)	0.0008	6.3 (2.2-17.9)	0.0008

OR, odds ratio; CI, confidence interval; IQR, Interquartile range; STI, sexually transmitted infections *seroprevalences weighted by age and urbanization degree DISCUSSION

Acknowledgements

We would like to thank Mariet Feltkamp for critically reading the manuscript.

Key messages

• In addition to 12-year-old girls targeted for routine HPV vaccination, girls up to 16 years are likely to benefit substantially from HPV16/18 vaccination.

• Older age and a former diagnosis with sexually transmitted infections were associated with HPV seropositivity.

• Sero-epidemiological studies performed before and after introduction of HPV vaccination is a tool to monitor the impact of vaccination on the frequency of HPV-infection.

Contributors

MK conducted the data analysis and the drafting of the manuscript. LM, GS, HB, HM, FK contributed to the PIENTER study design and contributed to collection of clinical data and samples. LM contributed to the data analysis. GS contributed to laboratory testing. All authors critically revised the manuscript and approved the final version.

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Emily J Tweed
email: emilytweed43@gmail.com

Antony Hale

Martin Hurrelle

Ruth Smith

Valerie Delpech

Murad Ruf

Paul E Klapper

Mary E Ramsay

Lisa J Brant

Monitoring HIV testing in diverse healthcare settings: results from a sentinel surveillance pilot

Keywords: HIV, screening, sentinel surveillance, feasibility, diagnosis Word count: 2, 923

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

An estimated 20,700 of 73,300 adults (28%) living with HIV in the UK are unaware of their infection. Almost one-third of newly-diagnosed individuals are diagnosed late, with a CD4 cell count below 200 cells/mm 3 and at risk of poorer treatment response and increased mortality [START_REF]HIV in the United Kingdom: 2008 report[END_REF].

Recent research has highlighted missed opportunities to detect HIV infection among patients presenting at a range of non-genitourinary medicine (GUM) health services [START_REF] Burns | Missed opportunities for earlier HIV diagnosis within primary and secondary healthcare settings in the UK[END_REF][START_REF] Chadborn | The late diagnosis and consequent short-term mortality of HIV-infected heterosexuals (England and Wales, 2000-2004)[END_REF][START_REF] Sudarshi | Missed opportunities for diagnosing primary HIV infection[END_REF]. Scaling-up of voluntary confidential HIV testing is increasingly recognised as a key tool in preventing new infections and reducing morbidity and mortality among infected individuals [START_REF] Hamill | Time to move towards opt-out testing for HIV in the UK[END_REF].

In the UK, there is a high uptake of HIV testing at GUM clinics and as part of routine antenatal care [START_REF]HIV in the United Kingdom: 2008 report[END_REF]. In September 2007, a letter from the Chief Medical Officer (CMO) to healthcare providers encouraged diagnostic testing in non-HIV specialties among people with a recognised risk factor for infection or presenting with a clinical indicator condition such as malaise, weight loss or oral candidiasis [START_REF] Chief | Improving the detection and diagnosis of HIV in non-HIV specialties including primary care[END_REF]. Recent national guidelines have built on this by recommending scaling up the universal offer of an HIV test among people presenting with defined indicator conditions and in specific health care settings such as TB and hepatitis clinics. In addition, the guidelines recommend the expansion of HIV testing to all men and women aged 15 to 59 years registering in general practice or admitted to a general medical ward in areas of high HIV prevalence (defined as an area where the diagnosed prevalence of HIV exceeds 2 per 1000 population) [START_REF]British HIV Association, British Association for Sexual Health and HIV[END_REF]. In the United States, Centres for Disease Control and Prevention guidelines recommend opt-out screening of all patients aged 13 to 64 years in all health-care settings [START_REF] Bernard | Revised Recommendations for HIV Testing of Adults, Adolescents, and Pregnant Women in Health-Care Settings[END_REF].

The Health Protection Agency Centre for Infections (CfI) receives voluntary surveillance reports from clinicians and virology laboratories for all persons newly diagnosed with HIV in the United Kingdom. These are cross-checked with other surveillance systems such as the Survey of Prevalent HIV Diagnosed Individuals (SOPHID) and CD4 surveillance to maximise completeness. In addition, the HPA collects data on the offer and uptake of HIV testing in GUM and antenatal clinics. Little is known about testing practices and yield outside these services. A recent study of HIV testing uptake in general practices found low rates of testing in this setting [START_REF] Evans | Trends in HIV testing and recording of HIV status in the UK primary care setting: a retrospective cohort study 1995-2005[END_REF].

The sentinel surveillance of hepatitis testing has been a valuable adjunct to routine hepatitis surveillance systems, providing data on the demographic characteristics of people tested (and testing positive) for viral hepatitis, place of testing, prevalence in the population tested, and longterm trends in testing [START_REF] Brant | Sentinel laboratory surveillance of hepatitis C antibody testing in England: understanding the epidemiology of HCV infection[END_REF][START_REF] Brant | Where are people being tested for anti-HCV in England? Results from sentinel laboratory surveillance[END_REF]. Using similar methods we carried out a twelve-month pilot study at one sentinel centre to assess the feasibility and utility of this form of surveillance for monitoring HIV testing.

METHODS

The Leeds Teaching Hospitals Trust (LTHT) laboratory is part of the Leeds and Bradford Pathology Partnership which performs testing for two teaching hospitals, the primary care services provided by Leeds Primary Care Trust and some primary care services within Bradford and Airedale Teaching Primary Care Trust. It serves an overall population of approximately 1.1 million people. Demographic data for the cities of Leeds and Bradford are described in the latest census report from 2001 [START_REF]Census data for Yorkshire and the Humber[END_REF]; the diagnosed prevalence of HIV per 1000 people aged 15-59 is 1.7 and 1.0 in Leeds and Bradford & Airedale respectively. This site was chosen for the pilot because the computer and communications infrastructure for the hepatitis sentinel surveillance scheme was already well established there.

Laboratory test results and demographic information (such as date of birth and sex) for all samples tested for anti-HIV antibody at this laboratory were extracted electronically from laboratory information systems for the period 1 st April 2007 to 31 st March 2008. These data were collated, cleaned and checked for consistency by the sentinel surveillance project co-ordinator, with patient names replaced by pseudononymised soundex codes [START_REF] Mortimer | Soundex' codes of surnames provide confidentiality and accuracy in a national HIV database[END_REF] and individuals identified using a unique unnamed reference number. The dataset was then forwarded to CfI for analysis.

De-duplication to identify individuals tested more than once under different reference numbers or in different services during the study period was undertaken using date of birth, sex and soundex or GUM clinic number (where available). Age at first test was calculated for each individual, with children aged less than 1 year excluded since positive results in this age group may reflect passively-acquired maternal antibody rather than true infection.

Where an individual had been tested on more than one occasion, the number of tests were recorded, reviewed in chronological order and their HIV status classified using a hierarchical system [START_REF] Brant | Sentinel laboratory surveillance of hepatitis C antibody testing in England: understanding the epidemiology of HCV infection[END_REF]. If a negative result was followed by a positive result, the individual was classified as positive but flagged as a potential seroconversion: follow-up samples from these cases were then reviewed by the consultant virologist for confirmation.

The origin of the test request was used to define the services in which testing took place, such as general practitioners' (GP) surgeries, GUM clinics, antenatal care and various hospital specialties. These service types were further classified as primary care or secondary care, as shown in Table 1. Each individual was counted only once regardless of the number of tests: if an individual was tested more than once, their test location was classified according to the service type in which they were first tested during the study period. Clinical details accompanying the test request were used to identify women tested as part of antenatal care where the test request did not originate from an antenatal clinic, for example in general practice. 'Individuals tested outside antenatal and GUM settings' were defined as those individuals tested in a service type other than a GUM clinic or antenatal clinic who did not have antenatal screening mentioned in the clinical details field accompanying the test request. All samples referred for confirmatory testing or tested for quality control purposes were excluded.

Individuals who tested HIV positive during the study period were matched to the HPA national database of new HIV diagnoses, using a combination of GUM clinic number, date of birth, sex, soundex, origin of test request and region. A matched individual with a HIV laboratory test date before or up to 21 days after a new diagnosis report (to allow for delays in laboratory processing and data entry) was classified as being newly diagnosed.

Data were managed in MS Access and analysed in MS Excel and STATA (StataCorp, 2003).

Differences in the proportion testing positive for anti-HIV were assessed using chi-squared tests.

Mean ages were compared using an unpaired t-test.

The HPA has permission from the National Information Governance Board, under Section 251 of the NHS Act 2006, to handle personally identifiable data for the purposes of communicable disease surveillance. Sentinel laboratory surveillance has also been approved by the Northern and Yorkshire Multi-Centre Research Ethics Committee (MREC1/3/76) and the Public Health Laboratory Service Ethics Committee.

RESULTS

During the study period, 41,013 individuals over one year of age were tested at least once for anti-HIV antibody at the LTHT laboratory; 0.8% (n=312) were positive. 48 children aged less than 1 year were also tested, of whom two were positive: these individuals were excluded from all subsequent analyses.

Most HIV testing (77%) took place in GUM clinics and as part of antenatal screening (Table 1). A further 13% could be accounted for by routine screening of people undergoing fertility treatment, occupational health assessment or haemodialysis.

The proportion testing positive was highest among individuals tested in infectious disease services and general medical and surgical departments (Table 1). No individuals tested positive in renal units, occupational health services or Accident and Emergency departments.

Testing in GUM clinics

A total of 15,764 individuals were tested at three GUM clinics that send samples to the sentinel laboratory. Around half (51%; n=8,029) of individuals tested in GUM clinics were male, while the positivity rate for males and females was 1.2% (n=100) and 0.8% (n=58) respectively. The majority of those tested (82%; n=12,949) and 47% (n=82) of those testing positive were aged between 15 and 34 years. The proportion positive (3.5%; n=89/2,559) was highest in individuals aged 35-54 years.

Antenatal testing

In total, 15,754 women were tested as part of antenatal care, 37 (0.2%) of whom were positive.

Together this accounts for 58% of all females tested (38% of all individuals) and 60% of women aged between 15 and 44 years. The proportion positive among women screened through antenatal care was significantly lower than among those tested in other settings (0.2% v 1.2%; p<0.001).

Testing outside GUM clinics and antenatal screening

9,454 individuals were tested outside GUM clinics and antenatal screening, 102 (1.1%) of whom were positive. 1,568 individuals were tested in general practice, of whom 22 (1.4%) were positive.

Figure 1 shows the age and sex distribution of these individuals. Individuals tested outside GUM clinics and antenatal screening were on average 10.3 years older than those tested in GUM and antenatal settings (unpaired t test; CI=10.0-10.5 years, p<0.001); individuals testing positive outside GUM clinics and antenatal screening were on average 2.9 years older than those testing positive in these settings (unpaired t test; CI=0.8-5.0 years, p=0.008).

Repeat testing

Table 2 shows the extent of repeat HIV testing; 3,583 (8.7%) of the 41,013 people tested during the study period were tested more than once. The majority of repeat testing took place in GUM clinics and renal units: 8.5% (n=1,335) of individuals tested at a GUM clinic and 57.5% (n=692) of individuals tested in renal units were tested more than once over the study period.

Three seroconversions were identified among people tested more than once. All were male GUM clinic attendees, two aged between 35 and 44 years and one aged between 45 and 54 years. On follow-up with requesting clinicians, all were reported to be men who have sex with men (MSM). 1).

Feasibility

Establishment of the data collection system for HIV at the LTHT laboratory required a one-off period of three day's work by an IT engineer, since the majority of the necessary hardware, software and data extraction processes were already in place for hepatitis sentinel surveillance (as detailed in [START_REF] Brant | Sentinel laboratory surveillance of hepatitis C antibody testing in England: understanding the epidemiology of HCV infection[END_REF]). Data extraction by staff at the participating laboratory required approximately one hour's work each month, while data processing and analysis required approximately one day's work per month by an information officer. This pilot involved a set-up cost of approximately £1000 for labour and materials, with projected ongoing costs of approximately £330 per year.

DISCUSSION

This study indicates that the majority of HIV tests are performed in GUM clinics or as part of antenatal care. Furthermore, routine screening in occupational health services, renal units and fertility treatment services -aimed at ruling out infection rather than case detection -accounted for more than half of people tested outside these settings. Less than 4% of individuals were tested in general practice.

There is currently little discussion in the literature of HIV testing surveillance, although one behavioural survey from the US identified differences in self-reported HIV testing rates and locations according to ethnicity [START_REF] Rountree | HIV testing rates and testing locations, by race and ethnicity[END_REF]. We feel that this paucity of data reflects a gap in current surveillance activities which this study has made an important first step towards addressing.

This pilot study has demonstrated that sentinel laboratory surveillance is a feasible and informative means of monitoring HIV testing in different service types, trends in testing over time and the demographic characteristics of the population tested. Funding and agreement of existing sentinel laboratories to expand this pilot project has been secured and ethical approval is currently being processed.

One limitation of the study is the possibility of double-counting individuals tested more than once under different hospital or clinic numbers. Although a thorough de-duplication process was undertaken to identify such patients, a small number of duplicates may remain where details such as date of birth or soundex code were unavailable or incorrect. However, completion of patient details used in de-duplication is good in most hospital and primary care settings, and GUM clinic numbers tend to be used consistently within one clinic, so this problem is likely to predominantly affect the relatively small number of individuals moving between GUM and non-GUM services.

In this study, we did not have data on exposure category, ethnicity or country of birth of people tested, since these data are not currently routinely recorded in most laboratory information systems. However, integrated IT systems in some areas do allow access to ethnicity information and the planned upgrade of the NHS Care Records System may improve availability of these data in future.

One particular strength of the sentinel laboratory surveillance lies in the ability to match data with the national database of HIV and AIDS diagnoses and deaths to identify the service type in which an individual was first diagnosed. Currently, data on services in which case detection is taking place may be skewed by the fact that people testing positive in non-GUM services may only be reported to national surveillance after referral to GUM clinics for specialist care. The use of multiple identifiers (such as date of birth, sex and soundex/clinic number) in the matching process should allow for identification of such transfers of care.

The 26 individuals (of 312 testing positive in total) who could not be matched may reflect underreporting to national surveillance: a similar process of matching between sentinel surveillance and routine surveillance data demonstrated considerable under-reporting of hepatitis C diagnoses to national databases [START_REF] Brant | Sentinel laboratory surveillance of hepatitis C antibody testing in England: understanding the epidemiology of HCV infection[END_REF]. Alternatively, it may result from inadequate data for identifying individuals. 22 of the 26 unmatched individuals were tested in GUM clinics, where soundex codes are not usually available: although GUM clinic number can usually substitute for soundex in the matching process, it is possible that this identifier was incorrectly entered on the laboratory system or that they had been previously reported under a different clinic number or from a different clinic.

While this pilot study was limited to one laboratory, it offers proof of principle that such surveillance could be rapidly scaled up nationally by incorporation into existing hepatitis sentinel surveillance systems (currently active in 22 laboratories across England). This would enable automated collection of large amounts of data without additional work by the person requesting the test or the need for labour-intensive local audits. It would also provide the opportunity to monitor testing for co-infection with hepatitis B and C in HIV-positive individuals, something which is not currently possible with existing surveillance systems [START_REF] Dougan | Can current national surveillance systems in England and Wales monitor sexual transmission of hepatitis C among HIV-infected men who have sex with men?[END_REF]. Such a scheme may also be of value in other countries, particularly those with national health care systems where data collection from a network of representative sentinel laboratories is a feasible proposition.

As this paper has shown, sentinel surveillance can also help identify seroconversions among repeat testers: as data collection continues and longitudinal data accumulate, the potential of this aspect of the study will only increase. Although the roll-out of the serological testing algorithm for recent HIV seroconversion (STARHS) for all newly-diagnosed individuals will facilitate identification of recent infections, ongoing sentinel surveillance of testing provides the opportunity to monitor seroconversions in a range of healthcare settings without the cost and workload associated with additional laboratory testing. A similar system for identification of anti-HCV seroconversions has been successfully integrated into the sentinel surveillance of hepatitis testing study with minimal additional investment of time, providing valuable data on incidence of infection among individuals undergoing repeat testing and routes of transmission of recentlyacquired infections.

Recently published UK National Guidelines for HIV Testing [START_REF]British HIV Association, British Association for Sexual Health and HIV[END_REF] have identified levels of testing in primary and secondary care and the number of new HIV diagnoses made in different service types as key auditable standards. The study was conducted before the publication of these guidelines and therefore provides a baseline from which to monitor efforts to expand testing.

Current HIV testing surveillance focuses on GUM clinics and antenatal screening: sentinel laboratory surveillance data provides an opportunity to monitor trends in HIV testing and positivity rates in other health care services, such as general practices and prison clinics. Sentinel surveillance of HIV testing in multiple laboratories would also provide the opportunity to assess local and national trends in testing over time, both overall and in specific service types. Locally, such data may be used to investigate patterns of service use by different demographic groups and to evaluate changes in testing in response to local initiatives, such as awareness campaigns or new guidelines for healthcare professionals.

We conclude that sentinel laboratory surveillance of HIV testing should be prioritised in order to monitor and improve our understanding of patterns of HIV testing in the UK and to inform local and national policy.

KEY MESSAGES

• Little HIV testing is currently taking place outside GUM and antenatal settings in the UK.

• Sentinel laboratory surveillance is a feasible way of monitoring HIV testing in different service types, trends in testing and demographic characteristics of the population tested.

• Data on HIV testing could be incorporated into existing hepatitis sentinel surveillance, allowing rapid scale-up across England with minimal cost and effort.

Table 1 .

 1 Number of individuals tested, and testing positive, for anti-HIV antibody, by service type. In this table 'Antenatal screening' refers both to individuals tested in antenatal clinics and those tested as part of antenatal care in other services, as identified by clinical details accompanying the test request. *Includes 22 men and 19 individuals of unknown sex tested in antenatal services, none of whom tested positive: this may reflect partner follow-up or errors in data entry at the source laboratory.**Includes all hospital services not otherwise specified above: these can be broken down further into individual specialties but are grouped here in the interests of brevity. § These are hospital services where specific service type could not be identified, and may include any of the secondary care services listed above.The proportion positive among males tested was more than twice that of females (1.1% v 0.5%), though after excluding antenatal testing the proportion testing positive was similar (1.1% of males v 0.9% of females; p=0.174). People aged 15-34 years accounted for 76% (n=31,014) of those tested and 50% (n=157) of those testing positive. The proportion positive was highest in those aged 35-54 years (2%; n=144/8,196).The number of individuals tested for HIV by this laboratory increased by 5% overall in the sixmonth period following the Chief Medical Officer letter of September 2007, with a decrease in the number of people testing positive. The largest increase was seen in GUM clinics (17% increase, n=7,164 to 8,600) and other primary care settings (16% increase, n=1,366 to 1,624), while testing in renal units and other secondary care services declined.

						Number of new
	Service type	Number of individuals tested	% of all testing taking place in this service type	Number of individuals testing positive (% positive)	Number of individuals testing positive who could be matched to national surveillance report	diagnoses, among those matched to national surveillance (% new diagnoses among individuals tested in this
						service)
	Primary care
	Antenatal screening*	15,795	38.5	37 (0.2)	36	20 (0.1)
	Accident and Emergency	34	0.1	0 (0.0)	-	-
	General practice	1,568	3.8	22 (1.4)	22	17 (1.1)
	GUM clinics	15,764	38.4	173 (1.1)	151	88 (0.6)
	Occupational health	1,242	3.0	0 (0.0)	-	-
	Prison services	225	0.5	1 (0.4)	1	1 (0.4)
	Specialist drug services	422	1.0	2 (0.5)	2	2 (0.5)
	Secondary care
	General medical & surgical departments	164	0.4	12 (7.3)	12	9 (5.5)
	Infectious disease services	118	0.3	37 (31.4)	36	18 (15.2)
	Fertility treatment centres	3,091	7.5	2 (0.1)	2	1 (0.0)
	Obstetrics and gynaecology services	38	0.1	0 (0.0)	-	-
	Paediatric services	150	0.4	1 (0.7)	0	-
	Renal units	1,203	2.9	0 (0.0)	-	-
	Other hospital services**	1,191	2.9	25 (2.1)	24	17 (1.4)
	Unknown hospital services §	8	0.0	0 (0.0)	-	-
	Total, all service types	41,013	100.0	312 (0.8)	286	173 (0.4)
	N.B.

Table 2 .

 2 Number of occasions on which individuals in dataset have been tested for anti-HIV antibody.

	Number of tests within twelve-month period	Total number of individuals, all service types	Number of individuals tested in GUM clinics	Number of individuals tested in renal units
	1	37,430	14,429	512
	2	2,740	1,156	227
	3	558	149	241
	4	211	25	163
	5	56	3	46
	6	13	1	12
	7	3	1	1
	8	1	0	1
	9	1	0	1
	Total	41,013	15,764	1,204

ACKNOWLEDGEMENTS

We would like to acknowledge the contribution of the laboratory staff and clinicians of the Leeds and Bradford Pathology Partnership, who carried out testing and provided data. We would also like to thank Darren Lyons for his work on data extraction and processing, and Brian Rice for his assistance with the HIV new diagnoses database.

COMPETING INTERESTS

The authors declare they have no competing interests.

FUNDING

The resources for this pilot study were drawn from existing staffing and funds provided by the English Department of Health. The funding body did not have any involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

COPYRIGHT STATEMENT

I Emily Tweed, the corresponding author of this article (the Contribution) has the right to grant on behalf of all authors and does grant on behalf of all authors, a licence to the BMJ Publishing Group Ltd and its licensees, to permit this Contribution (if accepted) to be published in Sexually Transmitted Infections (STI) and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence set out at http://sti.bmj.com//ifora/licence.pdf.

STATEMENT OF CONTRIBUTORSHIP

This pilot study was conceived and designed by MH, PK and MR. Data extraction was carried out by AH, MH and ET. Data were analysed by ET and LB. RS matched sentinel surveillance data to the HPA new HIV diagnoses database, under supervision from VD. The paper was written by ET, with comments and contributions from all authors. All authors approved the final version submitted for publication.


Keywords: EPIDEMIOLOGY, HETEROSEXUALS, RISK BEHAVIOURS, STI CONTROL, SURVEILLANCE

Objective

Identification of STI risk groups is essential for optimal prevention and medical care. Until now, swingers, i.e. heterosexual couples who are practicing mate swapping, group sex, visit sex clubs for couples, are not considered as a specific risk group for STI in health care services and prevention. Here, we compare STI prevalence rates in swingers to other risk groups. Methods At the STI clinic, South Limburg, The Netherlands, we systematically register since 2007 whether an attendee is a swinger. STI clinic surveillance data are analyzed to assess swingers' share in consultations and STI diagnoses; here Chlamydia Trachomatis (CT) and/or Neisseria Gonorrhoea (NG). Results Of all 8971 consultations, 12% were comprised by swingers (median age 43 years, interquartile range 38-48). Overall, STI prevalence is highest in youth, MSM and swingers. Older swingers show a CT prevalence of 10% and a NG prevalence of 4%. In the older age group with STI diagnosis, swingers have with 55% and MSM with 31% the largest share in STI. Conclusions Swingers comprise substantial part of STI consultations. They are a mainly older group and have an important share in STI diagnoses. While other risk groups for STI, such as young heterosexuals and MSM, are systematically identified at STI health care facilities in order to provide them with fitting services, for swingers this is generally not the case. Swingers, as other groups with risk behaviours, need to be identified and treated as a risk group in STI prevention and care.

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 (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms).

Authors declare there are no competing interests

INTRODUCTION

The economic benefits of STI programs can be improved by effectively targeting the groups at risk. [START_REF] Chesson | Formulas for estimating the costs averted by sexually transmitted infection (STI) prevention programs in the United States[END_REF] Therefore, the continuous evaluation and identification of such groups is essential. Gains are highest when targeting those groups who most fuel the STI epidemic, i.e. sexual networks characterized by frequent partner change, concurrent partners, and less contact with the health care system. [START_REF] Wasserheit | The dynamic topology of sexually transmitted disease epidemics: implications for prevention strategies[END_REF][START_REF] Kretzschmar | Measures of concurrency in networks and the spread of infectious disease[END_REF] Until now, swingers, i.e. heterosexuals whom as a couple practice mate swapping, group sex, visit sex clubs for couples, are (inter)nationally not considered as a risk group for STI in health care services and prevention. However, since swingers connect in networks typed by multiple sex partners, concurrent partnerships and risk behavioura practices, [START_REF] Jenks | Swinging: a review of the literature[END_REF][START_REF] Van Der Poel | Swingers en hepatitis B. Onderzoek naar seksueel risicogedrag van swingers en kansen voor hepatitis-B-preventie[END_REF][6][START_REF] Niekamp | SWAP Study; Identification of STD spread within a sexual network of swingers. Oral presentation[END_REF][START_REF] Niekamp | Swingers, a hidden sexual culture at risk for STD. Oral presentation[END_REF] i.e. structures enabling quick spread of STI, swingers are a likely target for STI prevention and care. By addressing them properly, more tailored prevention and enhanced STI screening is likely to render gains on the individual and population (reduced STI burden) and economic (costs) level.

At our STI clinic, we started in 2007 to systematically register whether an attendee was a swinger. Here, we present surveillance data comparing STI prevalence among swingers, classic high risk and lower STI risk groups.

METHODS

The outpatient STI clinic of the South Limburg Public Health Service offers free-of-charge examination and treatment for STI. The clinic is spread over South Limburg (population 0.63 million) with three fixed visit locations. At every new consultation, attendees are tested for Chlamydia Trachomatis (CT) and Neisseria Gonorrhoea (NG) on first void urine (men) and self swab (women and men) by commercially available NAAT (SDA, Becton Dickinson ProbeTec ET system, Maryland, USA or PCR, Roche Cobas Amplicor, California, USA). Blood is tested on Treponema pallidum (TPPA), hepatitis B (HbsAg and AntiHBc, AXSYM, Abbott, Chicago, USA) and HIV (antiHIV (1/2), Axsym; Abbott, Chicago, USA; reactive samples are confirmed by Western Blot, HIVblot 2.2, Genelabs Diagnostics, Sciencepark, Singapore). Clients comprise persons with and without symptoms including those attending after partner notification. A client is registered as swinger when he or she identifies as heterosexual and as a couple has sex with other heterosexuals. For statistical analyses, the variable risk category was compiled hierarchically, with non overlapping categories, from heterosexual, female prostitute and MSM to swinger. We here analyse STI clinic surveillance data from January 2007 through December 2008, South Limburg, The Netherlands to assess STI prevalence and share in STI diagnoses of the risk groups and age categories. For the current analyses, STI is defined as a positive CT and/or NG diagnosis. We did not focus on syphilis, hepatitis B or HIV because of low number of positive diagnoses overall. Univariate and multivariate logistic regression analyses are used to assess nationality, risk category, age (and their interaction), as predictors for STI. We considered a p value <0.05 as statistically significant. Analyses were performed with the SPSS package version 14.0.2 (SPSS, Inc., Chicago, IL). The prevalence of CT, NG and CT and NG combined was 9.7%, 0.6%, and 10.1%, respectively, in heterosexuals; 10.2%, 6.3%, and 14.2% in MSM; 4.2%, 0.8%, and 4.8% in female prostitutes; and 6.4%, 4.3%, and 10.4% in swingers. STI prevalence was not associated with nationality, but was associated with age and risk group (both p<0.001). Resulting multivariate modeling including age, group and their interaction (p<0.001) STI prevalence showed a distinct pattern by age and by risk group (table 1): with older age STI prevalence decreased in heterosexuals and increased in swingers (both p<0.001) while remaining stable among MSM and prostitutes. In clinic attendees aging 45 years and over, a higher STI prevalence was observed in swingers (13.7%) and MSM (14.6%) compared to heterosexuals or prostitutes (2.9%). This was the case for both men and women (table 1). Older female swingers had the highest prevalence, even when compared to older male swingers (p 0.032).

RESULTS

DISCUSSION

We confirm previous UK observations suggesting older STI clinic attendees are a notable STI clinic population. [START_REF] Bodley-Tickell | Trends in sexually transmitted infections (other than HIV) in older people: analysis of data from an enhanced surveillance system[END_REF] Here, we add that swingers are a generally missed target for STI services. To our knowledge, no systematic data on STI diagnoses in these heterosexuals exist except the here presented data from our regional STI clinic showing that swingers considerably contribute to STI consultations and STI diagnoses. They have high STI prevalence, especially in older aged clients. The relative share of swingers in number of STI consultations and STI diagnosis comparable to those of the classic risk group of MSM.

Although exact estimates are unavailable, the swingers population is likely large. One of the largest dating websites for swingers (Swingers Date Club; www.sdc.com) estimate there are millions of swingers worldwide; in the Netherlands 15,000 couples (30,000 individuals) have a profile on this website.[10] The total number of swingers probably is larger. In the Netherlands, awareness of swingers being a risk group is growing, however slowly. The Dutch Ministry recently set up a method to register them in national STI surveillance. However, so far its national implementation is very limited (in 51% of consultations it is not registered), showing 3,296 consultations by swingers in 2008 (personal communication, F. Koedijk, RIVM, 2009). Swingers are probably not properly identified by STI care services; it is also possible that swingers are not optimally attending STI care. (Inter)national data on STI care seeking behaviour or STI rates of swingers however are lacking. To our knowledge, there is not a single country that mentions swingers as a special focus group in national STI prevention. Already in 1986 the Centres for Disease Control reported two female swingers to be infected with HIV in a club (New York Times November 14, 1986). Some outdated studies mention the fear of AIDS or STI among swingers [START_REF] Jenks | Swinging: a review of the literature[END_REF] but none were followed by more medical or scientific attention for swingers and STI.

Swingers differ from non-swinging heterosexual adults by their sexual network, typed by concurrent sexual partners and high rate of unprotected sex. [START_REF] Jenks | Swinging: a review of the literature[END_REF][START_REF] Van Der Poel | Swingers en hepatitis B. Onderzoek naar seksueel risicogedrag van swingers en kansen voor hepatitis-B-preventie[END_REF][6][START_REF] Niekamp | SWAP Study; Identification of STD spread within a sexual network of swingers. Oral presentation[END_REF][START_REF] Niekamp | Swingers, a hidden sexual culture at risk for STD. Oral presentation[END_REF] These risk behaviours make swingers more prone to STI [START_REF] Kretzschmar | Measures of concurrency in networks and the spread of infectious disease[END_REF] as confirmed by our study. Potentially, they may act as an STI transmission bridge to the entire population. By identifying and testing swingers (regularly) for STI, individual and population burden of STI as well as STI spread can be reduced. Yet, (inter)nationally, swingers are underrepresented in the STI services population.

Key messages

1. Swingers are a hidden risk group who need to be addressed in STI prevention and care services 2. Identification of high risk groups is essential for effective STI control 3. Older STI clinic clients should not be overlooked as a potential risk group in STI care services

  Of 8965 consultations, 89.1% were by Dutch attendees, 1.6% by German and 4.2% by Belgian attendees; 11.7% were by clients older than 45 years, 15.5% by clients aged between 35 and 45, 60% aged between 20 and 35, and 12.8% between 16 and 20 years.

	Overall, 11.6% of consultations were comprised by swingers (median age 43 years
	[interquartile range IQR: 38-48]), 74.8% by heterosexuals (median age: 24 [IQR: 21-29]),
	9.6% by MSM (median age 32 [IQR: 23-46]), and 4% by female prostitutes (median age:
	35 [IQR: 28-44]).

Table 1 :

 1 Proportion of CT and/or NG positive test results and risk estimates by age, risk category and gender following multivariate modelling, South Limburg Public health STI clinic, 2007 and 2008We further evaluated the relative share in STI diagnoses by age and risk categories. Figure2shows that in the youngest age groups, heterosexuals contribute the largest part of STI diagnoses. In the older age groups, STI is mostly diagnosed in swingers and MSM. The share in STI diagnoses is for swingers and MSM increasing with age (both p<0.001) reaching 55% for swingers and 31% for MSM in clients with STI who are older than 45 years. Collapsing all age groups, swingers comprised 12% of STI diagnoses (versus MSM; 13%).

			Age 45 or younger		Age older than 45
		STI Prevalence Multivariate	STI Prevalence Multivariate
			OR, 95% CI		OR, 95% CI
	MALES
	Heterosexuals 10.0	1**	2.4%	1***
	MSM	14.1%	1.48 (1.15-1.91)	14.6%	6.96 (2.84-17.08)
	Swingers	8.5%	0.83 (0.54-1.29)	10.4%	4.74 (1.89-11.88)
	FEMALES
	Heterosexuals 10.9%	1**	4.0%	1***
	Prostitutes	5.2%	0.45 (0.27-0.77)	2.9%	0.72 (0.14-3.83)
	Swingers	8.3%	0.74 (0.50-1.09)	17.9%	5.28 (1.99-14.01)
	*** p < 0.001; ** p < 0.01;

Acknowledgements

The authors thank the staff of the STI clinic.

Funding

Investigator initiated

Conflict of interest statement

There are no conflicts of interest. The corresponding author has had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Contributions of the authors

Contributions: ND analyzed the data and wrote the manuscript; EB and AN contributed to the data collection; AN and CH organized STI clinic procedures; all authors contributed to the final draft of the manuscript.

Word count

Abstract: 239 words Article: 1200 words (text only) Appendices:


Dr Frédéric Méchaï
email: mechaifrederic@wanadoo.fr

Bertille De Barbeyrac

Olivier Aoun

Audrey Mérens

Patrick Imbert

Doxycycline Failure in Lymphogranuloma venereum

The number of Lymphogranuloma venereum (LGV) cases is currently reemerging in the homosexual community, particularly in HIV seropositive patients. The standard treatment for this infection caused by Chlamydia trachomatis L1, L2 and L3 serotypes is a 3 week doxycycline regime. We report the case of a male patient presenting with an LGV and rapidly cured by moxifloxacin after an initial clinical failure following an extended therapy with cyclines. This fluoroquinolone is known to be highly active in vitro on the LGV pathogenic agent. Thus, it could represent a useful alternative to doxycycline in case of failure.

Introduction

Lymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) caused by

Chlamydia trachomatis L1, L2 and L3 serotypes. This infection is currently reemerging in Europe and is most often cured by an extended cycline antibiotic regimen. We report an original case of a successful fluoroquinolone treatment after an initial clinical failure following doxycycline therapy.

Clinical case

A 27 year old man was admitted to our infectious diseases department for anal pain and large, inflammatory and painful inguinal lymphadenopathies which was persisting despite a 2 week doxycycline regime. This treatment had been prescribed by his general practitioner for a proctitis with a positive Chlamydia trachomatis testing on a superficial anal sample (Roche technique PCR).

He reported unprotected anal intercourse two weeks prior with an irregular partner. He had no fever, nor urethral or anal discharge. No STI history nor previous doxycycline treatment were reported. Clinical exam found bilateral lymphadenopathies, approximately 3 cm large, stretched and very tender without any cutaneous lesion on the lower limbs. Proctological examination using rectoscopy showed a 5 mm diameter racket-like shaped ulceration on the anal margin, without discharge. Blood tests showed a mild inflammatory syndrome with a 39 mg/l CRP and a normal full blood count. A lymph node aspiration revealed a macroscopically purulent liquid. The culture was sterile, as the patient had already been taking doxycycline for trachomatis infection can be made by culture, antigen or nucleic acid detection (gene amplification) on a genital or lymph node sample [START_REF] Mc Lean | Treatment of lymphogranuloma venereum[END_REF]. A serology with high levels of antibodies can be helpful in case of suggestive clinical history [START_REF] Mc Lean | Treatment of lymphogranuloma venereum[END_REF]. Doxycycline remains the first line of LGV treatment at a bidaily 100 mg dose for 21 days.

This lengthy treatment duration is suggested as published studies have shown persistent

Chlamydia RNA in rectal samples after more than 2 weeks of doxycycline therapy in LGV patients while DNA was undetectable in less than 7 days in proctitis cases due to other Chlamydia serotypes [START_REF] Vries | Delayed microbial cure of lymphgranuloma venereum proctitis with doxycycline treatment[END_REF]. Macrolides represent an alternative [START_REF] Mc Lean | Treatment of lymphogranuloma venereum[END_REF].

C.trachomatis is naturally sensitive to cyclines, macrolides and fluoroquinolones. However, the concept of C. trachomatis acquired resistance is not clear [START_REF] Somani | Multiple drugresistant Chlamydia trachomatis associated with clinical treatment failure[END_REF][START_REF] Wang | Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: A meeting report[END_REF]. Some treatment failures related to less sensitive strains were reported in the literature without determining the genetic support [START_REF] Bébéar | Genital Chlamydia trachomatis infections[END_REF][START_REF] Lefèvre | Comparative in vitro susceptibility of a tetracyclineresistant Chlamydia trachomatis strain isolated in Toulouse[END_REF]. In vitro, it is possible to obtain resistant mutants, particularly to fluoroquinolones. Currently, little is known about the epidemiology of Chlamydia trachomatis acquired resistance. In general practice, evaluation of in vitro sensitivity is not routinely done due to some difficulties related to its compulsory intracellular feature, the lack of a standardized method and the poor reproducibility of the sensitivity tests [START_REF] Bébéar | Genital Chlamydia trachomatis infections[END_REF]. It also depends on the availability of the strain. Unfortunately, in this case, cell culture remained negative. It could be possible to look for a resistance gene as TetM which is responsible for tetracycline resistance. However, in clinical specimens containing polymicrobial flora, the presence of antibiotic resistance genes does not prove that they are located on the C.trachomatis strain. In addition, it is always difficult to assign clinical failure to treatment failure due to possible reinfection [START_REF] Somani | Multiple drugresistant Chlamydia trachomatis associated with clinical treatment failure[END_REF]. Our patient was compliant with the antibiotic therapy.

Moreover, his painful symptomatology made him unfit for new anal intercourse which could have explained the lack of recovery after more than three weeks of doxycycline therapy. Thus, a fluoroquinolone like moxifloxacine was chosen, due to its good intrinsic activity on C. trachomatis and to its possible use in genital infections. No study has shown its efficacy in

LGV treatment [START_REF] Mc Lean | Treatment of lymphogranuloma venereum[END_REF]. In vitro, Bébéar et al showed that moxifloxacine is the most active molecule against Chlamydia trachomatis with a MIC 90 higher than doxycycline, macrolides and other fluoroquinolones [START_REF] Bébéar | Activity of moxifloxacin against the urogenital mycoplasmas Ureaplasma spp., Mycoplasma hominis and Mycoplasma genitalium and Chlamydia trachomatis[END_REF]. In our patient, the spectacular regression of symptoms with this new fluoroquinolone seems to support the hypothesis of a good in vivo activity also.

To our knowledge, no cyclines' treatment failure was reported in LGV. In these situations, new fluoroquinolones such as moxifloxacine could represent a useful alternative.

  10 days. PCR Chlamydia trachomatis testing (Cobas TaqMan Roche technique) was positive in this sample and in the anal ulceration biopsy. The molecular analysis conducted at the national Chlamydiae reference center confirmed the L2 serotype LGV. Culture on McCoy cells remained sterile. No other STI was found (HIV, HBV, HCV, syphilis, gonorrhoea). C.trachomatis serology was positive with 1/512 IgG and 1/256 IgA titers for a 1/8 positivity threshold. Despite an extended 200 mg per day doxycycline regime for more than 3 weeks, the patient developed discharging inguinal sinuses requiring repeated drainage and the anal lesion was still observed on proctological examination. Following this clinical failure, doxycycline was replaced by a 400 mg daily moxifloxacine therapy allowing a spectacular recovery in 10 days. No relapse was observed after a 2 month follow up. Discussion Lymphogranuloma venereum is a sexually transmitted infection caused by Chlamydia trachomatis L1, L2 and L3 serotypes. Recent outbreaks were reported in Europe and in the United-States, mainly in the HIV seropositive homosexual community. Clinical presentation includes a genital or rectal ulceration with inguinal lymphadenopathy. The diagnosis of C.