Recently, in the county of Halland, Sweden, a new Chlamydia trachomatis variant (swCT variant) with a deletion of 377 bp in the cryptic plasmid has been reported [1,2]. This swCT variant has also been designated as new variant C. trachomatis (nvCT). The deletion was found in the target area for two commercial CT nucleic acid amplification tests (NAATs) (Roche and Abbott) leading to false negative results when screening patients infected with this new Swedish variant [2]. In some regions in Sweden, a 24-78% reduction in the CT prevalence was found when Roche diagnostics was used as compared to Becton Dickinson (BD) ProbeTec [1,2,3]. So far no epidemiological characteristics have been described.

Several studies [4,5] have been published regarding monitoring of the swCT variant outside Sweden and, as expected, the first cases have been described recently in Denmark and Norway [3,6]. Since false negative test results lead to under-treatment and continuing transmission, the detection of the swCT variant in the population is essential. This report describes the actions of public health experts in cooperation with specialists in sexually transmitted diseases (STD), epidemiologists and (molecular) microbiologists to investigate the possible introduction of the swCT variant in the Netherlands.

In December 2006, the Preparedness and Response Unit (LCI) of the Centre for Infectious Disease Control of the Dutch National Institute for Public Health and the Environment (RIVM) established a study group whose task has been to increase awareness among STD specialists, molecular biologists and microbiologists about the swCT variant and to define methods for investigating whether this new variant had been introduced in the Netherlands. "Inf@ct", an electronic message system operated by LCI, has been used as an important communication channel through which information could be shared instantly.

The study group proposed the following main actions and strategies to detect the potential presence of swCT variant in the Netherlands:
1. Investigating trends in CT epidemiology
2. Retesting samples by TaqMan PCR
3. Screening sample pools for the presence of the swCT variant

Methods

1. Investigating trends in CT epidemiology

According to laboratory data, in the Netherlands around 80% of C. trachomatis infections are diagnosed by Roche Diagnostics assays. This means that if the swCT variant were present in the Netherlands, it would go largely undetected. The other 20% of positive tests are performed mainly with GenProbe, BectonDickinson ProbeTec and so-called “in house” assays. A few years ago, one of these in-house assays, a Real-Time PCR (TaqMan), was developed by the VU University Medical Center, Amsterdam and subsequently implemented in 10 laboratories (both public and private hospitals) in the Netherlands for the diagnostics of C. trachomatis. Sequence analyses of the primer-probe region inside the CT-plasmid showed that this PCR detected the swCT variant, validating the diagnostic setting.

Sexually transmitted infections (STI) surveillance
The STI surveillance in the Netherlands is based on registration of STI consultations at STI clinics and public health services. Consultations have been registered in SOAP (an internet-based application) since 2003. The data reported includes demographic variables, history of STI and HIV testing, laboratory tests and diagnoses of STI. Based on these variables, positivity rates for STI, including chlamydia, can be calculated.

Laboratory surveillance
Laboratory surveillance (Infectieziekten Surveillance Informatie Systeem – ISIS) collects information on tests and test results with demographic characteristics from nine laboratories in the Netherlands.
For the purpose of this study, nucleic acid amplification tests (NAAT) ISIS-based positivity rates per year were calculated. A test that was performed during the two months following a former positive test in the same patient was not counted.

2. Retesting samples with TaqMan PCR

In the period January-May 2007, laboratories in the Netherlands using Roche diagnostic assays for CT detection were urged to send patient samples for retesting at the VU University Medical Center in Amsterdam in cases when clinical presentation in combination with sexual risk profile of the patient did not correspond with a CT negative laboratory result. The samples were retested for the presence of CT with the TaqMan Real-Time PCR which besides all standard CT strains detects also the new swCT variant strain. In addition, a real-time TaqMan assay detecting only the swCT variant was developed [7]. Results of this retesting were sent within a week to the laboratory which had provided the specimen.

3. Screening sample pools for the presence of the swCT variant variant

Sample pools were selected for retesting with the TaqMan PCR method detecting both the wild-type CT strains and the swCT variant [4], as well as with the new real-time swCT variant TaqMan [7].

Results

1. Investigating trends in CT epidemiology

STI surveillance
In Figure 1, the number of individuals tested for chlamydia in the period 2003-2006, and the positivity rates, are displayed. Since 2003, the number of tests for chlamydia has increased in all groups – heterosexual men and women, as well as men having sex with men (MSM). The positivity rates have slightly increased among heterosexual males but stayed more or less the same in MSM.

Laboratory surveillance
Figure 2 shows the positivity rates of NAAT in the laboratory surveillance from 2004 until mid-2007. Among men (M) the positivity rates were higher than among women (F); however, there was no clear trend in either men or women, suggesting a lower prevalence than observed in Sweden. Unfortunately, sexual preference was not registered in this system.

Based on the available STI surveillance data from STI clinics and laboratories, there are no indications for a declining number of chlamydia diagnoses in the Dutch population. This overall stable trend does not preclude a proportion of diagnoses being missed due to reduced diagnostic sensitivity, as the overall detected prevalence could also be affected by changes in populations (although no direct evidence for this was found) or by changes in registration systems.

2. Retesting samples through the TaqMan PCR

During the five months between January and May 2007, a total of 58 samples previously tested CT-negative with Roche (but suspected of being infected with CT on the basis of the clinical picture) were sent to the VU University Medical Center in Amsterdam and retested there for CT and swCT variant. All samples were CT-negative and also swCT variant-negative. This enhanced surveillance system therefore did not reveal the presence of swCT variant in the Netherlands.

3. Screening sample pools for the presence of the swCT variant variant

The following sample pools were retested for the presence of swCT:
1) Random samples of 515 patients attending an STD clinic in Amsterdam, including 75 samples tested CT-positive with Roche Diagnostics (not detecting the swCT variant).
2) 30 samples tested CT-positive in house real-time PCR (detecting also the swCT variant) were selected from the Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands (population: CT prevalence 1.8%).
3) 57 samples tested CT-positive with Becton Dickinson (BD) ProbeTec (detecting also the swCT variant) were selected from the Department of Infectious Diseases, South Limburg Public Health Service, Heerlen, the Netherlands (population: CT prevalence 7.3%).
None of the samples from the three above pools were positive for the swCT variant when tested using the new swCT variant TaqMan. (Table)

The following sample pool surveillance is still ongoing:
4) Since May 2007, in Groningen, during one week in each month all diagnostic clinical samples (n= around 400) have been screened specifically for the swCT variant. In May, 443 samples were tested with both the Abbott M2000 and the VUmc TaqMan PCR. There were 18 positive samples, and no discrepancies. In June-July, another 618 samples were tested in a similar way, with 43 positive test results in both tests and no discrepancies. (Table)

Discussion

Last year Sweden notified a new CT variant which was not detected by the regular Roche and Abbott PCR tests. Since these tests are also commonly used in the Netherlands, there was reason to monitor the potential introduction of this variant there, as well. A swCT study group, set up for this purpose, initiated a number of actions aimed at detecting the new variant in the Netherlands.

To date, however, neither the enhanced laboratory surveillance nor analyses of the epidemiology of CT indicate the presence of swCT in the Netherlands. Although its broad spread in the Netherlands within a short term is not likely, small scale introduction cannot be ruled out. As single cases of swCT variant have been detected outside Sweden, the introduction and further spread of swCT to other countries including the Netherlands is still a realistic scenario. The Dutch swCT study group therefore continues to monitor the situation with the following specific actions:

1) In Groningen, one week per month all diagnostic clinical samples (n=around 400) are being screened specifically for the swCT variant.
2) In Amsterdam, every three months a group of patients presenting at VU University Medical Center and Municipal Health Service (n=around 200, unselected) will be screened for the presence of the swCT variant.
3) Further trend analysis will be performed using the national surveillance dataset from STD clinics and laboratorial results (ISIS) for epidemiological changes in chlamydia cases.

This monitoring system will continue until the manufacturers Roche and Abbott have adapted their C. trachomatis diagnostic assays to detect the swCT variant.