During late summer 2006, the presence of a mutant variant of Chlamydia trachomatis (CT) was discovered in Sweden, after a decrease in the number of CT PCR positive urogenital specimens was observed in one county (Halland) between November 2005 and August 2006 [1]. The routine diagnostic method of this county’s laboratory was PCR targeting the cryptic plasmid. Subsequent analyses conducted with a different method (Artus) targeting the major outer membrane protein (MOMP) chromosomal area revealed a CT prevalence corresponding to the usual level. Successive sequencing of specimens found positive with the latter method showed that a new clone of CT had emerged, differing from the usual CT strains by a 377 base pair deletion in the cryptic plasmid.

Several commercially available test kits for the laboratory diagnosis of CT are unable to detect the new variant because they target this specific area of the cryptic plasmid: Roche Taqman 48, Roche COBAS Amplicor, and Abbott m2000. Two other diagnostic kits employ other targets and are therefore able to detect the new variant: Becton Dickinson ProbeTec, and GenProbe Aptima Combo2 and Aptima CT.

To date, several reports have documented the spread of the new variant to other counties in Sweden [2-4] and various initiatives have been undertaken to detect its possible spread to other countries. In Ireland [5] the new variant was not detected in any of 8,797 samples collected between July and December 2006. In the Oslo area, Norway, the new variant was diagnosed in two female patients, one Swedish and one Norwegian, among 409 patients who had been tested between late November 2006 and early February 2007 [6]. In Amsterdam, the Netherlands, a study published in 2007 reported no detection of the new variant among 515 visitors to an outpatient STI clinic [7].

In Denmark, the mandatory laboratory CT surveillance is based on quarterly submission of data on specimens found positive for CT at the diagnostic laboratories in each county. The data include the total number of samples examined per quarter and the number of positive results. For the latter, additional information is provided on patient’s sex and age, body site the sample was taken from (cervical, urethral, anorectal) and the date it was collected, as well as type of the health care provider and laboratory method used. An analysis of these data stratified by month and laboratory method for the period from January 2004 through December 2006 did not suggest a decline in the number of positive specimens that could be attributed to the diagnostic method as described above in case of Sweden. Nevertheless, in order to closely monitor the possible emergence of this variant in Denmark a surveillance plan was arranged in co-operation with some of the diagnostic laboratories.

Methods

Sampling
The 15 counties in Denmark, with a total of 5.4 million inhabitants, are served by 17 laboratories performing CT diagnostic assays. Nine of these employ methods incapable of detecting the new variant. From 1 November 2006 through 3 April 2007, i.e. for about five months, a voluntary surveillance system was employed in which specimens initially assayed in other laboratories were subsequently tested in our laboratory at Statens Serum Institut (SSI).
One laboratory using the ProbeTec method capable of detecting the new variant submitted 50 positive and 50 negative specimens in November 2006, and 50 positive ones in March 2007. Four other laboratories using Roche methods incapable of detecting the new variant submitted a total of 977 negative specimens and 23 positive specimens.
During the study period, further 2,620 samples were sent for routine testing at SSI directly by clinicians.

Diagnostic method
Our standard CT diagnostic method has been described previously [8]. In short, the cryptic plasmid was amplified with primers identical to those used in the Roche Amplicor assay and all positive results were confirmed with primers amplifying a part of the 16S rRNA gene. Both assays contained an internal control for inhibition. Samples were tested with both primer-sets, thus allowing detection of the new variant. For the purpose of documenting that the lack of amplification with the plasmid primer was due to the mutation present in the new variant, a PCR using primers flanking the deletion in the new variant was applied [9]. This assay was originally designed as a dual-probe real-time assay, but we used it without the probe in a conventional gel-based assay where the new variant and the wild-type could be distinguished by the difference in size of the amplified product.

Results

During the five-month period from 1 November 2006 through 3 April, 2007, a total of 3,770 specimens (2,620 samples received for routine testing and 1,150 submitted by other labs) were examined in our laboratory with both plasmid and 16S rRNA gene PCRs. Only one case of the new CT variant was detected (Table). It was found in late March in a specimen from a Danish 19-year-old woman from the Copenhagen Capitol area, who had tested positive also with the ProbeTec CT-assay (Becton Dickinson) at the primary laboratory. She reported having had a steady relationship for one year and no contact to Sweden. No specimens were available from the partner.

Among the 1,027 specimens that had tested negative at other laboratories, four were positive in both assays of our standard methods. Among the 123 specimens that had been diagnosed as positive for CT at other laboratories, all were positive with the 16S rRNA gene assay whereas the one new variant strain was negative in the plasmid PCR.

Discussion and conclusion

Sexually transmitted infections are unlikely to respect national borders, especially in an extended period of time. It was therefore an unexpected finding that only one case of the new CT variant was detected among 3,770 specimens tested during a five-month period. The samples were submitted from the whole of Denmark, although the majority came from the Copenhagen area. Considering the intense daily traffic between the Copenhagen area in Denmark and southern parts of Sweden, it is surprising that the spread occurred so late. One reason could be that the vast majority of CT testing in the Greater Copenhagen area is performed with assays capable of detecting the new variant, consequently leading to containment of the new variant. However, this explanation is not valid for Northern Jutland, where the new variant was not detected either and the traffic between Denmark and Sweden is also quite intense.

The emergence of a new bacterial variant capable of escaping laboratory diagnosis emphasises the need to avoid reliance on a single assay and to use genes of known and essential function as targets for NAATs. Although the prevalence of the CT variant outside Sweden is still low, its occurrence in Norway and Denmark indicates dissemination. It is therefore likely that it will also appear soon in other European countries.

Added in proof

In June 2007, i.e. after the submission of this report, the new CT variant was found in a first void urine specimen collected from a 62-year old man on Bornholm, a Danish island near Sweden.

Acknowledgements*

We are grateful to Dr. Henrik Westh, University of Copenhagen Hospital, Hvidovre, Denmark, for submitting the two specimens in which we detected the new variant Chlamydia trachomatis

*Authors' correction
The authors regret that the Acknowledgements section was initially missing from the article. This was corrected on 6 November 2007.