A Chlamydia trachomatis
variant has been identified in patients in Sweden that harbours a 377 base pair deletion in the ORF1 in the C. trachomatis
cryptic plasmid . The deletion is located in the target area of several commercial nucleic acid amplification tests (NAATs) for diagnosing urogenital C. trachomatis
infection. This includes the commercially available tests manufactured by Roche and Abbot . Nucleic acid amplification tests for C. trachomatis
that do not target the cryptic plasmid or target outside of this deletion region are not affected by the new variant, such as those from Becton Dickinson and Genprobe.
There is no evidence for the presence of this new C. trachomatis variant outside Sweden [2,3]. However, we wanted to assess whether this variant was circulating in Oslo, in order to implement new procedures for optimal specificity and sensitivity in detecting urogenital C. trachomatis infections. An observed decline in the trend of chlamydia cases and in the proportion of positive test result for C. trachomatis infections were reason to investigate the diagnostic methods leading to the identification of the Swedish C. trachomatis variant [1,4]. A retrospective study of 22 microbiology departments by the Norwegian Institute of Public Health, covering all of Norway in the first 10 months of 2006, showed that the proportion of positive test results for C. trachomatis was increasing, especially among those laboratories using Roche tests. This was in contrast to the Swedish findings, and it was concluded that the new C. trachomatis variant has not spread to a detectable extent in Norway . Given the extensive flow of guest workers and visitors between Norway and Sweden, we wanted to perform a more direct investigation to look for the presence of the new C. trachomatis variant in Norway. We thus performed a comparative study whereby a subgroup of patients attending an STI clinic in Oslo were tested on both Roche TaqMan 48 CT test and our in-house PCR test for C. trachomatis, which does not target the 377 bp deletion region in the cryptic plasmid of C. trachomatis.
DNA was purified from first void urine from male and female patients, and cervical swabs from female patients using MagnaPure LC (Roche). Patients presented at an STI clinic in Oslo with signs of urethritis or lower genital tract infection. Purified DNA was used for detecting C. trachomatis using Roche TaqMan 48 CT and a triplex in house real-time PCR assay that amplifies a segment of C. trachomatis cryptic plasmid , the MOMP gene of C. trachomatis  and an internal control on SDS7500 (Applied Biosystems). To test for the 377 bp deletion, primers were designed to amplify a 189 bp DNA fragment across the deletion junction of the cryptic plasmid in the presence of deletion and a 566 bp amplicon, in the non-deletion variant, although the latter amplicon does not amplify in standard TaqMan PCR assay. The amplicons from the cryptic plasmid variant could be separated and visualised using a dissociation curve in the presence of SYBR Green or by electrophoresis. The amplicon from the deletion variants were sequenced using a nested primer to the amplification primers.
Between 29 November 2006 and 9 February 2007, we tested 409 patients for urogential C. trachomatis infection with Roche Taqman 48 CT assay and our in-house cryptic plasmid / MOMP gene real-time PCR assay. Forty-seven patients were found to be positive using TaqMan 48 CT test. Our in-house NAAT found two additional patients that were positive for C. trachomatis. Urine and cervical swab samples from these patients were retested on Taqman 48 CT test as well as the Abbott RealTime CT test and again found to be negative. Both patients were female, one being Swedish and the other Norwegian.
These two samples were tested to detect the specific 377 bp deletion in the cryptic plasmid reported in the Swedish C. trachomatis variant. Amplification from both samples gave a specific amplicon of expected size by agarose gel electrophoresis and a major peak with a Tm of 76.5°C in presence of SYBR Green in dissociation analysis (Figure). These amplicons were not present when amplification was performed on C. trachomatis negative sample or from samples infected with the non-variant C. trachomatis. The nucleotide sequence in the amplicons from both samples was determined using a primer nested to the amplification primers and found to harbour the same 377 bp deletion found in patients in Sweden (data not shown).
Figure. Dissociation analysis, in presence of SYBR Green, of amplicon amplified across the deletion junction of the Swedish C. trachomatis variant.
The Swedish C. trachomatis variant, which harbours a 377 bp deletion in the ORF1 of the cryptic plasmid, was found in two patients with lower tract genital infection in Oslo area, one of whom was of Swedish origin. It is the opinion of the authors that the cryptic plasmid of C. trachomatis remains a correct target for NAAT as it increases sensitivity of detection. However, in this interim period, while new tests are being designed, validated and CE-certified according to IVD regulations , laboratories that use commercial tests from Roche or Abbott should perform a second test to monitor and prevent the spread of the newly identified variant of C. trachomatis.
We thank Dr Björn Herrmann for information regarding the sequences deleted in the Swedish C. trachomatis variant, and Dr Harald Moi for valuable advice and partner information.
Editorial team (email@example.com), Eurosurveillance editorial office
In the 22 February article "Measles outbreak in the Barcelona Region of Catalonia, Spain, October 2006 to February 2007", the text in line 2. was changed to "She presented on 28 August 2006 with a fever of 38 ºC", rather than "on 26 September 2006" as stated in the original text.
In the paragraph below Figure 2. we stated mistakenly that "Seventy are still under investigation and 42 have been discharged from hospital." The text was corrected and now reads: "Seventy are still under investigation and 42 have been excluded as cases."
These changes were made in the text on 1 March 2007.