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A specific real-time polymerase chain reaction followed by melt curve analysis was developed for the detection of the Swedish variant (nvCT) strain of Chlamydia trachomatis (CT). Surveillance was performed on 476 CT-positive clinical specimens obtained from 15 laboratories around the United States using nucleic acid amplification test assays, which would not miss the nvCT. All were negative for nvCT; thus, there is no evidence of the nvCT in the United States.
Chlamydia trachomatis (CT) is the most common and most frequently reported sexually transmitted bacterial infection. Most infections are asymptomatic and often remain undiagnosed.1 A new variant (nvCT) strain of CT has been previously been described in Sweden and Norway.2 It has not been observed in Australia.3 Most of the other detections of the nvCT outside Sweden and Norway have been shown to have epidemiologic linkages to Sweden.4–9 No evidence of spread elsewhere has been reported.10–13 The nvCT infections in different counties in Sweden have been reported to represent from around 10% of all CT infections up to 64% in Dalarna county in 2006 to 2007. In Södra Älvsborg County, among the 789 screening samples, 69 (8.8% positivity rate) were positive for wild-type chlamydia as detected by Roche CTM48. An additional 24 were identified using a laboratory-developed polymerase chain reaction (PCR) that could detect nvCT. Thus, 25% of a total of 93 CT-positive samples were nvCT.7 As of yet, there has not been evidence of the nvCT in the United States, but surveillance has not been performed on a wide scale.
The nvCT is characterized by a 377-base-pair (bp) deletion in the cryptic plasmid, a common target for nucleic acid amplification test (NAAT) assays.4,5 The Aptima Combo 2 test, Aptima CT (Genprobe, Inc., San Diego, CA), ProbeTec Strand Displacement Assay (Becton Dickinson, Sparks, MD), and RealArt CT Kit (Qiagen, West Sussex, UK) target either a different gene or a different region of the cryptic plasmid. These assays remain unaffected by the deletion but only identify the nvCT as C. trachomatis and do not distinguish it as the nvCT.2,4,5 However, other older NAAT assays (Roche Cobas Amplicor, Roche TaqMan48, and the earlier version Abbott m2000 assay) that target the portion of the plasmid with the deletion produced false-negative results and were unable to detect the nvCT.4,5
Our objective was to develop a specific real-time PCR for the nvCT, followed by confirmatory high-resolution melt analysis (HRMA) for the detection of the nvCT and perform surveillance in the United States. As a pilot surveillance study, C. trachomatis clinical samples tested by existing NAAT assays that would not miss detection of the nvCT strain as a CT strain, from across the United States, were obtained and tested using our PCR assay. Such assays would not differentiate CT, however, as a nvCT but indicate it as positive for CT. Samples that had been collected for 3 different years, from 2008 to 2010, were obtained for this study as de-identified remnant samples and were from the following states: Ohio, North Carolina, Massachusetts, Indiana, Washington, Kansas, Georgia, Montana, Delaware, California, Maryland, Tennessee, Michigan, New Jersey, and Virginia. These states were volunteers wishing to participate after a request went out to the State Health Laboratories participating in the Infertility Prevention Program of the Centers for Disease Control and Prevention.
A total of 476 swab and urine samples previously tested positive by Aptima Combo 2 (Gen-Probe, Inc., San Diego, CA) or Becton Dickinson (Sparks, MD) for C. trachomatis were tested using our assay. The nvCT DNA extract for use as a positive control was kindly provided by Dr Kenneth Persson from Malmo, Sweden. Samples were de-identified for research purposes. The study was approved by The Johns Hopkins University institutional review board.
DNA was extracted from the samples using the Roche MagNA Pure robotic instrument (Roche Diagnostics, Indianapolis, IN). A specific primer pair flanking the deletion region, chlamydia mutant forward (CtMF) and chlamydia mutant reverse (CtMR) (Table 1), modified from a previously described primers,3,4 and a novel Taqman 26-bp probe, which covered the sequences on either side of the deletion (Table 1) were designed and used. The assay was designed to only generate a positive signal when the probe binds to the nvCT target. Polymerase chain reaction included the following: 50 μL total volume 10 μL of DNA template, 1 μL of 50 μM CtMF and CtMR primers, 12.8 μL molecular-grade water, 0.2 μL of 50 μM probe, and 25 μL of 2× Taqman Universal PCR mix (PE Applied Biosystems, Foster City, CA) The cycling conditions were 2 minutes preincubation at 50°C, denaturation at 95°C for 10 minutes and 50 repeats at 95°C for 15 seconds, and annealing/extension at 60°C for 60 seconds.
Subsequently, another specific PCR was performed, without use of the probe, but coupled with melt curve analysis of the amplicon. Melt analysis was performed on the Lightscanner instrument (Idaho Technology, Salt Lake City, UT) for confirmation of a possible nvCT. A 10-μL volume of PCR mixture comprised 2 μL of DNA template, 1 μL of 1.5 μM CtMF and CtMR primer, 2 μL molecular-grade water, and 4 μL of 2× Universal PCR mix and LC Green dye (Idaho Technology) was tested in triplicate in a Geneamp Thermocycler (PE Applied Biosystems) using denaturation at 95°C for 30 seconds, followed by 45 cycles at 95°C for 30 seconds and annealing/extension at 60°C/72°C for 60 seconds, and 1 cycle at 95°C for 30 seconds and 28°C for 30 seconds. Each post-PCR sample amplicon was subjected to HRMA on the LightScanner instrument, version 2.0. Melting temperatures ranged from 60°C to 95°C. Fluorescence data acquisition was obtained for every 0.1°C increase in temperature. Confirmatory melting profiles generated by HRMA for the nvCT and the wild-type CT were 73.19°C and 82.79°C, respectively.
Of the 476 samples tested, all 476 were negative for the nvCT using the real-time PCR with probe detection and PCR coupled with HRMA. The Chlamydia-positive samples were mostly from laboratories serving sexually transmitted disease and family planning clinics. Continued surveillance for this variant may not be as necessary in the future because the older assays are being phased out of use. The use of the real-time PCR coupled with melt analysis confirmation not only allowed detection of the nvCT but may also allow for detection of an unknown or emerging variant of C trachomatis, if different melt curve temperature results are detected.
Although many of the currently used NAATs, such as m2000 PCR (Abbott Molecular) and COBAS TaqMan CT v2.0, will not miss the nvCT,14,15 there are reasons for some continued surveillance because of the possibility of new variants that may arise. US public health laboratories perform approximately 3.6 million CT tests per year.16 Most (64.4%) are NAATs, many of which target the CT plasmid, and NAAT assays are increasingly being used.16 Monitoring of test capacity for detection of possible new molecular variants of CT should continue.
Thus far, surveillance has demonstrated that the nvCT persists in Sweden7,8 and Norway.9 There has been no evidence of spread to Australia,3 England,10,11 Wales,11 Ireland,12 or the Netherlands13; and we, too, find no evidence that the new variant has spread to the United States.
We thank all the laboratories that provided specimens for testing. Funded, in part, by U54EB007958 and HPTN NIAID/NIH U01-AI 068613.
These data were presented, in part, at the Proceedings of the Sixth Meeting of the European Society for Chlamydia Research. P-63, pp. 260, Aarhus, Denmark, July 1–4, 2008.