Bacterial strains

To investigate the analytical specificities of the primers and probes sixteen isolates were used representing the serovars and serovariants of Chlamydia trachomatis (Table 3), and additionally C. muridarum, C. pneumoniae, C. pecorum, C. psittaci and twenty other microorganisms that normally reside in the human oropharynx, the urogenital and perianal tract. These microorganisms corresponded to the gram-positive and gram-negative bacteria and yeast previously described by Morré and coworker [19]: Acinetobacter baumannii, Campylobacter jejuni, Candida albicans, Enterococcus faecalis, Escherichia coli, Streptococcus agalactiae, Haemophilus influenzae, Klebsiella pneumoniae, Lactobacillus casei, Mycoplasma genitalium, M. hominis, M. pneumoniae, Neisseria meningitidis, Pasteurella multocida, Pseudomonas aeruginosa, Salmonella enteritidis, Shigella sonnei, Staphylococcus aureus, Ureaplasma parvum and U. urealyticum. Chlamydial DNA was kindly provided by Servaas Morré and DNA extracts of the other microorganisms derived from our diagnostic laboratory, each covering not less than 10 ng DNA/2.5 μl.

Clinical specimens

The specimens with a request for detection of C. trachomatis originated from different departments of the University Clinic of Duesseldorf. Sample collection for routine diagnostic procedures was conducted in adherence with the guidelines of good clinical practice under approval of the Institutional Review Board of the University Clinic Duesseldorf. Written informed content was obtained from all patients. Less than 10% were ocular samples. 43% of the samples emanated from women with clinical symptoms and 7% from women for routine screening in pregnancy. 32% of the specimens derived from departments specialising in sexual transmitted diseases especially of men (andrology (16%); dermatology (6%) and infectious diseases, which also includes the treatment of HIV-infected men, (10%)).

The ocular, urethral, cervical and rectal swabs were transported to the Medical Microbiology in 3 ml M4-transport medium (Remel, UK) which contains 3–4 glass beads. Upon arrival, the specimens were resuspended in the medium by vortexing, and a portion of 1 ml centrifuged at 15,000 × g for 15 min. The supernatant was removed and the sediment washed in 500 μl phosphate-buffered saline, pH 7.3. After further centrifugation the sediment was resuspended in 25 μl 10 mM Tris-(hydroxymethylaminomethan) (Tris)/HCl, pH 7,5. Thereafter 50 μl Proteinase K-solution (100 μg/ml Proteinase K - 0,5% Tween 20 in 10 mM Tris/HCl, pH 7.5) was added and the samples were incubated for 60 min at 56°C followed by inactivation of the Proteinase K at 95°C for 30 min. After a short centrifugation, the samples were ready to be used in PCR. If not tested immediately, they were stored at -20°C.

Design of Primers and Probes

Multiple-sequence alignments were performed with the MegAlign program of the sequence analysis software of DNASTAR (see Figures 1 and 3). The Primer Express Software of Applied Biosystems (Foster City, CA, USA) was used for the final design of specific primers and probes. The omp-1-sequences (Figure 3) and pmp-H sequences (serovars A (AY184155), B (AY184156), C (AY184158), J (AY184165), K (184166), L1/2/3 (AY184167/8/9)) were retrieved from the database at the National Center for Biotechnology Information (see Availability and requirements section for URL). Primers and probes derived from MWG-Biotech (Ebersberg, Germany), metabion (Planegg-Martinsried, Germany) or Eurogentec (Seraing, Belgium).

PCR assays

The CT/LGV-real-time PCR assay was carried out in a total volume of 25 μl consisting of 1 × Eurogentec MasterMix without ROX, 5 mM MgCl₂, Amperase (Eurogentec, Seraing), 150 nM CT-F primer, 115 nM each CT-R (-R1 and -R2) primer and 100 nM FAM-labelled CT-probe, 150 nM of the LGV primers LGV-F and -R and 100 nM of the HEX-labelled LGV-probe and 2.5 μl of template DNA. As internal inhibition control 10³ copies of IK_C plasmid and 100 nM TexasRed-labelled Dros-probe (for the detection of internal control plasmid amplification) were added.

Thermal cycling conditions for all real-time PCR assays were as follows: 1 cycle at 50°C for 10 min, 1 cycle at 95°C for 10 min followed by 45 cycles at 95°C for 15 sec and 60°C for 1 min. Cycling, fluorescent data collection and analysis were carried out with an iCycler thermocycler from BioRad according to the manufacturer's instructions.

The internal control plasmid IK_C was constructed essentially as described previously by Rosenstraus et al. [29]. The IK_C-F and -R primers (Table 1) used in the PCR were composed of the respective 5'-sequence of the CT primers R1 and R2 fused to 3'-sequences of the ninja transposon of Drosophila simulans (not found in Chlamydia trachomatis). The plasmid pSARM [30] was used as template which consists of a 276 bp sequence of the ninja transposon and contains the 88 bp target sequence. This target sequence is made up of a 44 bp region of the transposon, containing the 29 bp region recognised by the Dros-probe, and flanked by the primer sequences CT-R1 and CT-R2.

IK_C PCR was conducted in 100 μl 1 × Biorad iQ-Supermix with 3 mM MgCl₂, 1 μM primer pair IK_C-F and -R and 10 ng pSARM plasmid DNA under the following thermal cycling conditions: 1 cycle at 95°C for 5 min followed by 40 cycles at 95°C for 15 sec, 48°C for 30 sec and 72°C for 30 sec.

Conventional L2-PCR was conducted in 100 μl 1 × Biorad iQ-Supermix with 3 mM MgCl₂, 1 μM primer pair L123-F and L2-R under the same cycling conditions of the real-time PCR.

IK_C amplicon and the L2-PCR amplicon were isolated using the PCR purification kit of Macherey and Nagel and 10–20 ng amplicons were ligated into 25 ng pGemT-easy using the rapid DNA ligation kit from Roche following the manufacturer's instructions and subsequently propagated in E. coli DH5α . After cultivation on LB-Amp agar plates, insert-positive clones were identified by restriction analysis and the sequence determined by sequencing using an ABI sequencer using the method of Sanger et al. [31].

Two genes for a species- and LGV-specific C. trachomatis detection

Over a period of several years we evaluated the usefulness of targeting a species-specific region of the omp-1 gene for C. trachomatis detection by comparing real-time PCR data with those produced by a commercial immuno-fluorescence test (IFT) from BioMerieux for perianal, urogenital and ocular swabs [32]. The specificity of the TaqMan primers and probe was verified by alignment of the amplified omp-1 gene regions of all known 20 serovars. To compensate for the two variant nucleotides within the reverse primer binding site (marked by stars in Figure 1(a)) we used a mixture of reverse primers, CT-R1 and -R2 (see Table 1). In 2005, 26 of 985 specimens were positive in the CT-real-time PCR whereas only 24 were IFT-positive. Both methods showed 91.7 % concordance (data not shown).

To enable a simultaneous detection of LGV-serovars which, in Europe, have increasingly lead to infections in men who have sex with men, we supplemented the CT reaction with LGV detection. As the polymorphic membrane protein H gene, pmp-H has a unique deletion in all LGV-serovars we chose this gene as a target for LGV detection. The LGV-primers used corresponded to those designed by Morré and co-workers [19] whereas the deletion-spanning MGB-probe was replaced by a HEX-labelled standard TaqMan probe (Figure 1).

Combined in a duplex real-time PCR assay we first determined the sensitivity and the lower limits of the CT- and LGV-detection using the pGemT-cloned amplicons as template DNA in serial dilutions. All values were measured in duplicate and reproduced in a second run. As shown in Figure 2, the quality of the CT- and LGV-PCR assays was similar in terms of efficiency (93,7% and 90,5%), r² values (0,988 and 0,993), linear detection range between 10⁹ and 10² copies per reaction and detection limits of about 50 genome copies per reaction.

Differentiation of the LGV serotypes L1, L2 and L3

In order to develop a real-time PCR assay to detect and discriminate between L1-, L2- and L3-serotypes, a multiple sequence alignment of the omp-1 genes revealed a less conserved region which showed a 3'-overlap with the formerly described variable segment VS2 of omp-1 [7]. As shown in a phylogenetic tree of this gene region (Figure 3(a)), the serotypes of C. trachomatis cluster in three main branches:

• the L1-cluster containing the serotypes L1, D, Da, E, F and G;

• the L2-cluster containing the L2-serotypes (L2, L2a and L2b) and B/Ba; and

• the L3-cluster comprising the serotypes A, C, H, I/Ia, J/Ja, K and L3.

The designed L1-, L2- and L3-TaqMan PCR assays differed slightly in their relative positions within that gene region (Figure 3(b)). We tested the sensitivity on amplicons of the respective PCR, which had been cloned into a plasmid. The monoplex L1-, L2- and L3-PCR assays were similar in terms of efficiency (92.6% (L1), 95.3% (L2) and 93.5% (L3)), r² values (0,987, 0,998 and 0,995 resp.), and linear ranges between 10⁹ and 10² copies per reaction. The detection limits corresponded with 50 genome copies per reaction to those of the CT/LGV duplex PCR. Analysing DNA of the serovars L1, L2 and L3 the Ct-values remained nearly un-affected when duplexing the HEX-labelled L1- with the FAM -labelled L2-reaction; and the TET-labelled L3- with the FAM-labelled GAPDH-PCR (Table 2). It should be noted that the fluorescence of the TET-labelled probe was detectable in the HEX-channel and in addition of nearly the same intensity in the FAM-channel. The amplification of human GAPDH provided a control of the quality of the specimen. When duplexed with the L3-PCR the value of the GAPDH-reaction was increased slightly in the presence of a positive L3-reaction, whereas the L3-value remained unaffected. To circumvent this problem in a multiplex assay, another fluorophore (e.g. TexasRed) should be used.

Specificity of the real-time PCR assays

The analytical specificity of the multiplex CT/LGV-, L1/L2- and L3/GAPDH- real-time PCR assays was investigated by testing the C. trachomatis serovars and other bacteria that normally reside in the human perianal and urogenital tract (see Materials and Methods). There was no cross-reaction with DNA of non-chlamydial microorganisms (data not shown) or with chlamydial species other than C. trachomatis (Table 3). Analyses of the different C. trachomatis serovars revealed a high specificity of the newly developed real-time PCRs. However the serovars B, I-/Ia and L1 cross-reacted slightly, in that serovar B reacted in the LGV-reaction, I-/Ia in L3-PCR and L1 in L2-PCR. As the Ct-values of the specific and non-specific reactions differed by a factor of six or more, cross-reactions can be readily identified. Thus, the basic prerequisite for LGV-serotyping is a positive LGV-PCR with a Ct-value comparable to that of the CT-reaction. Weak cross-reactivity of L1-serovar in L2-PCR was a disadvantage when duplexing the typing assays. A highly positive L1-reaction and weak L2-PCR could either be based on a double infection with a high load of L1-serovar and a low load of L2-serovar or the result of the cross reacting L1-DNA in the L2-PCR. To avoid misinterpretation in this case the specimen must be analysed either in monoplex L1- and L2-PCR assays or subsequently in monoplex L2-real time PCR.

Retrospective analysis of C. trachomatis -positive specimens

From 2004 to 2006 2381 ocular, urogenital and rectal specimens were tested by monoplex CT-real time PCR for C. trachomatis in the diagnostic laboratory. More than 90% of the tests were conducted on specimens from patients with clinical signs of infection. Of the 67 CT-positive specimens 52 samples were still available for retrospective analysis of the LGV status. We analysed these specimens in the CT/LGV duplex real-time PCR assay and found 50 positive for C. trachomatis. They consisted of ten conjunctival swabs, two poch of Douglas aspirations, eleven cervix swabs, 2 tubal swabs, fourteen urethral swabs and eleven rectal swabs. Two specimens, one urine and one cervix swab were negative. This discrepancy may be either due to a false-positive IFT or more probably to degradation of the DNA as an in-house nested PCR targeting 800 bp of the omp1 gene was also negative.

As summarised in Table 2, seven of the 50 C. trachomatis positives were also positive in LGV detection, all from men (six rectal and one urethral swab). With comparable Ct-values in both reactions a double infection with LGV- and non-LGV-serovars could be excluded. The rectal swabs were taken from HIV-positive patients, four of whom had ulcerating proctitis. The urethral specimen came from a HIV-negative patient with an urethral ulcer. LGV typing revealed that six were of L2-type. One rectal specimen revealed a double-infection with L2- and L3-serovars (Table 2). Sequencing of the L3-amplicon of the double positive specimen confirmed the presence of L3-DNA. As the sequence of the L2-amplicon is identical in L2-, L2a- and L2b-serovars and therefore unqualified for further sub-typing the L2-positives were subjected to a conventional PCR amplification of an extended region of omp-1 including point mutations between the subtypes. Sequence analysis of the PCR-products revealed that they were all of L2b-subtype.