Characterization of ompA Genotypes by Sequence Analysis of DNA from All Detected Cases of Chlamydia trachomatis Infections during 1 Year of Contact Tracing in a Swedish County

Setting.Värmland County has 274,000 inhabitants residing in 16 municipalities. Testing for C. trachomatis is performed in a range of different settings, including hospital-based clinics for venereology, gynecology, and infectious diseases; antenatal, family planning, primary care, and youth clinics; and services provided by military medical officers.

Clinical samples.Urogenital or urine samples were obtained from patients visiting clinics that provide examination of genital chlamydia infections. Samples were sent to a single laboratory at the county hospital in Karlstad, Sweden, and no alternative laboratory was used in the catchment area. Specimens were collected according to the instructions with the COBAS Amplicor C. trachomatis test (Roche Diagnostic Systems, Inc., Branchburg, N.J.). C. trachomatis-positive samples (original urine and processed DNA specimens from urine or swabs) were frozen at −70°C and transported on dry ice to the University Hospital, Uppsala, Sweden, for further analysis.

DNA extraction.DNA was extracted according to the manufacturer's instructions, but when the ompA PCR (see below) was negative, DNA was purified by using a QIAamp DNA minikit (Qiagen, Hilden, Germany) for urogenital swabs or a QIAamp viral RNA minikit for urine. The urine was concentrated by centrifugation for 10 min at 13,000 × g prior to extraction.

PCR.Amplification of an approximately 990-bp fragment of ompA was performed by PCR based on the primer pair P1 (5′-ATG AAA AAA CTC TTG AAA TCG G-3′; nucleotides [nt] 1 to 22 in the sequence with accession number X52557 )-OMP2 (5′-ACT GTA ACT GCG TAT TTG TCT G-3′; nt 1124 to 1103) (18), and if reamplification was necessary, the inner pair MOMP87 (5′-TGA ACC AAG CCT TAT GAT CGA CGG A-3′; nt 87 to 111)-RVS1059 (5′-GCA ATA CCG CAA GAT TTT CTA GAT TTC ATC-3′; nt 1079 to 1050) (30) was used.

The first PCR step was carried out with primer pair P1-Omp2 and 10 μl of DNA extracted from urine or swabs. Amplification was performed in a final reaction volume of 50 μl containing a 0.4 μM concentration of each primer, 2.0 mM MgCl₂, 200 μM deoxynucleoside triphosphates, and 1.5 U of HotStar Taq DNA polymerase (Qiagen). Amplification conditions consisted of initial polymerase activation at 95°C for 15 min; 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s; and a final elongation step at 72°C for 7 min. A negative water control was included in each run. In nested PCRs, 3 μl of product from the first PCR step was added to a final volume of 50 μl. PCR conditions were as described above except that the annealing temperature was 60°C. The amplified products were visualized on an agarose gel stained with ethidium bromide.

DNA sequencing.The ompA fragment obtained was purified by using a QIAquick PCR purification kit (Qiagen), and both strands of a 480-bp segment were sequenced by using a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.). The reaction mixtures were loaded onto a 310 Genetic Analyzer (Applied Biosystems). The primers used for sequencing were RVS1059 and 191S (5′-GCT YTS TGG GAR TGT GGR TGT GC-3′; nt 598 to 620). For a subset of 188 PCR products, sequencing was also performed on separate strands with primers MOMP87 and C214 (5′-TCTTCGAYTTTAGGTTTAGATTGA-3′; nt 671 to 648), resulting in the sequence of almost the entire ompA gene. (See also the indicated primers in Fig. 1.) Each month, 12 samples were selected for sequencing of the 990-bp fragment. After 6 months, serovars E, F, and K were given less priority than other serovars, since they are more conserved in sequence than other serovars. In addition, some samples were selected because they were included in contact-tracing trees of interest. Thus, the selection criteria for sequencing of the 990-bp fragment were mainly random, and the distribution is shown in Table 1.

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FIG. 1.

Description of sites with nucleotide changes in ompA. Arrows indicate nucleotide changes compared to the reference sequence. Dashed arrows indicate positions with changes compared to D/B120 but not seen in the Da strain D/IC-Cal8. Percentages indicate the proportions of sequences with nucleotide changes in each serotype. Sequence primers are indicated as dotted arrows; thus, the 480-bp fragment is flanked by primers 191S and RVS1059 and the 990-bp fragment is flanked by primers MOMP87 and RVS1059.

BLAST analysis and alignments.The consensus sequences were compared to known C. trachomatis strains by using the BLAST search tool at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov ). The sequences were assembled into alignments by using the reference strains A/Sa1 (accession number M58938 ), B/TW-5 (M17342 ), B/IU-1226 (AF063208 ), C/TW3 (M17343 ), D/B-120 (X62918 ), D/IC-Cal8 (X62920 ), E/Bour (X52557 ), F/IC-Cal3 (X52080 ), G/UW57 (AF063199 ), H/Wash (= H/UW4) (X16007 ), I/UW-12 (AF063200 ), Ia/IU-4168 (AF063201 ), J/UW36 (AF063202 ), Ja/IU-A795 (AF063203 ), K/UW31 (AF063204 ), L1/440 (M36533 ), L2/434 (M14738 ), L3/404 (X55700 ), and, as an outgroup, Chlamydia muridarum MoPn^T (M64171 ).

Phylogenetic analysis.Residues corresponding to flanking primers were excluded from analysis. Sequences were manually aligned and adjusted to prototype sequences as described previously (38). Neighbor-joining trees with the Kimura two-parameter correction were produced in PAUP* 4.0b8-10 (34). Phylogenetic reconstructions were also performed by parsimony analysis. Confidence levels for the branching pattern were estimated by a bootstrap resampling of the data based on 1,000 randomly generated data sets.

Statistical analysis.We used chi-square tests to look for evidence of serotype fluctuation during the study.

Ethics.The study was approved by the Research Ethics Committee of the Medical Faculty of Uppsala University, Uppsala, Sweden.

Genotyping.Sequence analysis of the 480-bp fragment of the ompA gene comprising VD3 and VD4 was achieved from amplified DNA in 678 (94%) of the clinical samples. Genotypes K and Ia showed no variation and were identical to the reference strains used. Genotype E was the most prevalent (39%), followed by F (21%), G (11%), D and Da (9%), K (9%), J (7%), H (2%), B (1%), and Ia (1%). For a subset of 188 samples, the complete 990-bp fragment was sequenced and all four VDs were covered. There were 25 genotype variants in the 480-bp fragments and 29 in the entire 990-bp fragments. Among the 29 variants, there were 34 positions with point mutations, and 32% of the mutation sites were located in the constant domains (Fig. 1). Seven substitutions (64%) in the constant domains were silent, but in the VDs the reverse was seen, with 22 of the mutations (96%) resulting in nonsynonymous substitutions.

The predominant genotype E was highly conserved. Although five subtypes were found, the four diverging variants accounted for only 4% of the total number of genotype E cases, and all nucleotide substitutions in the four variants were clustered towards the end of VD4 (dispersed over four positions) and resulted in amino acid changes (Fig. 1 and Table 2). Our D strains comprised seven variants with variations in the 990-bp fragment, and 50% differed from the reference strain D/B120. The D2, D3, and D6 variants have nine discrepant positions between nt 247 and 364 in conserved domain 1 (CD1) compared to D/B-120, but they are identical (D2) or very similar to strain D/IC-Cal8. For other D genotype variants, the substitutions were found in CD1 and VD4. Minor variation was observed for serotype B: out of nine samples, only one differed from the reference strain. For genotypes G (87%) and H (88%), a majority of the sequences differed from the reference strains, and four genotype variants of each serotype were found. Serotype G had point mutations at four positions situated in CD1, VD2, CD3, and VD4, while the closely related F serotype only had one base substitution in 1 out of 139 cases. Serotype H substitutions were observed at seven positions, i.e., two in VD1, two in VD2, two in CD4, and one in VD4. The nucleotide substitutions in positions 440 and 1018 were present in all H variants with mutations. Most J strains (81%) were identical to the reference strain, but sequence alignment revealed three variants. For genotype J2, substitutions were observed at 12 different positions distributed in three different CDs and three VDs, whereas the J3 variant had a substitution only in CD2.

There was no evidence of concordance in mutational distribution between the 29 genotype variants. Mutational hot spots were not found and in most cases mutations could not be found at corresponding positions in different serotypes. However, the variants D2, D3, D6 (nt 343), and G3 (nt 228) had a silent T→A substitution in the same position in CD1. Genotypes G2 and G3 (nt 1003) and H3 (nt 1178) had substitutions in VD4 that differed by one nucleotide only, involving a codon for serine that was replaced by alanine (G2), threonine (G3), or asparagine (H4) (Table 2). Similarly, genotypes G3 and G4 (nt 487) had a mutation including the same codon as the nucleotide substitution seen in D6 (nt 600), which for G3 and G4 resulted in a change from glycine to serine, whereas in D6 asparagine was replaced by serine.

Phylogenetic analysis of the 990-bp PCR product was performed on the 29 genetic variants from 9 serotypes obtained in our study, 18 reference sequences from 14 serotypes of C. trachomatis, and 1 strain of C. muridarum as an outgroup (Fig. 2). As expected, three main clusters were identified, according to previous grouping in the B, F-G, and C complexes (13, 30). The two genetic variants of serotype B in our study population differed at one nucleotide; one was identical to the reference strain B/IU-1226, which is also a urogenital strain, while the B prototype strain B/TW-5 was isolated from conjunctiva and is genetically more distant. Noteworthy is the clade of the Da variants, including genotypes D2, D3, D6, and the D/IC-Cal8 strain. As anticipated from Table 2, the five E variants form a closely related cluster, where the E1 variant is identical to the prototype strain E/Bour. The two F genotypes in our population differed in only one nucleotide, and F1 was identical to the reference strain F/IC-Cal3. Among the four variants of serotype G, the G1 genotype was identical to G/UW57 and the other variants were closely related. Likewise, in the C complex H1 was identical to H/Wash, and the other three variants were similar. Our J1 genotype is identical to the reference strain J/UW36, while the J2 variant is more distant than J3. The only K strain in this study was identical to K/UW31.

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FIG. 2.

Phylogenetic neighbor-joining tree based on the C. trachomatis ompA nucleotide sequences from 29 genetic variants in the clinical material and 18 reference sequences available from GenBank. The sequence from C. muridarum is used as an outgroup to root the tree. Branch lengths are proportional to the amount of sequence that diverged between taxa in the tree, as illustrated by the bar. Relevant bootstrap values (as percentages of 1,000 replicates) are given.

Temporal and clinical associations with serotypes.Fluctuation of serotypes over a 1-year period was modest. Serotype E predominated throughout the year, except for January, when more cases of type F were detected (E, 21 cases; F, 26 cases). There was evidence of variation in the proportion of F strains, which decreased until May and then increased again (P = 0.0133). Trends for other types could not be examined owing to small numbers.

We did not find any evidence of an association between genotype and clinical manifestations (Table 3). Most infections were asymptomatic. Of 587 individuals with chlamydia who provided clinical information, 33% had urethral irritation and/or genital discharge. Fourteen percent of the females had abdominal pain, fever, or other symptoms that could be related to pelvic inflammatory disease. The prevalence of symptoms was similar in males and females. We did not find any dual infections in our samples, although such infections probably occur in low numbers.

Sexual networks.Genotype variants were linked to sexual networks identified through contact tracing. Examples of networks for which sequencing provided information in addition to that obtained from contact tracing alone are shown in Fig. 3. The analysis of minority variants (B2, D3 to -7, E2, E4, E5, F2, G4, H1, H3, H4, J2, and J3) derived from persons who had mentioned at least one sexual contact comprised 33 samples. Of these, 23 (70%) could be correlated to sexual contacts outside the county or the country. Thus, detection of a new genotype variant suggests a probable importation of a new strain into the population, as exemplified in Fig. 3A. Of all 397 sexual networks, 146 (37%) resulted in at least two ompA sequences, and of these, different serotypes were seen in 32 networks (22%). In only two networks were different variants of the same main serotype detected. In one network the genotypes E1 and E5 were detected at the same time point, and a single point mutation would explain the variation in the tree. In the other tree the two different genotypes G2 and G3 were detected 6 months apart in two individuals, and three point mutations would be required for such a change. Here the most likely explanation is infection with two different strains.

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FIG. 3.

Examples of sexual networks. Arrowheads indicate stated contacts. Squares, males; circles, females. (A) Uncommon genotype suggesting import of a new strain into the population. Filled circles indicate untraced contacts outside the country. The male examined on 25 June 2001 and found to have a genotype J3 strain had two contacts abroad, and this genotype was subsequently transmitted locally. (B) Example of an infection with possible persistence. Filled squares indicate untraced contacts. The female examined on 13 June 2001 and found to have a genotype F1 strain is suggested to have transmitted chlamydia to the male who was negative in a test in July. In an examination 6 months after the first contact (19 December 2001), he had an F1 strain when his other contacts were infected with G1.