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J Clin Microbiol. 2004 Apr; 42(4): 1641–1647.
PMCID: PMC387550

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

Abstract

In this study we aimed to characterize the ompA gene by sequencing DNA from all detected cases of Chlamydia trachomatis infection in a Swedish county during 2001, in order to improve the efficiency of contact tracing. Approximately 990 bp of the ompA gene was amplified, and sequence analysis was achieved in 678 (94%) of 725 C. trachomatis-positive cases in this unselected population. The most prevalent genotype was serotype E (39%), followed by F (21%), G (11%), D (9%), K (9%), J (7%), H (2%), B (1%), and Ia (1%). Serotype E was found in five genotype variants, with the reference sequence comprising 96% of all E cases. Serotype D was the most variable, and of seven sequence variants, three were identified as recombinants with serotype E. Altogether 29 genetic variants were detected, and mutations and recombination events are discussed. Clinical manifestations were not associated with genotypes. Sequence variation was linked to sexual networks identified by contact tracing and improved epidemiological knowledge but was of limited benefit.

Genital infections with Chlamydia trachomatis are associated with a wide spectrum of diseases: urethritis, epididymitis, cervicitis, salpingitis, pelvic inflammatory disease, ectopic pregnancy, and infertility (7). In Sweden, reporting of all cases of genital infection to the county medical officer for communicable disease control has been mandatory since April 1988. The reported incidence declined to a low and constant level at the beginning of the 1990s in all Swedish counties. Contact tracing, screening, and treatment of asymptomatic people were considered important contributory factors to the decline. In 1994, however, an increase was reported in one county (15), and this was later confirmed by national data. In 2001 the reported incidence was 250 cases per 100,000 inhabitants, an increase of 15% compared to 2000 and the same rate as 10 years previously (33).

The major outer membrane protein (MOMP) of C. trachomatis is one of the principal cell wall components and is responsible for the structural integrity of the extracellular infectious elementary body and the developmental conversion to the plastic and fragile intracellular reticulate body (6, 14). The MOMP is encoded by the ompA gene and exhibits extensive DNA sequence variation that is localized mainly to four discrete regions, termed variable domains (VD1 to -4) (28, 38). Three of the VDs are surface exposed and contain antigenic peptides (1, 29). Serotyping of MOMP with antibodies directed to these VDs differentiates between at least 15 serovars: A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, and L3. Sequencing of the ompA gene is the method of choice for strain differentiation because it is more discriminating than genotyping with restriction fragment length polymorphism and can also differentiate between strains (21, 22, 37).

Characterization of C. trachomatis strains can provide important epidemiological knowledge and contribute to improved control measures. In this study we aimed to increase the effectiveness of contact tracing and to characterize the ompA gene by sequence analysis of DNA from all detected cases of genital C. trachomatis infections in the county of Värmland during 1 year.

MATERIALS AND METHODS

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. Fig.1.)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 Table11.

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 ...
TABLE 1.
Distribution of 480- and 990-bp sequence fragments in different serovars of C. trachomatis

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 MoPnT (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.

RESULTS

In total, 725 of 12,761 samples (5.6%) were positive for C. trachomatis in the routine Amplicor PCR test. Males contributed 17% of the samples but comprised 42% of positive cases. Of 1,389 partners identified by contact tracing, 73% were examined for C. trachomatis. The remaining individuals were not examined owing to residence outside the county or abroad (17%) or to incomplete information (10%).

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. (Fig.1).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. (Fig.11 and Table Table2).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.

TABLE 2.
Nucleotide changes found in 29 genetic variants of 678 clinical C. trachomatis specimens compared to reference sequencesa

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 (Table2).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. (Fig.2).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 Table2,2, 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.

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 ...

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 (Table3).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.

TABLE 3.
Association between serotype and clinical manifestations

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. Fig.3.3. 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. Fig.3A.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.

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 ...

DISCUSSION

In this study we used gene sequencing of C. trachomatis to link molecular information to contact tracing data and to characterize ompA by sequencing DNA from all detected cases of C. trachomatis in the county of Värmland in 1 year. In this unselected population, 5.6% were positive in chlamydia tests and 678 sequences were analyzed, resulting in 29 genetic variants of nine different serotypes.

Out of 725 C. trachomatis-positive tests in routine analysis, in 6.5% DNA was not amplified by the ompA PCR. This compares favorably with failure rates of 1 to 24% in similar studies (17, 18, 31). The serotype distribution in our study was similar to that found in other investigations based on the use of monoclonal antibodies (23, 35), restriction fragment length polymorphism (25, 36), or gene sequencing (17, 18). The study of Jurstrand et al. (18) took place in a neighboring county less than a year prior to our study, so it is noteworthy that only 2.5% of their cases, compared with 11% of ours, were of serotype G. This difference is probably due to fluctuations of serotypes within the population over time, since genotype G constituted 4% of the strains in Värmland in January and 20% in December (P < 0.001).

Sequence analysis of ompA and other genes has been used previously as a tool to differentiate chlamydia strains and gain epidemiological knowledge. The variability in ompA sequences in previous studies, measured as genetic variants as a proportion of all chlamydia cases, has ranged between 10 and 81% (4, 9, 22, 31, 32). However, in these studies the numbers of cases were low (in four of the five studies, fewer than 70) and/or the study populations were recruited from high-risk groups. We found 29 genetic variants (4.3%) among the 678 ompA sequences, and in serotypes F, I, and K there was variation in fewer than 1% of the cases. In the most prevalent serotype, E, only 3.6% diverged from the reference sequence, which contrasts with a previous study in which 16% of 67 E strains represented genetic variants (8). It may be argued that our results are explained by bias, since the 990-bp fragment, for example, was sequenced in only 56 out of 264 serotype E cases. However, assuming that the mutation rate is the same in the entire gene (although it is higher in the VDs), the probability of observing no mutations, as we do, in the upstream part is 11.5% (P = 0.115). Thus, our low mutation rate for serovar E strains is reasonable, and the limited number of sequenced 990-bp fragments is unlikely to lead to erroneous interpretations of the results. To conclude, this indicates that the sequence variation in an unselected community population is much lower than that reported from previous studies.

Common to all genotypes was the lack of mutations in VD3. This was not surprising, since this domain does not induce neutralizing antibodies but contains T-cell epitopes involved in the cell-mediated immune response (16, 27). The most immunoreactive parts of the MOMP that induce neutralizing antibodies are found in VD1, -2, and -4 (2), and therefore point mutations are expected to be found in these domains. In fact, 68% of the nucleotide changes in our study were found in these three VDs, and almost all (96%) of the mutations resulted in amino acid changes. The domain harboring most mutations is VD4, but as seen in Fig. Fig.11 the occurrence of mutations in different VDs varied among the serotypes, and for strains in the C complex most mutations were found in VD1 and -2. The majority of substitutions in CDs were silent, reflecting the importance of these domains in maintaining the structural integrity of the protein.

The highest sequence heterogeneity was observed for strains linked to serotype D. Three of the seven genetic variants were closely related to the Da subtype (26), and the CD1 is similar to the sequence in serotype E. This suggests that a genetic recombination with a crossover point immediately upstream of VD1 has occurred. Recombination in ompA of Chlamydia has recently been investigated (20), and it was concluded that recombination was more common in the downstream half of the gene and that there is a higher degree of recombination for the C class than for the B class. Thus, the genesis of the Da variant was a rare event having led to a successful strain type that constitutes a substantial proportion of strains related to serotype D. Recombination is considered to increase the genetic diversity required to evade immune surveillance. In this case, however, it appears to have influenced only CD1, which is involved in maintaining the protein structure rather than in interacting with the immune response.

A similar recombination seems to have taken place between serotypes B and D, since in the Bb variant CD1 is very similar to the corresponding sequences in D/UW3 and H/UW4 (11). This difference in CD1 in Bb strains is characteristic of urogenital strains of serotype B, and there is a difference in 11 positions compared to the prototype strain B/TW5 isolated from conjunctiva. Our nine B strains were similar to the Bb strains described by Frost et al. (11), and when compared to sequences covering the entire ompA, eight of our strains were identical to B/IU/1226 (30).

In the C complex, most serotype J strains were identical to the reference strain, but our J2 variant, comprising 13% of the J strains, had 12 discrepant nucleotide positions and is identical to the Ja type previously reported from the United States (30). A very similar type, Jv, has been reported from The Netherlands (21), indicating that Ja-Jv variants are widely spread. Considering that there were only 16 strains of serotype H, the finding of four genetic variants indicated that this serotype is variable. Strains with sequences identical to the reference sequence were found, but most strains differed in two to five nucleotide positions. The only variant of serotype I in our study was identical to Ia/IU-4168, which has been suggested to be a recombinant of I and H (19), but is distant from the prototype strain I/UW12.

Serotypes H, J, Ia, and K all belong to the C complex. There is little difference between the sequences of these serotypes, especially in the second half of the gene covering VD3 and -4. This high degree of similarity is considered to increase the probability of recombination between serotypes (20), and 4 to 32% of isolates have been found to have sequence patterns in VD1, -2, and -4, suggesting them to be recombinants of I/H, C/J (37), or H/L3 or L variants (4). However, the patients in those studies were high-risk core groups, and in our unselected population 188 fragments of 990 bp (678 fragments covering VD3 and -4) were analyzed, but no apparent case of previously undescribed recombination was detected. Thus, although recombination is most likely essential for the genetic diversity and successful survival of Chlamydia, the rate of genetic shift is very low.

Phylogenetic analysis of the 29 detected genotypes and reference sequences indicated three main clusters. The cluster with serotypes B, D, and E showed larger genetic distances than the other clusters. As discussed above, recombination appears to have occurred, and subvariants such as Da, Bb, Ja, and Ia show some distance from prototype strains of each serotype. Lateral gene transfer leading to recombination may be important in transferring virulence traits and has been suggested to occur in chlamydiae (10), and this later was supported by statistical analysis (20). However, lateral gene transfer has been rejected as an explanation for transfer of virulence between species of the Chlamydiacae, since no correlation was seen between chlamydial evolution and virulence characters when ompA and four other genes were analyzed (5). In a phylogenetic analysis of ompA in C. trachomatis, it was concluded that evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiological success) could not be found (30). This supports our finding of a lack of association between serotype and clinical symptoms. Previous conflicting data have suggested weak support for an association between symptoms and some serotypes or complexes (3, 9, 35). The two largest studies, however, found no (24) or only weak (12) evidence for such an association.

Introduction of gene sequencing in contact tracing contributed to the available epidemiological information. We described networks in which ompA genotypes indicated that individuals were linked and may have had coinfections. Genotyping also showed the likely introduction of a new genetic variant. In other scenarios, genotyping suggested that there were unmentioned contacts and could have helped differentiate between a reinfection and a persistent infection. The only case of genotype D5 in our study was from a male referring to a contact in Örebro, a town in a neighboring county. This rare sequence was also detected in 2 out of 26 D strains in the study by Jurstrand et al. (18), which was conducted in Örebro at the same time as our D5 variant was sampled. This suggests a link in a sexual network that could not be further traced.

In conclusion, sequencing of the ompA gene provides discrimination of C. trachomatis strains and provided additional information when applied to contact tracing. We have shown that in unselected populations the genetic variability and hence the benefits are too limited to recommend the use of sequence-based genotyping in routine work. Technical developments and cost reduction may render it cost-beneficial in the future.

Acknowledgments

This work was funded by the National Institute of Public Health (NIPH), Sweden.

We thank Nicola Low for linguistic revision and insightful comments on the manuscript.

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