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Genotyping of Urogenital Chlamydia trachomatis in Regional New South Wales, Australia

Mossman, David*†; Beagley, Kenneth W.†‡; Landay, Alan L.§; Loewenthal, Mark*†; Ooi, Catriona*†; Timms, Peter; Boyle, Michael*†

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Sexually Transmitted Diseases: June 2008 - Volume 35 - Issue 6 - p 614-616
doi: 10.1097/OLQ.0b013e31816b1b80
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CHLAMYDIA TRACHOMATIS IS THE MOST common cause of bacterial sexually transmitted infections worldwide. Pelvic inflammatory disease, ectopic pregnancy, and infertility in women, and occasionally epididymitis in men, complicate infection. Chlamydia also acts as a marker of increased risk for human immunodeficiency virus infection and may directly contribute to human immunodeficiency virus infection.1,2 The prevalence of C. trachomatis has been rising rapidly in most developed countries, including Australia. In New South Wales where this study was conducted, the population rate of C. trachomatis infection doubled between 1996 and 2000, and then doubled again between 2001 and 2005 (national center report).

There are 19 recognized serovars of C. trachomatis, as defined by serological responses to the outer membrane protein (OmpA), of which D, Da, E, F, G, Ga, H, I, Ia, J, and K are associated with infection of the genital tract. These serovars can be conveniently defined by sequence analysis and genotyping of the ompA gene.3–5 There has been significant interest in whether these genotypes are differentially associated with clinical manifestations, geographic locations, or gender.6–8 The current study was undertaken to explore these issues in a cohort of patients referred from a wide geographic area of regional Australia.

Four hundred clinical samples (first void urines) sent to the Hunter Area Pathology Service for assessment between January 2005 and November 2006 were screened as positive for C. trachomatis by polymerase chain reaction (PCR) using the COBAS Amplicor system (Roche Diagnostic Systems, Indianapolis, IN). DNA remaining from the original extraction was stored at −2°C before amplification by nested PCR. Two hundred and three (50%) of the samples yielded sufficient DNA for sequence analysis of the ompA gene.

Nested PCR was used to produce a 1314-bp amplicon of the ompA gene of C. trachomatis. The first PCR reaction used primers OP1 (5′-GGACATCTTGTCTGGCTTTAACT-3′) and OP2 (5′-GCGCTCAAGTAGACCGATATAGTA-3′) to amplify a 1502-bp fragment of the ompA gene, as previously described.9 The primary PCR reaction was carried out in a volume of 25 μL, containing a final concentration of 200 μM dNTPs, 1× PCR buffer, 1.5 mmol/L MgCl2, 0.6 μM of each OP1 and OP2 primers with 1 U of Platinum Taq Polymerase (Invitrogen, Carlsbad, CA). The reaction was performed with an initial denaturation step of 94°C for 120 seconds. Amplification was achieved with 40 cycles of 94°C for 30 seconds, 52°C for 60 seconds, and 72°C for 60 seconds. A final elongation step of 72°C for 180 seconds was also used. In the secondary PCR, 2 μL of primary PCR product was used as template in a 25 μL reaction volume. PCR thermal cycler conditions were the same as used in the first round of PCR with the exception of the primers; inner primers IP1 (5′-GTGCCGCCAGAAAAAGATAG-3′) and IP2 (5′-CCAGAAACACGGATAGTGTTATTA-3′). The final product of 1314 bp was purified using Ampure PCR purification system (Agencourt Biosciences, Beverly, MA) before the sequencing reaction.

The entire coding region of the ompA gene was sequenced using BigDye Terminator Version 3.1 chemistry and primers S1 (5′-ATAGCGAGCACAAAGAGAGC-3′) and S2 (5′-TGGGATCGTTTTGATGTATT-3′). Sequencing reactions were purified with CleanSEQ purification system (Agencourt Biosciences) and sequenced on an ABI 3730 genetic analyzer (Applied Biosystems, Rockville, MD). DNA sequences obtained were aligned to obtain full-length sequence information of each sample using the aligner tool hosted at www.justbio.com. Aligned sequences were queried against the BLAST database (www.ncbi.nlm.nih.gov/BLAST/) on the National Centre for Biotechnology Information Website, which allowed experimentally obtained sequences to be matched with existing records in Genbank (www.ncbi.nlm.nih.gov/Genbank/). Based on the matches obtained, individual samples were then classified as a particular genotype of C. trachomatis. Upon completion, sequences of all samples exhibiting the same genotype were aligned with each other to identify substrain variations.

The association between age and genotype was examined using the Kruskal-Wallis test because the assumptions required for analysis of variance were violated. The relationship between gender and genotype was tested using Pearson’s chi-square. All P values are two-sided. Multivariate analysis of the association of genotype with age and sex using polytomous logistic regression did not materially alter these findings and was not reported.

The average age of patients with C. trachomatis infection in this cohort was 22.6 years (SD 7.29; Fig. 1) where 98 were women (48%) and 105 men (52%). Fifty-seven of the isolates were from the Newcastle area, with 29 from the Hunter valley excluding Newcastle, 37 from the Northern Rivers area, 34 from the New England area, 20 from southern New South Wales, and 25 from the mid coast of New South Wales. Genotyping of isolates revealed genotype E to be the most common. The frequency of isolation of the individual genotypes is given in Table 1. Mixed infections were identified in only 3 patients. Genotype D isolates could be divided into D/UW-3/CX (7 isolates) or Da/TW-448 and D/IC-CAL8 strains (12 isolates). G isolates were evenly distributed between the consensus strain G-UW57, and a G-UW57 strain with a single G-to-A substitution at nucleotide position 487 of the ompA gene (N = 17). This missense mutation resulted in a glycine-to-serine substitution. H isolates were also evenly distributed between the H consensus sequence and a novel H variant, with an A-to-G nucleotide substitution at position 272 of the ompA gene (n = 5). This missense mutation resulted in an asparginine-to-serine substitution in the VS1 region of the ompA gene. There were 7 single nucleotide changes in 6 isolates identified in the consensus nucleotide sequence of the 86 E genotype C. trachomatis species typed in this study. The mutation was silent in 3 of the 7 cases and missense in 4 (3 isolates had Asp-to-Glu substitution with a single nucleotide change at position 258; 1 isolate with Ser-to-Asn substitution with a single nucleotide change at position 512).

Fig. 1
Fig. 1:
Age distribution of patients recruited into study cohort.
TABLE 1
TABLE 1:
Distribution of Chlamydia trachomatis Types in Sample

The effect of age, gender, and geographic area on C. trachomatis type was examined. Patients with C. trachomatis type G had a mean age of 28.9 years, which was significantly older on average than the mean of the other groups combined of 22.6 years (P = 0.022). There was no association between gender or area of specimen collection and type (P values of 0.257 and 0.655, respectively.) Further analysis assessing for an association between strain variants and age, gender, and geography was undertaken. Again there was no significant association between strain variants of specific types of C. trachomatis and age, gender, or geographic location.

In common with studies from multiple countries, genotypes E, F, and G were the dominant strains of C. trachomatis found in our patient population 6,10–14. Moreover, the findings in this study closely resemble those of Lister et al.15 who found genotypes E (40%), F (19%), and G (17%) to be the most common strains in women in Melbourne, Australia. The evidence suggests that there is relatively little geographic variation in genotypic prevalence not only in New South Wales, but also more broadly across Australia and the globe.

Patients with C. trachomatis type G were significantly older (mean, 28.9 years) than the mean of the other groups of 22.6 years. It could be that the immune response to E drives a population switch to the G genotype with repeated exposure. This hypothesis suggests C. trachomatis infection would initially occur with the commonest strain circulating in the population, namely E. The immune response to this strain would develop and prevent further infection with this strain. However, the immune response is not broadly cross-protective, allowing the less common strains that are circulating in the population, including strain G, to occur with the repeated exposure to C. trachomatis that will occur if one continues to have multiple partners as one ages. Another possibility is that our data have been biased by the relatively high failure rate of our nested PCR approach to sequencing the ompA gene (50%). Other studies have reported similar difficulties with amplification of the ompA gene,3 and the concordance of the population genotype frequency with other studies makes such a hypothesis less likely.3,5,15 It is interesting to note that there was no association between age and the minor variations in the H and E variant genotypes identified in this study, even though most of these isolates had missense mutations in variable domains of the ompA gene. Further research will be needed to assess this association of older age and strain G, which has implications for vaccine design if confirmed.

It has been suggested that genotyping of C. trachomatis may be useful in contact tracing.4 The data presented here do not support that proposal. Genotypic variations were remarkably uniform across this wide geographic area, and most patients were infected with common genotypes. The only genotypic variant that seemed to have any geographic limitation, the single nucleotide variant of genotype H, was present in both Newcastle and the lower Hunter region. This strain was isolated in both the first and last months of the study. This genotypic variant is most likely a minor variant circulating in this population. The temporal spread of the specimen collections from the patients where the H variant was isolated make it unlikely that they form a sexual network. Clinical history would be a more robust and effective way of identifying sexual networks. Genotypic data would really only be of use as supportive evidence if an unusual genotype were identified.

In conclusion, this study confirms that strain E is the dominant strain of C. trachomatis in Australia. The association between age and strain G raises the possibility that immunity to strain E may drive a switch to strain G in older populations, previously infected with C. trachomatis. Further studies are needed to confirm this hypothesis, which would have significant implications for vaccine design if proven correct.

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