Evaluation of high-resolution typing methods for Chlamydia trachomatis in samples from heterosexual couples

Abstract

We aimed to compare conventional ompA typing of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). Previously used MLST and MLVA systems were compared to modified versions that used shorter target regions and nested PCR. Heterosexual couples were selected from among persons with urogenital C. trachomatis infections visiting the sexually transmitted infection outpatient clinic in Amsterdam, The Netherlands. We identified 30 couples with a total of 65 C. trachomatis-positive samples on which MLST and MLVA for eight target regions were performed. All regions were successfully sequenced in 52 samples, resulting in a complete profile for 18 couples and 12 individuals. Nine ompA genovars from D to K, with two variants of genovar G, were found. The numbers of sequence type and MLVA type profiles were 20 for MLST and 21 for MLVA, and a combination of MLST and MLVA yielded 28 profiles, with discriminatory indexes (D) ranging from 0.95 to 0.99. Partners in 17 couples shared identical profiles, while partners in 1 couple had completely different profiles. Three persons had infections at multiple anatomical locations, and within each of these three individuals, all profiles were identical. The discriminatory capacity of all MLST and MLVA methods is much higher than that of ompA genotyping (D = 0.78). No genotype variation was found within the samples of the same person or from heterosexual couples with a putative single transmission. This shows that the chlamydial genome in clinical specimens has an appropriate polymorphism to enable epidemiological cluster analysis using MLST and MLVA.

Fig. 1.

Graphical representation of the circular genome of Chlamydia trachomatis (reference strain D/UW-3/CX). Indicated are the locations of the regions used by Klint et al. (12) (hctB, CT058, CT144, CT172, pbpB, and ompA) and Pedersen et al. (20) (CT1291, CT1299, CT1335, and ompA). As can be seen, CT172 and CT1291 concern the same region.

Fig. 2.

Graphical representation of region CT172/CT1291. Indicated are the positions of regions on the genome. The upper representation is the modified version. It is designed in such a manner that it includes all significant variation of the original assay of region CT172 of Klint et al. (12). It also completely covers region CT1299 of Pedersen et al. (20). The primers are designed in such a way that the regions flanking the repeat region are at least 70 bp long. This improves the readability of the sequences. Outer primers are given in black, and inner primers are given in gray. The length of the amplified fragment is represented by the lines between the primer sets. The 5′ positions of the primers are given as in Table 1.

Fig. 3.

Minimum-spanning tree using the modified C. trachomatis MLST method with samples from 18 putative couples. The coding is by couple number and gender (♂, male; ♀, female). Genovars (ompA) are indicated with letters within the tree, and nodes with identical patterns indicate identical genovars. The sizes of the nodes indicate the number of samples included (1 to 8). The length of the branch indicates the number of alleles shared between connected sequence types (1 to 5). When multiple samples are available for one person, the number of included samples is given in parentheses. It can be seen that the partners of couple 18 do not share the same strain of Chlamydia trachomatis.

Fig. 4.

Minimum-spanning tree using the modified Chlamydia trachomatis MLST and VNTR analysis MLVA method combined on 18 putative couples. The coding is by couple number and gender (♂, male; ♀, female). Genovars (ompA) are indicated with letters within the tree, and nodes with identical patterns indicate identical genovars. The sizes of the nodes indicate the number of samples included (1 to 8). The length of the branch indicates the number of alleles shared between connected sequence types (1 to 7). When multiple samples are available for one person, the number of included samples is given in parentheses. It can be seen that the partners of couple 18 do not share the same strain of Chlamydia trachomatis.