MLVA subtyping of genovar E Chlamydia trachomatis individualizes the Swedish variant and anorectal isolates from men who have sex with men

Abstract

This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other molecular typing methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time-consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens.

Figure 1. MST of the MLVA-5 types of genovar E C. trachomatis isolates and positive specimens.

Each circle denotes a particular MLVA-5 type. The size of the circle is proportional to the number of isolates or specimens belonging to the indicated MLVA-5 genotype. The distance between neighbouring genotypes is expressed as the number of allelic changes and is outlined by short bold lines for one change. No cluster is emphasised as shown by the distance, equal to one, between neighbouring genotypes. ⁺ MLVA-5 types encompassing anorectal isolates and specimens. ^★ MLVA-5 type containing nvCT isolate and specimens.

Figure 2. Clustering dendrogram of 43 genovar E C. trachomatis isolates typed by four molecular methods.

The order in which the isolates are listed, from top to bottom, is based on the MLVA-5 dendrogram (left side of the figure). The dendrogram was constructed using a categorical coefficient and UPGMA clustering. Concomitant and sequential isolates from the same patients are highlighted in grey and are underlined, respectively. The MLVA-5 type 21, which included all anorectal isolates, is outlined in black. nd, not determined.