In this study, we present a new MLVA-based molecular typing system for the discrimination of genovar E and other genovar D to K of C. trachomatis strains. MLVA-5 is a reproducible and fast technique that does not require a sequencing step and can be standardised, thereby facilitating large-scale molecular epidemiological investigations. The application of multiplex PCR and capillary electrophoresis on a genetic analyzer enables a high-throughput analysis and allows easier interpretation of results in contrast to agarose gel electrophoresis, particularly for VNTRs with a small number of repeat units.
displays remarkable genome sequence similarity and synteny, considering the different diseases and tissue tropism exhibited by the different genovars. Of 202 TR loci identified, only five were polymorphic on genovar E isolates, confirming the high genetic homogeneity of this species. In comparison, eight VNTRs were found in C. psittaci
among 20 TRs and five VNTRs were identified among 34 TRs for C. abortus
The marker Ct-51, located in the hctB
gene, showed six different allele sizes with a repeat copy number ranging from 2.5 to 4.5. This gene was one of the targets of the MLST technique developed by Klint et al..
All of the nvCT isolates displayed the allelic variant hctB-21 with MLST and a 2.5 repeat for the marker Ct-51 with MLVA-5. Other genovar E isolates shared one of these two allelic variants, although they did not have the plasmid deletion. The marker Ct-531, located in the tarp
gene, was the least discriminatory of the VNTRs identified for the genovar E. These results confirm the data of Lutter et al.
, who showed that among 14 urogenital genotype E clinical isolates collected from different areas, 12 had Tarp sequences identical to that of the E/Bour reference strain 
. However, the marker Ct-531 allowed distinction of the nvCT isolates from the other isolates harbouring identical allelic profiles on the four other VNTRs. Although the Ct-719 displayed two allele sizes among genovar E isolates, this marker was monomorphic among the urogenital non-E genovar specimens analysed. However, more specimens need to be tested to confirm these results.
The stability of the method was assessed in vitro by ten-passage culturing for five isolates and in vivo using sequential isolates and specimens from 11 patients on a time interval of 1–28 months. Moreover, in 17 cases, concomitant specimens or isolates harboured identical MLVA-5 types. Taken together, both the in vitro and the in vivo data showed no MLVA-5 type variation over time or within individuals, implying that our MLVA-5 scheme is genetically stable for short-term applications.
In our study, nine of the 11 anorectal C. trachomatis
genovar E isolates displayed the same unique MLVA-5 type 21. All of these patients were MSM and were mainly from the same region in France, suggesting clonal spread of a strain. Interestingly, the available genome of the E/150 rectal isolate shared the MLVA-5 type 21 
. Unfortunately, no information about the sexual behaviour and geographical origin of the patient from whom the E/150 strain was isolated was available. Using whole-genome sequencing along with comparative genomics, Jeffrey et al.
showed that there were only 1130 substitutions and 54 insertions or deletions between the E/150 male rectal and the E/11023 cervical genovar E isolates 
. In their work, the authors identified three ORFs that were statistically correlated with rectal tropism only in genovar G, while none of these ORFs were statistically associated with rectal tropism in genovar E isolates. We genotyped six anorectal isolates from MSM using MLST and MLVA-3. All of the isolates except one belonged to ST-56 and MLVA-3 type 8.6.1. These two genotypes were associated with both anorectal and urogenital isolates, while the MLVA-5 type 21 was only associated with all anorectal isolates except one. Indeed, one male urethral isolate displayed the MLVA-5 type 21, but the sexual behaviour of the patient was unknown. The association of a unique MLVA-5 type with anorectal genovar E specimens from MSM must be confirmed by further analysis of additional specimens. In contrast, the 31 anorectal non-E genovar specimens were distributed among 15 MLVA-5 types and there was no relation between MSM anorectal specimens tested and a MLVA-5 type.
Our results confirmed a clonal spread of the nvCT, in accordance with other reports 
. The nvCT specimens tested in our study were previously genotyped by MLST, and all of them displayed the same unique MLST profile ST-55 and an identical ompA
sequence to that of the E/Bour reference strain 
. All of these specimens also displayed the same unique MLVA-5 type 14.
Recently, using ompA
genotyping, it was shown that repeated C. trachomatis
infections in adolescent women were due to reinfection in 54% of the cases, treatment failure in 14% of the cases and persistence without documented treatment in 2% of the cases 
. In our study, we obtained, C. trachomatis
-positive specimens from five patients before and after treatment and these were for each patient of an identical MLVA-5 type, suggesting treatment failure and persistence of infection. However, we could not exclude recontamination as no information on partner treatment was available.
In several studies, the molecular epidemiology of chlamydial infections was based on analysis of the ompA
gene. However, to discriminate within genotypes, a single marker is inadequate because of the risk of recombination events 
. Therefore, more discriminating molecular techniques are needed 
. Klint et al.
developed an MLST scheme targeting the five most variable regions of the chlamydial genome, while Pedersen et al.
developed an MLVA method based on single nucleotide repeats, which are a type of VNTR, within three loci combined to ompA
. MLST and MLVA-3 are time-consuming techniques that require a sequencing step. In our work, we compared MLVA-5 to ompA
sequencing, MLST and MLVA-3 for 43 genovar E isolates. Results from ompA
sequencing confirmed a high level of conservation of genovar E isolates 
. Using MLST, 42 isolates exhibited17 STs, of which 12 were new. For one isolate, an ST could not be assigned because we failed to amplify one target region. Using MLVA-3, all isolates were amplified, and we described only eight MLVA-3 types of which one was new. It should be noted that it is difficult to interpret the MLVA-3 results, as more than 11 single nucleotide repeats can be observed at one single locus, associated with the strong probability that the DNA polymerase, routinely used in PCR, may have generated sequence errors 
. Furthermore, the stability of this technique has not been fully assessed 
. Our data were in agreement with those reported by Bom et al.
who observed that for 13 genovar E specimens, the number of STs described (eight) was higher than the number of MLVA-3 types (five) 
. Moreover, also in our study, nearly 60% of the STs that were identified were new. In contrast, the results obtained by Ikryannikova et al.
conflict with ours and those of Bom et al.
, as for 12 genovar E isolates, five MLVA-3 types but only two STs were described 
According to HGDI values, MLVA-3 showed the lowest discriminatory power (0.622), in contrast to other studies that found a better HGDI for genovar E, ranging from 0.782 to 0.902 
, while MLVA-5 and MLST presented similar HGDI values (0.913 and 0.860, respectively). Ideally, the HGDI should be 1.00, but in practice, it should be at least 0.90–0.95 for a typing system to be relatively optimal 
. A combination of MLVA-5 and MLST resulted in the highest HGDI (0.949).
The application of the five VNTRs identified within genovar E permitted to describe 13 additional MLVA-5 types among non-E genovar specimens. However, to optimize the method, it would be interesting to look for new VNTRs for non-E genovar.
In summary, MLVA-5 has been developed for the genovar E C. trachomatis and used to type a large collection of French isolates and C. trachomatis-positive specimens belonging to genovar E and urogenital non-E genovars. Our findings show that MLVA can be used to discriminate between C. trachomatis isolates and can be directly applied to clinical specimens, thereby providing a high-resolution molecular epidemiological tool. Accordingly, the overall performance of the MLVA-5 assay would facilitate large-scale molecular epidemiological investigations of sexually transmitted C. trachomatis infections.