Comparison of reverse hybridization and ompA sequencing methods applied on Chlamydia trachomatis strains from Tunisia

Abstract

Two techniques based on ompA amplification of Chlamydia trachomatis were compared, being reverse hybridization (RHM) and ompA sequencing (OSA), to investigate the concordance between them and to study the epidemiological relevance of each method. In addition, phylogenetic analysis was performed on the ompA sequences. One hundred and seven C. trachomatis positive samples from Tunisian patients and female sex workers were analyzed using both the RHM and ompA sequencing. The overall genovar distribution obtained with both techniques was very similar. The RHM identified nine genovars, being B, D, E, F, G, H, I, J and K, where B, I, J, and K were only found in mixed infections versus 7 types for the OSA being D, E, F, G, H, I, and K. The agreement between both typing techniques was 87.8%. Both methods showed that genovar E was the most predominant type. In 24.3% of the analyzed samples, mixed infections were detected. In 96.1% of these, the genovar identified by OSA was also detected using the RHM. OmpA sequencing allowed determination of six genovar types that could not be typed using RHM. The analyses of ompA nucleotide variation in the 107 clinical specimens detected ompA genovar variants with distinct ompA mutational patterns for types D2, G1, G2, and H1. In conclusion, RHM and OSA showed a high agreement in C. trachomatis genotyping results with each having their specific benefits.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

“…Notably, the most prevalent genotype of isolates from patients attending an STI clinic in Tel Aviv is genotype E, which accounts for about half of the isolates. Interestingly, the genovar's distributions are similar to the report from European countries [24,27] and Tunisia [38], but present differences from Asian countries such as China, where the genotypes D and G were the most common [39], and India where genotype D was found to be most prevalent [40].…”

Identification and characterization of mixed infections of Chlamydia trachomatis via high-throughput sequencing

“…In our study, C. trachomatis genotype E and F were more frequently detected in the mixed-genotype infections ( Table 2 ), which may be due to the extensive distribution of these two genotypes in China. Gallo Vaulet and Gharsallah, et al also reported that 86.7 and 76.9% of the mixed-genotype infections contained genotype E, respectively ( Gallo Vaulet et al, 2016 ; Gharsallah et al, 2018 ). Interestingly, we found that genotype G only accounted for 5.94% of the total C. trachomatis infections in our study population, but was detected in 25.0% of the mixed-genotype infections.…”

“…Sanger sequencing is a powerful tool for genotyping and has been used to determine C. trachomatis genotypes. However, it may not be suitable for identifying mixed-genotype infections of C. trachomatis since Sanger sequencing only produces one consensus sequence according to the alignment results of multiple sequencing data and the intensity of sequencing signals at each point; hence, only the dominant genotype sequence could be identified ( Gharsallah et al, 2018 ). Although the mixed-genotype infections can be identified by PCR-RFLP, hybridization methods and DNA microarray assay, these assays are not sensitive enough to identify all the mixed-genotype infections and cannot quantify genotype proportions in mixed-genotype infections.…”

“…Sanger sequencing is the most widely used technique for genotyping, but is not adequate to detect mixed-genotype infections of C. trachomatis since it only provides one consensus sequence ( Quint et al, 2007 ; Ruettger et al, 2011 ; Gharsallah et al, 2012 ). Although PCR-RFLP, hybridization methods and DNA microarray assay can identify mixed-genotype infections to some extent ( Ruettger et al, 2011 ; Gharsallah et al, 2018 ), their sensitivity and reliability remain to be improved. In contrast, next-generation high-throughput sequencing (NGHTS) targeting variable regions of ompA gene can not only inherit the advantage of ompA Sanger sequencing to identify C. trachomatis genotypes, but also determine mixed-genotype infections and the proportion of different genotypes in the mixed-genotype infections due to enough sequencing depth and large number of sequencing reads ( Kawada et al, 2016 ), which are more suitable for the detection of co-infections or super-infections of different C. trachomatis genotypes.…”