Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Abstract

Aim To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification. Methods and Results The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases. Conclusion The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies. Significance and Impact of the Study This technique allowed rapid C. trachomatis genotype identification.

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

“…A possible limitation of RHM is that cross‐hybridization may occur, which would also be visible as faint spots and which lower the specificity of the assay. The RHM used in this study was previously validated before use, and its specificity was very high (Gharsallah, Frikha‐Gargouri, Besbes, et al., ).…”

“…Mixed infections may result from two separate episodes of infection and the lack of immunological cross protection between genovars (Hsu et al., ). Reverse hybridization methods have proved to be of use in many studies to detect multiple infections (Gharsallah, Frikha‐Gargouri, Besbes et al., ; Li et al, ; Quint et al., ; Xiong et al., ; Zheng et al., ). High frequencies of mixed infections were reported in studies using hybridization methods.…”

“…The reverse hybridization method, developed in our laboratory, was used for the detection of specific C . trachomatis genovars (Gharsallah, Frikha‐Gargouri, Besbes, et al., , Gharsallah, Frikha‐Gargouri, Sellami, et al, ). Briefly, the nylon membrane was spotted with probes hybridizing specifically to genovars A to L3 at specific positions.…”

“…However, sequencing techniques were not really adequate to detect mixed genovar infections. In contrast, hybridization methods targeting the variable regions of the ompA gene are very well suited to detect co‐infections or mixed infections, and several hybridization based assays are available nowadays (Gharsallah, Frikha‐Gargouri, Besbes, et al., ; Quint et al., ; Ruettger et al., ; Xiong, Kong, Zhou, & Gilbert, ; Zheng et al., ). These hybridization methods are suitable for epidemiological studies and are preferred because of their low costs and low technology requirement.…”

Diagnosis of Chlamydia trachomatis genital infections in the era of genomic medicine

“…These techniques are based on hybridization of the amplified DNA with probes labeled on nylon membranes or carboxylated beads and results are noted either by blot formation or by analyzing the signals generated [9]. Studies conducted using these methods have highlighted their role in revealing multiple genotypes from a single clinical specimen and effective genotyping in large epidemiological studies [88][89][90]. However, poor resolution and inability to identify genovariants are major drawbacks [9].…”