Molecular Detection Methods

Several DNA amplification based techniques that detect H. ducreyi directly in patient samples have been developed and significantly improved the sensitivity of laboratory diagnostic tests for chancroid. Specific DNA primers have been designed to target and identify the bacteria in these techniques, which include probe hybridization and PCR amplification assays.

Probe Hybridization

Two probe hybridization assays for detection of H. ducreyi have been developed (Parsons et al., 1989; Rossau et al., 1991). In one assay, three 32P-labeled DNA probes designed on basis of encoding H. ducreyi-specific proteins have been demonstrated to react strongly with H. ducreyi DNA in both bacterial suspensions as well as in infected rabbit lesion material blotted onto nitrocellulose membranes (Parsons et al., 1989). The sensitivity of this probe hybridization assay is around 103-104 CFU of H. ducreyi in both pure and mixed cultures. The other assay approach was based on the development of specific rRNA-derived oligonucleotide probes for H. ducreyi (Rossau et al., 1991). Hybridization probes were chemically synthesized on eight oligonucleotide sequences complementary to different regions in the 16S and 23S rRNA molecules. This DNA-RNA hybridization assay has been demonstrated to show high specificity on culture grown isolates, but the sensitivity of the technique has not been provided. There has been no complete evaluation on the usefulness of these DNA or RNA probe hybridization techniques in the diagnosis of chancroid by H. ducreyi detection using clinical specimens.

Several PCR techniques have been developed to improve on the sensitivity of laboratory diagnosis of chancroid (Johnson et al., 1994; Parsons et al., 1995; Orle et al., 1996). Target regions of the primers of these assays include 16S rRNA gene (Orle et al., 1996), the rrs (16S)-rrl (23S) ribosomal intergenic spacer region (Gu et al., 1998), an anonymous fragment of cloned DNA (Johnson et al., 1994), and the groEL gene encoding the GroEL heat shock protein (Parsons et al., 1995). As mentioned previously for T. pallidum, a multiplex PCR (M-PCR) assay with colorimetric detection has been developed for the simultaneous amplification of DNA targets from H. ducreyi, T. pallidum, and herpes simplex virus (HSV) type 1 and 2 (Orle et al., 1996). Sensitivity and specificity of M-PCR detection of H. ducreyi was 98.4% and 99.6%, respectively, as compared with 74.2% and 100% for culture. Expectedly, the sensitivity of culture is relatively low in comparison with PCR. Provided adequate clinical correlation studies can be carried out, the PCR assay has the potential to become an accurate and easily available reference method for the detection of H. ducreyi.

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