Development of a Polymerase Chain Reaction Assay for the Detection of Haemophilus ducreyi

Abstract

Background and Objectives: Haemophilus ducreyi, the causative agent of chancroid is a fastidious organism difficult to culture and identify. Consequently, culture is an insensitive method for diagnosis. The polymerase chain reaction (PCR) offers a sensitive and specific nonculture method for the detection of bacterial pathogens. Study Design: A polymerase chain reaction (PCR) assay was developed to detect the presence of Haemophilus ducreyi. A pair of primers was selected from sequences of an anonymous fragment of DNA cloned from H. ducreyi. The primers were tested in amplification reactions with both purified DNA and lysed organisms for their ability to detect H. ducreyi, and with DNA from a variety of different bacteria for their specificity. The utility of the primers for the detection of H. ducreyi in samples taken from genital ulcers was also tested and compared with culture. Results: PCR was positive for 62% of the specimens that were culture-positive, however, PCR was also positive for 49% of the culture-negative specimens. Comparison of specimens that were dark field positive for T. pallidurn and PCR-positive with those that were culture-positive for herpes simplex virus and PCR positive suggested that PCR was giving true and not random positive results. Additional studies demonstrated that the failure of PCR to detect H. ducreyi in all of the culture-positive specimens probably resulted from inhibitors of the Taq DNA polymerase that were present in the nucleic acids extracted from the clinical specimen. Conclusion: PCR should be a useful method for the detection of H. ducreyi in genital lesions, especially where culture sensitivity is poor. However, the presence of unidentified Taq polymerase inhibitors in some ulcers specimens require the development of improved methods for specimen handling.