DNA probes for the identification of Haemophilus ducreyi

Abstract

Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32p, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 107 to 6.0 x 107 CFU was tested. When 105 to 106 CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 104 CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 103 CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen.

Development of the polymerase chain reaction for diagnosis of chancroid

“…With the introduction of DNA diagnostic methods (17) and the ability to amplify the signal with the polymerase chain reaction (PCR), newer tests should improve the sensitivity and specificity of diagnostic tests for chancroid. First-generation DNA probes for H. ducreyi have been reported with sensitivities of 103 to 104 organisms and 100% specificity (25,26). We report here our results with the development of primer sets and probes for the diagnosis of chancroid by DNA amplification using PCR.…”

“…First, rRNA genes exist in multiple copies in most eubacteria and therefore increase the signal for amplification. Second, the conserved regions can be used for broad specificity primers which should give positive a Culture + 25 9 Probe 6. Detection of H. ducreyi in 100 clinical specimens from male patients with suspected chancroid.…”

The Laboratory Diagnosis of Haemophilus ducreyi

“…Additional identification tests to consider include oxidase (positive for H ducreyi), catalase (negative for H ducreyi) and X factor nutritional requirement (H ducreyi requires X factor for growth and this can most easily be evaluated using the porphyrin test). For confirmation of species identification, the isolate can be sent to the National Microbiology Laboratory (Winnipeg, Manitoba) for H ducreyi-specific molecular tests (8,14,15).…”

“…A variety of genetic probe (14,15) and amplification (16,17) methods have been developed for culture confirmation or direct detection of H ducreyi in clinical samples. Of particular relevance is the Roche-developed multiplex-PCR test (Roche Diagnostics Canada) that simultaneously detects H ducreyi, T pallidum and herpes simplex virus (17).…”

Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid

“…Several such methods have been reported, including direct examination by Gram stain, which has been shown to be less than 50% sensitive (15), use of monoclonal or polyclonal antibodies in immunofluorescence tests (7,12), and use of DNA probes (17). None of these methods is widely used for clinical diagnosis, since these techniques have not been sufficiently sensitive or specific.…”