Development of a Rapid Immunodiagnostic Test for Haemophilus ducreyi

Abstract

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted disease that increases the rate of transmission of human immunodeficiency virus. Chancroid ulcerations are difficult to distinguish from those produced by syphilis and herpes. Diagnosis based solely on clinical grounds is inaccurate, and culture is insensitive. Highly sensitive PCR has largely superseded culture as the preferred method of laboratory diagnosis; however, neither culture nor PCR is feasible where chancroid is endemic. We developed a rapid (15-min) diagnostic test based on monoclonal antibodies (MAbs) to the hemoglobin receptor of H. ducreyi, HgbA. This outer membrane protein is conserved in all strains of H. ducreyi tested and is required for the establishment of experimental human infection. MAbs to HgbA were generated and tested for cross-reactivity against a panel of geographically diverse strains. Three MAbs were found to be unique and noncompetitive and bound to all strains of H. ducreyi tested. Using an immunochromatography format, we evaluated the sensitivity and specificity of the test using geographically diverse strains of H. ducreyi, other Haemophilus strains, and other bacteria known to superinfect genital ulcers. All H. ducreyi strains were positive, and all other bacteria were negative, resulting in a specificity of 100%. The minimum number of CFU of H. ducreyi detected was 2 ؋ 10 6 CFU, and the minimum amount of purified HgbA protein detected was 8.5 ng. Although this level of sensitivity may not be sufficient to detect H. ducreyi in all clinical specimens, further work to increase the sensitivity could potentially make this a valuable bedside tool in areas where chancroid is endemic.Chancroid, caused by the gram-negative bacterium Haemophilus ducreyi (for reviews, see references 2, 47, and 60), is one of the sexually transmitted genital ulcer diseases. Although outbreaks are more prevalent in developing countries, sporadic outbreaks have occurred in the United States. Up to 50% of patients visiting sexually transmitted disease clinics in subSaharan Africa may have chancroid (12,23). Additionally, it is an independent risk factor for the heterosexual transmission of human immunodeficiency virus (HIV) (34; F. A. Plummer, M. A. Wainberg, P. Plourde, P. Jessamine, L. J. DCosta, I. A. Wamola, and A. R. Ronald, Letter, J. Infect. Dis. 161:810-811, 1990); therefore, the interest in chancroid has recently intensified. In the last 10 years, several laboratories have begun to define the molecular biology of this pathogen. The study of virulence factors, the body's immune response to infection, and potential vaccines for H. ducreyi have all made significant contributions to the understanding of this bacterium (3-10, 13-16, 22, 24, 26-28, 32, 33, 36, 38, 39, 46, 50, 53, 54, 59, 61, 64). With the genome sequence completed, the process of annotation will, undoubtedly, further accelerate progress on this strictly human pathogen (www.microbial-pathogenesis.org).At present, there are several laboratory methods for the d...

An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding

“…Plasmids expressing hgbA with a leader sequence are unstable in E. coli (data not shown). Moreover, these inclusion body-derived (IBD) rHgbA proteins are not recognized by conformation-dependent antinHgbA monoclonal antibodies (20), IBD rHgbA does not bind Hb, and antibodies elicited to IBD rHgbA do not recognize whole cells of H. ducreyi (data not shown). To avoid these issues, we decided to express hgbA from plasmids in H. ducreyi.…”

Haemophilus ducreyi Outer Membrane Determinants, Including DsrA, Define Two Clonal Populations

“…HgbA is also required for expression of full virulence in the temperature-dependent rabbit model (59) and the human model of chancroid (5). Patterson et al (44) reported that HgbA was highly conserved, since three antiHgbA Mabs bound to all 26 strains tested. The sole exception was MAb 4.23, which bound all strains except HMC112, identified as a class II strain in the present study.…”

“…One-liter cultures of pUNCH 1293 (dsrA I ), pUNCH 1294 (dsrA II ), and pUNCH 1295 (ncaA I ) in BL21(DE3)pLysS were grown in liquid broth with appropriate antibiotics ( Table 1). As previously described (14,44), protein expression was induced from these plasmids with IPTG. The cultures were subsequently treated with rifampin to inhibit bacterial RNA polymerase.…”

Immunization with theHaemophilus ducreyiHemoglobin Receptor HgbA Protects against Infection in the Swine Model of Chancroid

“…In Western blots, HgbA was weakly recognized with HgbA-immunized pig sera, probably due to its change in conformation after denaturation or poor transfer from the gel to nitrocellulose. Previously, we found that mouse monoclonal antibodies (44) and rabbit antisera (36) elicited to native HgbA also failed to bind in Western blots, suggesting that the conformation of HgbA is complex and critical to antibody binding. In Western blots, we also found that anti-HgbA sera recognized an unknown component of approximately 20 kDa.…”

“…Except for FX504, all strains were grown under heme-limiting conditions (GCB with 1% IsoVitaleX and 5% FBS) as described previously (19). Since the FX504 mutant does not grow under the described heme-limiting conditions, 5 g of hemin per ml was added to the above-described broth medium (44).…”