Experimental infection with Haemophilus ducreyi in persons who are infected with HIV does not cause local or augment systemic viral replication.

We infected 11 HIV-seropositive volunteers whose CD4(+) cell counts were >350 cells/ microL (7 of whom were receiving antiretrovirals) with Haemophilus ducreyi. The papule and pustule formation rates were similar to those observed in HIV-seronegative historical control subjects. No subject experienced a sustained change in CD4(+) cell count or HIV RNA level. The cellular infiltrate in biopsy samples obtained from the HIV-seropositive and HIV-seronegative subjects did not differ with respect to the percentage of leukocytes, neutrophils, macrophages, or T cells. The CD4(+):CD8(+) cell ratio in biopsy samples from the HIV-seropositive subjects was 1:3, the inverse of the ratio seen in the HIV-seronegative subjects (P<.0001). Although CD4(+) cells proliferated in lesions, in situ hybridization and reverse-transcription polymerase chain reaction for HIV RNA was negative. We conclude that experimental infection in HIV-seropositive persons is clinically similar to infection in HIV-seronegative persons and does not cause local or augment systemic viral replication. Thus, prompt treatment of chancroid may abrogate increases in viral replication associated with natural disease.

Localization of Haemophilus ducreyi in naturally acquired chancroidal ulcers.

Haemophilus ducreyi causes the sexually transmitted genital ulcer disease chancroid. In human inoculation experiments, bacteria colocalize with neutrophils and macrophages but remain extracellular. The organism also colocalizes with collagen and fibrin but not with keratinocytes, fibroblasts, laminin, or fibronectin. These relationships are established by 48 h postinoculation and persist through the pustular stage of disease. To extend these observations to the ulcerative stage of disease, and to compare results in the human model with those of natural disease, we obtained biopsies from patients with naturally acquired chancroid. All ulcers were culture positive for H. ducreyi and histologically very similar to pustules from the human model. Staining with H. ducreyi-specific monoclonal antibodies demonstrated H. ducreyi within 5 biopsies. The organism was chiefly found within the granulocytic infiltrate of the ulcer. Dual staining for H. ducreyi and eukaryotic tissue components showed that H. ducreyi colocalized with neutrophils and fibrin at the ulcerative stage of disease. No bacteria were associated with keratinocytes, fibroblasts, or collagen. Overall, these findings are consistent with results from the human model. This is the first reported study to localize bacteria specifically identified as H. ducreyi within naturally acquired chancroid.

Prevalence of, antibody response to, and immunity induced by Haemophilus ducreyi hemolysin.

Haemophilus ducreyi, the etiologic agent of chancroid, a genital ulcer disease, produces a cell-associated hemolysin whose role in virulence is not well defined. Hemolysin is encoded by two genes, hhdA and hhdB, which, based on their homology to Serratia marcescens shlA and shlB genes, are believed to encode the hemolysin structural protein and a protein required for secretion and modification of this protein, respectively. In this study, we determined the prevalence and expression of the hemolysin genes in 90 H. ducreyi isolates obtained from diverse geographic locations from 1952 to 1996 and found that all strains contained DNA homologous to the hhdB and hhdA genes. In addition, all strains expressed a hemolytic activity. We also determined that hemolysin is expressed in vivo and is immunogenic, as indicated by the induction of antibodies to hemolysin in both the primate and rabbit disease models as well as in human patients with naturally acquired chancroid. Wild-type strain 35000 and isogenic hemolysin-negative mutants showed no difference in lesion development in the temperature-dependent rabbit model. However, immunization of rabbits with the purified hemolysin protein reduced the recovery of wild-type H. ducreyi, but not hemolysin-negative mutants, from lesions. Our study indicates that hemolysin is a possible candidate for vaccine development due to its immunogenicity, expression in vitro and in vivo by most, if not all, strains, and the effect of immunization on reducing the recovery of viable H. ducreyi in experimental disease in rabbits.

Antibodies specific to surface antigens are not effective in complement-mediated killing of Haemophilus ducreyi.

The bactericidal activity of serum is an important primary host defence against gram-negative bacteria. Little is known regarding such antibodies that are specific to outer membrane (OM) antigens as pili and lipooligosaccharides (LOS) in the bactericidal killing of Haemophilus ducreyi. Presence of serum antibodies with specificity to a 430 kDa protein (polymer of the 24 kDa protein, named fine-tangled pili) and LOS in serum from chancroid patients and healthy individuals were investigated by ELISA. Using a bactericidal assay, we investigated the role of human and rabbit antibodies with the aforementioned specificity. Accessibility of LOS and of OM antigens, as well as the deposition of components of the complement (C) system on the surface of the bacteria, was further investigated by whole-cell ELISA and immunoelectron microscopy. Immunoglobulin G (IgG) antibodies specific to the 430 kDa polymer and to LOS were demonstrated in the majority of sera from chancroid patients and healthy individuals. However, sera from chancroid patients did not significantly enhance the C-mediated killing of H. ducreyi compared with normal human serum (NHS). Similar results were demonstrated using rabbit sera to whole bacteria, specific to the 430 kDa protein and LOS of H. ducreyi. However, using the same assay noncapsulatedH. influenzae was totally killed, as were H. influenzae type b in presence of specific antibodies. This suggests a limited effectiveness of antibodies specific to surface antigens in C-mediated killing of H. ducreyi. LOS was detectable on the surface of H. ducreyi with a specific monoclonal antibody in white-cell ELISA. However, a significant enhancement of LOS detection was demonstrated on washed bacteria. OM antigens of 26, 40, 45 kDa and the major outer membrane protein (MOMP) of 43 kDa were not detectable on the surface of nonwashed and washed bacteria by specific monoclonal antibodies, indicating a lack of accessibility of these antigens on the bacterial surface. However, the C6 to C9 components of C were detected on the bacterial surface, suggesting capacity of forming the membrane attack complex. Altogether, these findings imply that antibodies specific to surface antigens, such as the 430 kDa protein and LOS, are not capable of enhancing killing of bacteria. The demonstrated relative resistance is probably due not to a lack of deposition of the membrane attack complex components, but rather to a blocking of LOS accessibility and OM proteins as potential targets of bactericidal antibodies and C action.

Specificity of the immune response to Haemophilus ducreyi.

The specificity of the antibody response to Haemophilus ducreyi in sera from patients attending a sexually transmitted disease clinic in South Africa has been studied using immunoblotting. Patients with chancroid were shown to have higher levels of IgG (mean 0.74, SD 0.34) to H. ducreyi than those with no history of chancroid (mean 0.34, SD 0.19). The pattern of the antibody specificity was highly variable between patients with culture proven chancroid but there was no observed strain specificity. In comparison, the patterns obtained using sera from patients without known exposure to H. ducreyi showed less variation between patients and were of less intensity at the dilution used. Sera from patients with chancroid recognised epitopes on proteins that varied in molecular weight between strains, particularly of 60-66kDa (10 of 36 patients) and 25-27kDa (8 of 36 patients). In addition epitopes were recognised on the GroEL and/or DnaK heat shock proteins in 13 of 36 sera tested. There was no apparent change in the epitopes recognised on proteins between the homologous and heterologous strains. Patterns of antibody specificity in sequential sera only varied in one of six patients tested.

Failure of treatment for chancroid in Rwanda is not related to human immunodeficiency virus infection: in vitro resistance of Haemophilus ducreyi to trimethoprim-sulfamethoxazole.

A comparative open study was performed to evaluate the efficacy of single doses of ciprofloxacin (500 mg) and trimethoprim-sulfamethoxazole (TMP-SMZ; 640 mg/3,200 mg) for the treatment of culture-proven chancroid. Clinical cure or improvement was observed 7 days after treatment in 32 (76.2%) of the 42 patients who received ciprofloxacin and 21 (52.5%) of the 40 patients who received TMP-SMZ (P = .04). Cultures for one (4.5%) of 22 patients not cured with ciprofloxacin and 16 (59.3%) of 27 patients not cured with TMP-SMZ were still positive for Haemophilus ducreyi 7 days after treatment (P < .001). Although 77 (71.3%) of the 108 patients tested were seropositive for HIV-1 antibody, HIV infection and the degree of CD4+ lymphocyte depletion had no effect on clinical and bacteriologic outcome. All isolates of H. ducreyi were highly susceptible to ciprofloxacin (MIC, 0.004-0.06 mg/L). In contrast, resistance to TMP-SMZ (MIC, > or = 4/76 micrograms/mL) was observed in 48.9% of isolates (22 of 45) and was significantly associated with treatment failure. Therefore, the administration of TMP-SMZ, in single or multiple doses, is no longer indicated for the treatment of chancroid in Rwanda.

Seroepidemiological studies of Haemophilus ducreyi infection in Ethiopian women.

BACKGROUND AND OBJECTIVES: To measure prevalence of anti-Haemophilus ducreyi antibodies in sera from Ethiopian female attendees, and to determine significant socioeconomic associations. STUDY DESIGN: A modified ELISA immunoassay was used to test sera of 1,831 Ethiopian women attending gynecological, obstetric, and family planning clinics in Addis Ababa. RESULTS: Overall seropositivity was 19.4%. Prevalence rates for seropositivity for antibodies to H. ducreyi were significantly associated with ethnic group and religion, older age (> or = 50 years: 28%), early age at first coitus (< 13 years: 28%) and first coitus before the menarche (25%), being divorced (27%) or a prostitute (24%), longer duration of marriage (> 20 years: 27%) and sexual life (> 20 years: 24%), number of lifetime sexual partners (2 to 5 partners: 27%) and self-reported history of both syphilis and gonorrhea (31%). Of these factors, the two most significant were first coitus before the menarche (P < 0.0001) and not being still married to the first husband/sexual partner (P < 0.001). Differences in seropositivity according to ethnic group and religion may be explained by the number of women within each group who had only one lifetime sexual partner. Women with serological evidence of exposure to another sexually transmitted disease (STD) had a greater risk of exposure to H. ducreyi. The odds ratio for H. ducreyi seropositivity in women with syphilis or gonorrhea was 3.6, for women with genital chlamydial infection, 2.3, and for those with HBV or HSV-2, 1.4 and 1.3 respectively. CONCLUSIONS: This study illustrates the usefulness of the modified ELISA immunoassay for measuring exposure to H. ducreyi, and the usefulness of H. ducreyi as a marker for cumulative sexual exposure. Further studies on the association of HIV transmission and H. ducreyi in Ethiopia are now indicated.

PIP: Genital ulcerated disease (GUD), which includes chancroid, has been identified as a risk factor for HIV transmission. This study reports the prevalence of anti-Hemophilus ducreyi (chancroid) antibodies in 1831 Ethiopian women and looks at the behavioral and social factors which might affect the incidence and potential spread of chancroid. Patient data regarding ethnic and socioeconomic aspects were collected from detailed questionnaires. Blood collection was performed under medical surveillance. Complete gynecological examinations were performed. Papanicolaou stained smears were used as the basis of the cytological data. Serological studies utilized an enzyme immunoassay (EIA) test for STD detection. Statistical tests used included the Chi-square test, the multivariate analysis technique, and the Cochran-Mantel-Haenszel General Association Statistic Test. Antibodies to H. ducreyi were found in 335 women (19.4%). Prevalence of H. ducreyi was significantly associated with Amhara or Tigre ethnic heritage; older age; first coitus before beginning menstruation; history of STDs; divorced status; being a prostitute; longer duration of married and sexual life; and younger age at first coitus. Logistic regression demonstrated that 3 factors were significant when associated with H. ducreyi seropositivity. First coitus before beginning menstruation was highly significant (OR 1.95; 95% CI, 1.49-2.57; P 0.0001). Not being still married to the first husband was also significant (OR 1.68; 95% CI, 1.23-2.30; P 0.001). Being of the Ethiopian Orthodox religion was significant (OR 2.11; 95% CI, 1.21-3.68; P 0.005). Prevalence in women with 2-5 lifetime husbands was higher than in women with only 1 husband.

The suppressive effect of serum samples from patients with chancroid on human mononuclear cells correlates with the clinical picture and is interleukin-2-dependent.

Plasma samples from patients with chancroid diagnosed both on clinical and microbiological grounds were assessed for their ability to inhibit mitogen-induced proliferation of human lymphocytes from healthy donors. All serum samples analysed suppressed phytohaemagglutinin A (PHA) blastogenic response. A significant difference in the observed extent was seen when serum samples from patients with and without associated lymphadenopathy were compared (P < 0.05). Using an interleukin-2 (IL-2)-dependent cell line it could be demonstrated that the addition of patients' plasma to cultured cells markedly depressed mitogen-induced IL-2 synthesis. Results presented suggest that cell-mediated mechanisms play a role in the pathogenesis of infection due to Haemophilus ducreyi.