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Haemophilus ducreyi 35000HP contains a cluster of homologues of genes required for the synthesis of enterobacterial common antigen (ECA), suggesting that H. ducreyi may express a putative ECA-like glycoconjugate. WecA initiates the synthesis of ECA by transferring N-acetylglucosamine to undecaprenyl-P, to form lipid I. A wecA mutant (35000HPwecA) was constructed, and 5 volunteers were inoculated at 3 sites with fixed doses of 35000HP on one arm and at 3 sites with varying doses of 35000HPwecA on the other arm. 35000HPwecA caused pustules to form at 3 sites inoculated with a dose 2.5-fold higher than that of 35000HP. However, at sites inoculated with similar doses of 35000HP and 35000HPwecA, pustules developed at 46.7% (95% confidence interval [CI], 23.3%-70.0%) of 15 parent-strain sites and at 8.3% (95% CI, 0.01%-23.6%) of 12 mutant-strain sites (P = .013). Thus, the expression of wecA contributes to the ability of H. ducreyi to cause pustules in humans.

Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%-75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%-73.9%) at 18 mutant-strain sites (P = .51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine.

In experimentally infected human volunteers, the cutaneous immune response to Haemophilus ducreyi is orchestrated by serum, polymorphonuclear leukocytes, macrophages, T cells, and myeloid dendritic cells (DC). This response either leads to spontaneous resolution of infection or progresses to pustule formation, which is associated with the failure of phagocytes to ingest the organism and the presence of Th1 and regulatory T cells. In volunteers who are challenged twice, some subjects form at least one pustule twice (PP group), while others have all inoculated sites resolve twice (RR group). Here, we infected PP and RR subjects with H. ducreyi and used microarrays to profile gene expression in infected and wounded skin. The PP and RR groups shared a core response to H. ducreyi. Additional transcripts that signified effective immune function were differentially expressed in RR infected sites, while those that signified a hyperinflammatory, dysregulated response were differentially expressed in PP infected sites. To examine whether DC drove these responses, we profiled gene expression in H. ducreyi-infected and uninfected monocyte-derived DC. Both groups had a common response that was typical of a type 1 DC (DC1) response. RR DC exclusively expressed many additional transcripts indicative of DC1. PP DC exclusively expressed differentially regulated transcripts characteristic of DC1 and regulatory DC. The data suggest that DC from the PP and RR groups respond differentially to H. ducreyi. PP DC may promote a dysregulated T-cell response that contributes to phagocytic failure, while RR DC may promote a Th1 response that facilitates bacterial clearance.

We examined the susceptibility of Haemophilus ducreyi to antimicrobial peptides likely to be encountered in vivo during human infection. H. ducreyi was significantly more resistant than Escherichia coli to the bactericidal effects of all peptides tested. Class I and II H. ducreyi strains exhibited similar levels of resistance to antimicrobial peptides.

Chancroid is a sexually transmitted infection associated with genital ulceration and lymphadenopathy caused by Haemophilus ducreyi. Localized skin infections, in the absence of genital lesions, have not been previously reported. We report 3 cases of lower limb ulceration in children caused by H. ducreyi and postulate that H. ducreyi may be a previously unrecognized cause of chronic skin ulceration.

The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.

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.

The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

Haemophilus ducreyi express two porin proteins, termed OmpP2A and OmpP2B. To test whether expression of OmpP2A and OmpP2B was necessary for virulence in humans, eight volunteers were experimentally infected with the parent (35000HP) in one arm and a double OmpP2A OmpP2B mutant (35000HP::P2AB) in the other arm. The pustule formation rates were 58.3% (95% CI, 33.2-83.5%) for the parent and 41.7% (95% CI, 19.3-64.0%) for the mutant (P=0.25). Biopsy of 35000HP and 35000HP::P2AB-infected sites yielded similar amounts of bacteria in quantitative culture. These results indicate that expression of OmpP2A and OmpP2B is not necessary to initiate disease or to progress to pustule formation in humans.

Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

Objectives: Development of single dose antibiotic treatments for chancroid has been followed by drug-resistant Haemophilus ducreyi in endemic areas. We examined the activity and interactions of antimicrobial agents and combinations against H. ducreyi., Methods: We evaluated the in vitro susceptibility of three virulent strains of H. ducreyi to ceftriaxone, azithromycin, rifabutin and streptomycin, and each two-drug combination by the agar dilution method. We then tested each two-antibiotic combination for activity by the chequerboard method. Lastly, we chose the antibiotic combination with the lowest fractional inhibitory concentration index (FICI) and tested combined sub-therapeutic doses, the highest doses which had no effect alone on lesion healing compared with controls, for in vivo interaction in the temperature-dependent rabbit model of H. ducreyi infection., Results: Each H. ducreyi strain was susceptible in vitro to each antibiotic and two-antibiotic combination, and combined ceftriaxone and streptomycin had the lowest FICI at 0.63. In five treated animals versus three untreated controls, combined sub-therapeutic doses of ceftriaxone (0.05 mg/kg) and streptomycin (10 mg/kg) reduced mean (SD) duration of culture positivity from 7.3 (1.1) to 2.6 (1.7) days (P<0.001), time to 50% reduction in lesion size from 9.7 (1.5) to 5.8 (0.8) days (P<0.005), and time to resolution of ulcer from 11.7 (2.3) to 6.6 (1.7) days (P<0.05)., Conclusions: Ceftriaxone and streptomycin have in vivo synergic interaction against H. ducreyi lesions in the temperature-dependent rabbit model of infection. Antibiotic combinations may be evaluated clinically as single-dose therapy for chancroid.

Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Neither naturally occurring chancroid nor experimental infection with H. ducreyi results in protective immunity. Likewise, a single inoculation of H. ducreyi does not protect pigs against subsequent infection. Accordingly, we used the swine model of chancroid infection to examine the impact of multiple inoculations on a host's immune response. After three successive inoculations with H. ducreyi, pigs developed a modestly protective immune response evidenced by the decreased recovery of viable bacteria from lesions. All lesions biopsied 2 days after the first and second inoculations contained viable H. ducreyi cells, yet only 55% of the lesions biopsied 2 days after the third inoculation did. Nearly 90% of the lesions biopsied 7 days after the first inoculation contained viable H. ducreyi cells, but this percentage dropped to only 16% after the third inoculation. Between the first and third inoculations, the average recovery of CFU from lesions decreased approximately 100-fold. The reduced recovery of bacteria corresponded directly with a fivefold increase in H. ducreyi-specific antibody titers and the emergence of bactericidal activity. These immune sera were protective when administered to naïve pigs prior to challenge with H. ducreyi. These data suggest that pigs mount an effective humoral immune response to H. ducreyi after multiple exposures to the organism.

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 x 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.

The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10(6)CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100microg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.

Background: Naturally occurring chancroid is usually more prevalent in men than in women., Goal: To examine whether there were gender differences in susceptibility to Haemophilus ducreyi infection by analyzing the papule and pustule formation rates for men and women who were experimentally inoculated with Haemophilus ducreyi., Study Design: Ninety volunteers were included in the analysis. A total of 189 sites were available for estimation of the papule formation rate, and 166 sites for estimation of the pustule formation rates using logistic regression modeling., Results: Although there were no gender differences in papule formation rates, the women had significantly lower rates of pustule formation than the men after adjustment for the estimated delivered dose., Conclusions: In women the disease will resolve and not progress to the pustular stage of disease as often as in men. The high male-to-female ratio in naturally occurring chancroid may in part reflect biological differences in gender susceptibility to disease progression, although the mechanisms responsible for this difference are unclear.

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10(6) CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.

Detection of Haemophilus ducreyi in genital ulcer specimens by culture lacks sensitivity. To enhance detection, a heminested polymerase chain reaction (PCR) assay was developed targeting the nucleotide sequence of a gene, designated p27, which encodes for a 27 kDa H. ducreyi-specific protein. The p27 PCR assay detected all (37/37) H. ducreyi strains tested and gave no amplified product from DNA extracts of any of 31 other microorganisms, from 30 non-genital ulcer specimens, or from 29 urethral and vaginal swab specimens collected from non-chancroid STD patients. In genital ulcer disease specimens, compared to combined positive results obtained by culture and a previously described PCR assay, the p27 PCR assay showed a sensitivity of 91% (48/53). The p27 PCR assay provides a specific and a sensitive detection of H. ducreyi in clinical specimens.

The aim of this study was to investigate the presence of the three cdtABC genes responsible for production of cytolethal distending toxin (CDT) in Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains. Of 100 H. ducreyi strains from the culture collection of the University of Göteborg (CCUG), 27 strains with low or intermediate cytotoxic titre (< 1 in 10(4)) and 23 of the remaining isolates with a high cytotoxic titre (> or = 1 in 10(4)) were selected. Twenty-nine strains of H. ducreyi were isolated recently from patients with chancroid and 50 A. actinomycetemcomitans strains from patients with periodontitis. The cytotoxic activity on HEp-2 cells and the presence of cdtABC genes were studied by cytotoxicity assay of bacterial sonicates and PCR with primers specific for individual cdtA, B, and C genes of H. ducreyi in bacterial DNA preparations, respectively. All strains that manifested a cytotoxic titre in sonicate > or = 1 in 100 possessed all the three cdt genes. Eighteen of the 50 strains selected from the culture collection were negative and 32 positive for cdt genes. As all strains with a high cytotoxic titre gave positive PCR results, it can be assumed that the remaining 50 strains, which have high cytotoxic titre, would have been positive as well. Thus, it can be estimated that 82% of the culture collection strains had cdtABC genes. Similarly, 24 (83%) of 29 recent H. ducreyi isolates expressed the CDT activity and displayed all cdtABC genes. Forty-three (86%) of 50 strains of the closely related A. actinomycetemcomitans, expressing a cytotoxic activity > or = 1 in 100, also possessed all three genes. Furthermore, the nucleotide sequence of the cdtABC genes was highly conserved among H. ducreyi strains from different geographic areas. These results indicate that the majority of pathogenic H. ducreyi and A. actinomycetemcomitans strains express a CDT activity encoded by all three cdtABC.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.

Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyi were subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, and lspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.

Haemophilus ducreyi expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that momp and ompA2 are transcribed independently. Sequences of the momp open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Omega-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into H. ducreyi 35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Omega-Km2 cassette in momp. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (P = 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by H. ducreyi in the human model of infection.

We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from Haemophilus ducreyi strain 35000 were cloned, overexpressed, and purified from Escherichia coli for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors.

To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.

The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.

A randomized, double-blind, placebo-controlled clinical trial was conducted in Nairobi, Kenya, to compare single-dose ciprofloxacin with a 7-day course of erythromycin for the treatment of chancroid. In all, 208 men and 37 women presenting with genital ulcers clinically compatible with chancroid were enrolled. Ulcer etiology was determined using culture techniques for chancroid, serology for syphilis, and a multiplex polymerase chain reaction for chancroid, syphilis, and herpes simplex virus (HSV). Ulcer etiology was 31% unmixed chancroid, 23% unmixed syphilis, 16% unmixed HSV, 15% mixed etiology, and 15% unknown. For 111 participants with chancroid, cure rates were 92% with ciprofloxacin and 91% with erythromycin. For all study participants, the treatment failure rate was 15%, mostly related to ulcer etiologies of HSV infection or syphilis, and treatment failure was 3 times more frequent in human immunodeficiency virus-infected subjects than in others, mostly owing to HSV infection. Ciprofloxacin is an effective single-dose treatment for chancroid, but current recommendations for empiric therapy of genital ulcers may result in high treatment failure due to HSV infection.

Cutaneous lesions of the human sexually transmitted genital ulcer disease chancroid are characterized by the presence of intraepidermal pustules, keratinocyte cytopathology, and epidermal and dermal erosion. These lesions are replete with neutrophils, macrophages, and CD4(+) T cells and contain very low numbers of cells of Haemophilus ducreyi, the bacterial agent of chancroid. We examined lesion formation by H. ducreyi in a pig model by using cyclophosphamide (CPA)-induced immune cell deficiency to distinguish between host and bacterial contributions to chancroid ulcer formation. Histologic presentation of H. ducreyi-induced lesions in CPA-treated pigs differed from ulcers that developed in immune-competent animals in that pustules did not form and surface epithelia remained intact. However, these lesions had significant suprabasal keratinocyte cytotoxicity. These results demonstrate that the host immune response was required for chancroid ulceration, while bacterial products were at least partially responsible for the keratinocyte cytopathology associated with chancroid lesions in the pig. The low numbers of H. ducreyi present in lesions in humans and immune-competent pigs have prevented localization of these organisms within skin. However, H. ducreyi organisms were readily visualized in lesion biopsies from infected CPA-treated pigs by immunoelectron microscopy. These bacteria were extracellular and associated with necrotic host cells in the epidermis and dermis. The relative abundance of H. ducreyi in inoculated CPA-treated pig skin suggests control of bacterial replication by host immune cells during natural human infection.

Chancroid, a sexually transmitted disease caused by Haemophilus ducreyi, is one of the most common genital ulcer diseases in developing countries. In the United States, while less common, the disease has been associated with outbreaks in inner cities, particularly among persons who engage in sex for drugs or money. Two outbreaks of chancroid were recently studied in the United States, one in New Orleans (from 1990 to 1992) and one in Jackson, Mississippi (from 1994 to 1995). By use of ribotyping, plasmid content, and antibiotic susceptibility, the chancroid cases in New Orleans were found to be due to a limited number of strains, consistent with a limited introduction of H. ducreyi into this community. The H. ducreyi isolates from New Orleans and Jackson had different ribotype patterns, suggesting that the two outbreaks were probably not linked.

Two groups of human volunteers were inoculated with 2 doses of live Haemophilus ducreyi 35000HP. The reinfection group consisted of 7 subjects who previously had participated in experimental infection with 35000HP to the pustular stage of disease. The control group consisted of 7 naive subjects. Papules developed at 92.8% (95% confidence interval [CI], 66.1%-99.8%) of sites inoculated with live bacteria, in the reinfection group, and at 85.7% (95% CI, 57.2%-98. 2%) of sites in the control group. Sixty-nine percent (95% CI, 36. 8%-90.9%) of papules evolved into pustules in the reinfection group, compared with 41% (95% CI, 15.2%-72.3%) in the control group. The recovery rates of H. ducreyi from surface cultures and the histopathology of biopsies obtained from both groups were similar. Thus, experimental infection to the pustular stage of disease does not provide protective immunity against subsequent challenge.

The potent cytolethal distending toxin produced by Haemophilus ducreyi is a putative virulence factor in the pathogenesis of chancroid. We studied its action on eukaryotic cells, with the long-term goal of understanding the pathophysiology of the disease. Intoxication of cultured human epithelial-like cells, human keratinocytes, and hamster fibroblasts was irreversible, and appeared as a gradual distention of three- to fivefold the size of control cells. Organized actin assemblies appeared concomitantly with cell enlargement, promoted by a mechanism that probably does not involve small GTPases of the Rho protein family. Intoxicated cells did not proliferate. Similar to cells treated with other cytolethal distending toxins, these cells accumulated in the G2 phase of the cell cycle, demonstrating an increased level of the tyrosine phosphorylated (inactive) form of the cyclin-dependent kinase p34(cdc2). DNA synthesis was not affected until several hours after this increase, suggesting that the toxin acts directly on some kinase/phosphatase in the signaling network controlling the p34(cdc2) activity. We propose that this toxin has an important role both in the generation of chancroid ulcers and in their slow healing. The toxin may also be an interesting new tool for molecular studies of the eukaryotic cell- cycle machinery.

The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396-402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.

Several species of the genus Haemophilus are well known etiological agents of pneumonia, meningitis, conjunctivitis, epiglottitis and chancroid. However, identification and speciation of Haemophilus is both time consuming and labor intensive. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) has been used by several investigators to profile proteins from intact and disrupted bacteria; consequently, MALDI/TOF-MS has emerged as a powerful tool in diagnostic bacteriology. This paper reports the use of MALDI/TOF-MS as a technique for the rapid identification and speciation of Haemophilus. This technique was used to not only identify the pathogen, H. ducreyi, but also to determine strain differences from different isolates. Mass spectral 'fingerprints' were obtained which permitted the rapid speciation of not only pathogenic forms of Haemophilus, but also those bacteria which are normally regarded as non-pathogenic and members of the normal flora. MALDI/TOF mass spectra can be acquired in 10 min, allowing the identification of Haemophilus spp. within 24 h rather than the 48 h or more needed for traditional bacteriological methods. In addition, these are the first mass spectral fingerprints available in the literature for many of these organisms.

Haemophilus ducreyi causes the genital ulcerative disease chancroid. One putative virulence factor of H. ducreyi is a pore-forming hemolysin that displays toxicity against human fibroblasts and keratinocytes. In order to test the role of the hemolysin in pathogenesis, an isogenic hemolysin-deficient mutant was constructed, designated 35000HP-RSM1. The lipooligosaccharide, outer membrane protein patterns, and growth attributes of 35000HP-RSM1 were identical to its parent, 35000HP. Human subjects were challenged on the upper arm with the isogenic isolates in a double-blinded, randomized, escalating dose-response study. Pustules developed at a similar rate at sites inoculated with the mutant or parent. The cellular infiltrate and bacterial load in lesions were also similar. These results indicate the hemolysin does not play a role in pustule formation. Due to the limitations of this model, the role of the hemolysin at later stages of infection could not be determined.

Human subjects were infected with Haemophilus ducreyi. All subjects developed papules and were randomized to treatment with a single dose of azithromycin (1 g) or ciprofloxacin (500 mg). At weekly intervals, volunteers were reinoculated with H. ducreyi, and drug concentrations were measured in peripheral blood mononuclear cells (PBMC). When papules developed, the subjects were treated with antibiotics and dismissed from the study. Eight of the ciprofloxacin-treated subjects developed papules 1 week after the initial treatment, and the ninth subject developed disease 2 weeks after treatment. The 9 azithromycin-treated subjects developed papules 4-10 weeks (mean, 6.8) after the initial treatment (P < .001). Azithromycin was detected in PBMC for 3-6 weeks (mean, 4). Pre- and posttreatment lesions had histology typical of experimental chancroid or were culture positive. Azithromycin prevents experimental chancroid for nearly 2 months. These findings have implications for strategies to prevent chancroid.

The intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.

Haemophilus ducreyi is the causative agent of the genital ulcer disease Chancroid. Chancroid has been shown to increase the risk of heterosexual transmission of HIV. Little is known regarding the attachment or localization of this organism to human cells in either the dermal or epidermal layer. In this study the attachment of H. ducreyi to human foreskin fibroblast (HFF) cells was further characterized. Attachment was mediated by more than one mechanism. Proteinase K treatment but not trypsinization of H. ducreyi significantly reduced attachment suggesting protein involvement. In addition, purified lipooligosaccharide (LOS) was able to inhibit attachment in a dose dependent manner. It appeared that the organism binds to fibronectin in the extracellular matrix of HFF cells, since competition studies using fibronectin showed that it was able to significantly reduce attachment in a dose dependent manner whereas collagen did not. We hypothesize that the attachment of H. ducreyi involves both a protein mediator of attachment (likely pili) as well as LOS and that one or both of these bacterial components interacts with fibronectin in the extracellular matrix to mediate attachment to HFF cells.

Chancroid, the most common cause of genital ulceration in Africa, is known to be associated epidemiologically with heterosexual transmission of human immunodeficiency virus (HIV). The pathophysiological mechanisms by which chancroid might facilitate the spread of HIV are obscure. To investigate the role of chancroid in HIV transmission, the authors studied the histological features of biopsies from 11 men with penile chancroid lesions including five who were serologically positive for HIV. The histomorphologic and immunophenotypic nature of the inflammatory infiltrates suggests that there is a significant role for cell-mediated immunity in the host response to Hemophilus ducreyi infection. This response may be critical to the role of chancroid in HIV transmission.

Of 22,274 patients 12 years of age or older attending a primary health care clinic in Nairobi, 1076 (4.8%) complained of symptoms suggesting a sexually transmitted disease (STD). Of these, 518 females and 462 males underwent complete clinical evaluation, and 78% had objective microbiologic or clinical evidence of STD, including 168 (17.1%) with genital ulcer disease (GUD). Presumptive specific clinical diagnoses on initial physical examination in cases of GUD were chancroid (131 patients), syphilis (25), genital herpes (15) and lymphogranuloma venereum (LGV) (1). Clinical diagnoses correlated only weakly with microbiological and serological diagnoses. Haemophilus ducreyi was isolated from 51 (41%) of the 125 with a clinical diagnosis of chancroid, and 4 (22%) of 18 with a diagnosis of syphilis, herpes, or LGV (P = 0.13). The rapid plasma reagin (RPR) test was reactive in 6 (24%) of 25 with a clinical diagnosis of syphilis, 18 (12.3%) of 146 with a diagnosis of chancroid or herpes, and 37 (4.7%) of 786 without a genital ulcer (P < 0.001, GUD vs no GUD). Sensitivity, specificity, and positive predictive value for presumptive clinical diagnosis of chancroid, relative to H. ducreyi isolation, were 93%, 16%, and 41%; and for diagnosis of syphilis, relative to reactive RPR, were 25%, 88% and 25%. Specific treatment based on presumptive specific clinical diagnosis frequently was inadequate for syphilis among patients with GUD and reactive RPR test. Syndromic treatment of GUD with antimicrobial combinations active against both chancroid and syphilis would be preferable to treatment with single drugs based on presumptive specific clinical diagnoses for this population.

Human subjects were experimentally infected with Haemophilus ducreyi for up to 2 weeks. Bacterial suspensions were delivered into the epidermis and dermis through puncture wounds made by an allergy-testing device. Subjects developed papular lesions that evolved into pustules resembling natural disease. Some papular lesions resolved spontaneously, indicating that host responses may clear infection. Bacteria were shed intermittently from lesions, suggesting that H. ducreyi may be transmissible before ulceration. Host responses to infection consisted primarily of cutaneous infiltrate of polymorphonuclear leukocytes, Langerhans cells, macrophages, and CD4 T cells of alpha beta lineage. Expression of HLA-DR by keratinocytes was associated with the presence of interferon-gamma mRNA in the skin. There was little evidence for humoral or peripheral blood mononuclear cell responses to bacterial antigens. The cutaneous infiltrate of CD4 cells and macrophages provides a mechanism that facilitates transmission of human immunodeficiency virus by H. ducreyi.

One-hundred thirteen men (mean age, 23 years) who presented with inguinal buboes to a government-operated hospital for sexually transmitted diseases (STDs) in Bangkok were studied between February 1987 and February 1989. The median duration of preceding symptoms was 7 days (range, 1-62 days). The majority of patients (74; 65%) had received treatment previously; 31 (27%) were febrile, 13 (12%) had extrainguinal lymphadenopathy, and 31 (27%) had concurrent active genital ulcers. There was no history of genital ulceration in 66 (58%) of the patients. Pus was obtained from 51 of the 110 buboes aspirated for culture; 21 (41%) of these cultures yielded Haemophilus ducreyi, and 2 (3.9%) were positive for Chlamydia trachomatis on immunofluorescence microscopy. Saline (1 mL) was injected and reaspirated from the buboes of 35 of the other 59 patients; 3 buboes yielded H. ducreyi and 9 were positive for C. trachomatis. All cultures for other aerobic and anaerobic bacteria and viruses in intact buboes were negative. Syphilis serology was positive in only one case. Patients attending STD clinics in this region who have large, fluctuant, edematous inguinal buboes containing pus should receive presumptive treatment for chancroid. If there is no pus, then the bubo is more likely to be caused by lymphogranuloma venereum.

Isolation rates of Haemophilus ducreyi from cases of chancroid are low. Experts recommend that isolation media be incubated at 33 to 35 degrees C, but the possible effect of this temperature range on the recovery of H. ducreyi has not been evaluated. We inoculated two sets of agar plates with material from genital ulcers and incubated one set at 33 degrees C and one at 35 degrees C; incubation at 33 degrees C identified 21% more cases than did incubation at 35 degrees C (109 versus 85 cases, respectively, of the 116 cases from which an isolation was made; P < 0.01).

Haemophilus ducreyi is a strict human pathogen that causes sexually transmitted genital ulcer disease. We infected domestic swine with H. ducreyi 35000, resulting in the development of cutaneous ulcers histologically resembling human chancroid lesions. Intraepidermal lesions progressed from pustules to ulcers containing polymorphonuclear leukocytes and were accompanied by a dermal inflammatory infiltrate containing T cells and macrophages. H. ducreyi was recovered from lesions up to 17 days after inoculation, and pigs did not develop immunity to reinfection with the challenge strain. Features of the model include inoculation through abrasions in the epidermis, ambient housing temperatures for infected pigs, the ability to deliver multiple different inocula to a single host, and the availability of monoclonal antibodies against porcine immune cells permitting immunohistochemical characterization of the host immune response to H. ducreyi infection.

Epidemiologic, clinical, and microbiologic data were collected from 299 men with nonsyphilitic genital ulcer disease. One hundred eighteen (39%) were culture-positive for Haemophilus ducreyi, 57 (19%) were culture-positive for herpes simplex virus, and 124 (41%) were culture-negative. Patients with chancroid were significantly more likely than those with genital herpes to have been frequent users of alcohol (44% vs. 23%, P = .006). They were also more likely recently to have used cocaine (25% vs. 9%, P = .013), had sex with a prostitute (17% vs. 5%, P = .035), traded drugs for sex (16% vs. 2%, P = .005), and had a sex partner who used drugs (38% vs. 13%, P = .001). Culture-negative patients were similar to chancroid patients with respect to most epidemiologic risk factors. Despite the epidemiologic similarities, the clinical features of culture-negative ulcers resembled those of culture-proven herpes ulcers more closely than they did those of culture-proven chancroid ulcers. These data establish a link between chancroid in the United States and the use of crack cocaine.

We conducted a randomized, unblinded, prospective study designed to determine the efficacy of single-dose azithromycin for the treatment of chancroid. Men and women 16 years of age and older who had darkfield-negative genital ulcers that were clinically suspected to be caused by Haemophilus ducreyi and who attended urban sexually transmitted disease clinics or presented to hospital emergency departments were enrolled in the study. Patients were randomized to receive 250 mg of ceftriaxone im or 1 g of azithromycin orally, both given as a single dose. They were followed for up to 23 days after treatment. For 65 patients, cultures were positive for H. ducreyi; there were 68 patients whose cultures were negative for both H. ducreyi and herpes simplex virus and who had no evidence of syphilis. All 133 patients returned for at least one follow-up visit. At the time of the last follow-up visit, all 32 patients whose cultures were positive for H. ducreyi and who were treated with azithromycin were clinically cured. In all 33 culture-positive cases in which ceftriaxone was used, there was either clinical improvement or cure at the time of the patient's last follow-up visit. In addition, azithromycin and ceftriaxone were equally effective in healing ulcers for which cultures were negative. We conclude that a single 1-g oral dose of azithromycin is as effective as a 250-mg im dose of ceftriaxone for the treatment of chancroid.