Penile carcinoma presenting as inguinal bubo, masquerading as a venereal disease.

We report a case of penile carcinoma presenting as an inguinal bubo in a young man. The associated painful genital ulcer and history of high-risk sexual behaviour raised a strong suspicion of a sexually transmitted infection. We review the literature for similar cases, highlight the similarities with venereal disease and discuss the differential diagnosis of inguinal bubo.

Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles.

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2-DE and by the 16-BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE two-detergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921.

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Improving the accuracy of syndromic diagnosis of genital ulcer disease in Malawi.

OBJECTIVES/GOAL: Most resource-poor settings rely on syndromic criteria to diagnose genital ulcer disease (GUD). However, the etiologic pathogens of GUD vary temporally and geographically, and current criteria may not reflect changes in the prevalence of specific pathogens. STUDY: In 1999, we estimated the prevalence of Treponema pallidum (Tp), herpes simplex virus (HSV), and Haemophilus ducreyi (Hd) in Malawi. We then used regression coefficients of independent correlates of HSV and Hd to develop weighted diagnostic algorithms, in which weights were beta-coefficients corresponding to each factor. RESULTS: Overall, a decrease in the proportion of sexually transmitted disease attributable to GUD was noted in 7 years. Thirty-five percent were attributable to HSV, 30% to H. ducreyi, and 4% to T. pallidum. Areas under the receiver operating characteristic curves for weighted and unweighted HSV diagnostic algorithms were 67.6% and 66.5%, respectively. There was no significant difference in the explanatory performance of the weighted and unweighted algorithms. CONCLUSIONS: Unweighted algorithms can therefore be used to improve diagnostic accuracy of GUD.

DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers.

Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P = 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

Expression of cytolethal distending toxin and hemolysin is not required for pustule formation by Haemophilus ducreyi in human volunteers.

Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and beta1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats.

To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection.

Haemophilus ducreyi expresses a conserved hemoglobin-binding outer-membrane protein (HgbA). To test the role of HgbA in pathogenesis, we infected 9 adults with isolate 35000 and its isogenic hgbA-inactivated mutant (FX504) on their upper arms in a double-blinded, escalating dose-response study. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule-formation rate was 55% (95% confidence interval [CI], 30. 8%-78.5%) at parent sites and 0 (95% CI, 0-10.5%) at mutant sites (P<.0001). The recovery rate of H. ducreyi from surface cultures was 16% (n=142) from parent sites and 0 (n=213) from mutant sites (P<. 0001). H. ducreyi was recovered at biopsy from 6 of 7 parent sites and from 0 of 3 mutant sites. The results indicate that hemoglobin may be a critical source of heme or iron for the establishment of H. ducreyi infection in humans.

A pilus-deficient mutant of Haemophilus ducreyi is virulent in the human model of experimental infection.

Haemophilus ducreyi expresses fine tangled pili, which are composed predominantly of a major subunit (FtpA). Confocal microscopy showed that an FtpA-specific monoclonal antibody bound to bacteria in biopsy samples obtained from infected human volunteers. To test the role of pili in pathogenesis, an isogenic mutant (35000HP-SMS1) was constructed by insertionally inactivating ftpA. 35000HP-SMS1 did not express FtpA and was nonpiliated but was otherwise identical to its parent, 35000HP. Seven healthy adults were challenged on the upper arm with the isogenic isolates in a double-blinded, escalating dose-response study. Sites inoculated with the mutant produced papules and pustules at rates similar to the rates observed at sites inoculated with the parent. The recovery rate of H. ducreyi from cultures and the histopathology of biopsy samples obtained from pustules inoculated with 35000HP or 35000HP-SMS1 were similar. Although pili are expressed in vivo, FtpA is not required for pustule formation in the human challenge model.

Etiology of genital ulcer disease in Dakar, Senegal, and comparison of PCR and serologic assays for detection of Haemophilus ducreyi.

We used PCR assays to determine the etiology of genital ulcers in patients presenting to a sexually transmitted disease clinic in Dakar, Senegal, and evaluated the ability of two PCR tests (groEL and recD) and two serological tests (adsorption enzyme immunoassay [EIA] and lipooligosaccharide [LOS] EIA) to detect current Haemophilus ducreyi infection. We found that in this population, H. ducreyi, T. pallidum, and herpes simplex virus HSV DNA were detected in 56, 15, and 13% of 39 genital ulcer specimens, respectively, and H. ducreyi DNA was detected in 60% (3 of 5) of samples from ulcerated bubos. Among 40 consecutive patients with genital ulcer disease and with sufficient sample for both PCR assays, the recD and groEL H. ducreyi PCR assays were 83% concordant, with the recD PCR assay detecting six (15%) additional positive specimens and the groEL assay detecting one (3%) additional positive specimen. Compared to PCR, the adsorption EIA and LOS EIA tests had sensitivities of 71 and 59% and specificities of 57 and 90%, respectively, for the diagnosis of current H. ducreyi infection. While these differences in specificity could be due either to previous infection with H. ducreyi or to the detection of cross-reacting antibodies, only 6% of patients from a nearby family planning clinic gave a positive reaction in both the adsorption EIA and LOS EIA assays, indicating that cross-reacting antibodies are not prevalent among clinic attendees in this city. Our studies indicate that the adsorption EIA detects both current and past infection, while the LOS EIA assay is more specific for current infection with H. ducreyi in this population.

Chancroid: from clinical practice to basic science.

Chancroid is a sexually transmitted disease caused by the bacterium Haemophilus ducreyi. It usually presents as a genital ulcer and may be associated with regional lymphadenopathy and bubo formation. H. ducreyi infection is predominantly seen in tropical resource-poor regions of the world where it is frequently the most common etiological cause of genital ulceration. Genital ulcer disease has been shown to be an extremely important co-factor in HIV transmission. With the advent of the AIDS epidemic, there has been increased research effort to elucidate those factors involved in the pathogenesis of chancroid. Several putative virulence factors have now been identified and isogenic H. ducreyi mutants constructed by mutagenesis of their encoding genes. This approach has facilitated investigations into the role each of these putative virulence factors may play in H. ducreyi pathogenesis through the use of in vitro and in vivo model systems. One major goal of current chancroid research is to identify antigens which are immunogenic and could form the basis of a vaccine against H. ducreyi infection. Such a vaccine, if shown to be effective in decreasing the prevalence of chancroid, could have the added benefit of slowing down the HIV incidence rates in those populations where chancroid is a major co-factor for HIV transmission.

Target cell range of Haemophilus ducreyi hemolysin and its involvement in invasion of human epithelial cells.

Haemophilus ducreyi, the causative agent of chancroid, produces a hemolysin, whose role in virulence is not well defined. To assess the possible role of hemolysin in pathogenesis, we evaluated its target cell range by using wild-type H. ducreyi 35000, nonhemolytic mutants with the hemolysin structural gene deleted, and isogenic strains expressing different amounts of hemolytic activity. The cytotoxicity of the various cell types was assessed by quantitating the release of lactate dehydrogenase into culture supernatants as a measure of cell lysis. In these experiments, human foreskin fibroblasts, human foreskin epithelial cells, and, to a lesser extent, HEp-2 cells were lysed by H. ducreyi hemolysin. Hemolysin also lysed human blood mononuclear cells and immune system cell lines including U937 macrophage-like cells, T lymphocytes, and B lymphocytes. In contrast, human polymorphonuclear leukocytes were not sensitive to hemolysin under the conditions tested. We also analyzed the effect of hemolysin on invasion of human epithelial cells and found that H. ducreyi strains expressing cloned hemolysin genes showed a 10-fold increase in invasion compared to the control strain. These data support the hypothesis that the H. ducreyi hemolysin is important in the pathogenesis of chancroid and may contribute to ulcer formation, invasion of epithelial cells, and evasion of the immune response.

HIV-1 subtype E incidence and sexually transmitted diseases in a cohort of military conscripts in northern Thailand.

OBJECTIVES: To determine the rate of and risk factors for HIV-1 seroconversion and describe sexually transmitted disease (STD) prevalence rates for young men in northern Thailand. METHODS: Data were collected from self-administered questionnaires and serologic testing at enrollment in a prospective study in 1991 and at follow-up after 6, 17, and 23 months on a cohort of 1115 men selected by lottery for military conscription. RESULTS: A total of 14 men seroconverted to HIV-1 envelope subtype E. The overall HIV-1 incidence rate was 1.1 (95% confidence interval [CI], 0.6-1.8) per 100 person-years (PY) of follow-up. However, the rate was 2.0/100 PY for conscripts from the upper northern subregion of Thailand compared with 0.5/100 PY from other regions (adjusted rate ratio [RR] = 2.69; 95% CI, 0.8-12.2). On multivariate analyses, the behavioral factors associated with HIV-1 seroconversion were frequency of sex with female sex workers (FSWs; p = .04), receptive anal sex (adjusted RR = 6.73; 95% CI, 1.8-21.7), and large amount of alcohol consumption (adjusted RR = 3.12; 95% CI, 1.0-10.9). Genital ulceration was the STD most strongly associated with seroconversion. The prevalence of serologic reactivity to syphilis, Haemophilus ducreyi, and herpes simplex virus type 2 increased with greater frequency of sex with FSWs and was generally higher for men from the upper north. CONCLUSION: Young men in northern Thailand are at high risk for HIV-1, primarily through sex with FSWs; and other STDs are highly associated with HIV-1 incidence. As HIV-1 infection extends into the general population, intervention programs are needed to address the problem of sexual transmission apart from commercial sex venues.

PIP: Findings are presented from a prospective study conducted to determine the rate of and risk factors for HIV-1 seroconversion, and to describe sexually transmitted diseases (STD) prevalence rates for young men in northern Thailand. Study findings are based upon data collected from self-administered questionnaires and serologic testing at enrollment in 1991 and at follow-up after 6, 17, and 23 months on a cohort of 1115 young men chosen by lottery for military conscription. Men in Thailand are generally eligible for conscription in the year of their 21st birthday. 6.9% of the men were HIV-1 seropositive at enrollment; 15.3% of men from the upper northern region compared with 2.5% of men from elsewhere. 14 subjects seroconverted to HIV-1 envelope subtype E over the course of the study. The overall HIV-1 incidence rate was 1.1/100 person-years (PY) of follow-up. However, the rate was 2.0/100 PY for conscripts from the upper northern subregion of Thailand compared with 0.5/100 PY from other regions. Multivariate analyses found frequent sex with female prostitutes, receptive anal sex, and high levels of alcohol consumption to be positively associated with HIV-1 seroconversion. Genital ulceration was the STD most strongly associated with seroconversion. The prevalence of serologic reactivity to syphilis, Haemophilus ducreyi, and herpes simplex virus type 2 increased with greater frequency of sex with female prostitutes, and was generally higher for men from the upper north.

Haemophilus ducreyi infection causes basal keratinocyte cytotoxicity and elicits a unique cytokine induction pattern in an In vitro human skin model.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen, H. ducreyi is present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions with H. ducreyi 35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observed H. ducreyi inside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures. H. ducreyi infection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1alpha levels were not elevated. IL-8 produced in response to H. ducreyi infection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.

Risk factors for sexually transmitted disease in Harare: a case-control study.

OBJECTIVE: To obtain information on risk factors and health-seeking behavior of men with sexually transmitted diseases (STDs) attending primary care clinics. STUDY DESIGN: Unmatched case-control. METHODS: Cases consisted of 256 consecutive men with genital ulcer disease (GUD) and 256 with other STDs. Control subjects (N = 256) were recruited from every third man with non-STD-related complaints. All subjects were at least 15 years of age. A structured questionnaire was administered. RESULTS: Genital ulcer disease cases reported more frequent sexual intercourse with a commercial sex worker (odds ratio [OR] = 17.4; 95% confidence intervals [CI] = 7.8-40.0) and a recent new sexual contact (OR = 6.7; CI = 3.3-14.1). Similarly, STD cases reported more frequent sexual contact with a commercial sex worker (OR = 3.4; CI = 2.0-5.6) and a recent new sexual contact (OR = 7.9; CI = 3.9-16.3). Reported condom use was less than 30% with all partner types. Of all STD cases, 80% sought treatment at the primary care clinics, with 35% delaying more than 7 days before seeking treatment. CONCLUSIONS: Culturally appropriate behavioral educational programs are advocated to reduce the risk of transmission and the period for seeking treatment for all STDs.