The demographic, sexual health and behavioural correlates of Mycoplasma genitalium infection among women with clinically suspected pelvic inflammatory disease.

OBJECTIVE: Mycoplasma genitalium has been identified as a cause of pelvic inflammatory disease (PID), a clinical syndrome associated with inflammation of the female upper genital tract and serious reproductive sequelae. As the demographic, behavioural and sexual risk profile of women with M genitalium-associated PID is not well understood, the characteristics of M genitalium-infected women presenting with clinically suspected PID were investigated. METHODS: Data from 586 participants in the PID Evaluation and Clinical Health Study were analysed. Demographic, sexual history and behavioural characteristics, including age, race, marital status, education level, sexual activity, number of sexual partners, history of sexually transmitted infection (STI), bacterial vaginosis and PID, contraception use, oral and anal sex, age at sexual debut, douching practices and drug, alcohol and tobacco use, were compared between 88 women testing positive and 498 women testing negative for M genitalium by PCR in the cervix and/or endometrium. Twenty-two women with M genitalium mono-infections were compared with 172 women who tested positive for Neisseria gonorrhoeae by culture and/or Chlamydia trachomatis by PCR. RESULTS: Age under 25 years, douching two or more times per month and smoking were independently associated with M genitalium. Women with M genitalium mono-infections were significantly less likely to be African-American (59.1% vs 86.0%, p = 0.001) than women with N gonorrhoeae and/or C trachomatis. CONCLUSIONS: Women infected with M genitalium had some characteristics commonly associated with PID and other STI. The demographic, sexual and behavioural characteristics of M genitalium-positive women were similar to women with chlamydial and/or gonococcal PID.

Clinical characteristics of bacterial vaginosis among women testing positive for fastidious bacteria.

OBJECTIVES: As the aetiology of bacterial vaginosis (BV) is not well understood, this study sought to determine the relationships between several fastidious microbes, BV and selected clinical characteristics of BV. METHODS: Endometrial and cervical specimens from 50 women with non-gonococcal, non-chlamydial endometritis were tested for Leptotrichia sanguinegens/amnionii, Atopobium vaginae, bacterial vaginosis-associated bacteria 1 (BVAB1), Ureaplasma urealyticum biovar 2 (UU-2) and Ureaplasma parvum using PCR. BV was categorised using Nugent's and Amsel's criteria. Odds ratios (OR) adjusted for age and race were estimated using multivariable logistic regression. RESULTS: Although elevated pH was a universal feature, other BV characteristics differed by pathogen, suggesting variable clinical presentation. Only UU-2 was strongly associated with vaginal discharge, but a positive whiff test and a 20% or greater classification of epithelial cells as clue cells were more common among women with L sanguinegens/amnionii, A vaginae and BVAB1. For each of these bacteria, there were trends towards associations with BV defined by Amsel's criteria (L sanguinegens/amnionii OR 2.9, 95% CI 0.5 to 15.7; A vaginae OR 2.6, 95% CI 0.6 to 11.4; BVAB1 OR 5.7, 95% CI 1.0 to 31.1) and significant associations with BV defined by Gram stain (L sanguinegens/amnionii OR 17.7, 95% CI 2.8 to 113.0; A vaginae OR 19.2, 95% CI 3.7 to 98.7; BVAB1 OR 21.1, 95% CI 2.2 to 198.5). CONCLUSIONS: L sanguinegens/amnionii, A vaginae and BVAB1 are associated with clinical characteristics consistent with BV and BV defined by Nugent's and Amsel's criteria. These fastidious bacteria may cause unrecognised infection, as none was associated with abnormal vaginal discharge.

Failure of cefoxitin and doxycycline to eradicate endometrial Mycoplasma genitalium and the consequence for clinical cure of pelvic inflammatory disease.

OBJECTIVES: As Mycoplasma genitalium is associated with pelvic inflammatory disease (PID), we examined the efficacy of a commonly used PID antimicrobial in treating M genitalium upper genital tract infection. METHODS: In the PID Evaluation and Clinical Health study of inpatient versus outpatient treatment, 682 women treated with cefoxitin and doxycycline for clinically suspected PID had stored cervical and endometrial specimens available for analysis. In the current sub study, we compared baseline endometritis, short term treatment failure (continued endometritis and pelvic pain 30 days following treatment) and sequelae among women with and without M genitalium, identified using PCR. RESULTS: Endometrial M genitalium was associated with baseline endometritis (adjusted OR 3.0, 95% CI 1.5 to 6.1). Among women with a positive baseline M genitalium test, 41% tested positive again 30 days following treatment. Women testing positive compared to those testing negative for M genitalium at baseline had an increased risk of short-term treatment failure (RR 4.6, 95% CI 1.1 to 20.1). Rates of sequelae, including infertility (22%), recurrent PID (31%) and chronic pelvic pain (42%), were high among women testing positive for endometrial M genitalium at baseline. There was a non-significant trend towards increased infertility, chronic pelvic pain and recurrent PID, and decreased pregnancy and live birth following M genitalium infection. CONCLUSIONS: M genitalium is associated with endometritis and short-term PID treatment failure. Cefoxitin and doxycycline, a Centers for Disease Control and Prevention recommended PID treatment regimen, is ineffective for the treatment of M genitalium upper genital tract infection.

Detection of Mycoplasma genitalium in women with laparoscopically diagnosed acute salpingitis.

OBJECTIVES: Mycoplasma genitalium has been associated with cervicitis, endometritis, and tubal factor infertility. Because the ability of this bacterium to ascend and infect the fallopian tube remains undefined, we performed an investigation to determine the prevalence of M genitalium in fallopian tube, endometrial, and cervical specimens from women laparoscopically diagnosed with acute salpingitis in Nairobi, Kenya. METHODS: Women presenting with pelvic inflammatory disease were laparoscopically diagnosed with salpingitis. Infection with M genitalium in genital specimens was determined by polymerase chain reaction (PCR). RESULTS: Of 123 subjects with acute salpingitis, M genitalium was detected by PCR in the cervix and/or endometrium in nine (7%) participants, and in a single fallopian tube specimen. In addition, those infected with M genitalium were more often HIV infected than women not infected by M genitalium (seven of nine (78%) v 42 of 114 (37%), p<0.03). CONCLUSIONS: M genitalium is able to ascend into the fallopian tube, but its association with tubal pathology requires further investigation.

Haemophilus ducreyi inhibits phagocytosis by U-937 cells, a human macrophage-like cell line.

Haemophilus ducreyi is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live H. ducreyi can be isolated from these lesions. To analyze the interaction of H. ducreyi with macrophages, we investigated the ability of H. ducreyi strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although H. ducreyi strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that H. ducreyi avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that H. ducreyi inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to H. ducreyi, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine H. ducreyi strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which H. ducreyi avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes.

Association of Mycoplasma genitalium with nongonococcal urethritis in heterosexual men.

Chlamydia trachomatis and Neisseria gonorrhoeae are universally acknowledged as urethral pathogens, yet the etiology in the majority of cases of urethritis is unclear. Our case-control study assessed the association of Mycoplasma genitalium, Ureaplasma urealyticum, and other potential pathogens with acute nongonococcal urethritis (NGU) in heterosexual men presenting to an urban sexually transmitted diseases clinic. M. genitalium was detected in 27 (22%) of 121 NGU case patients and in 5 (4%) of 117 control subjects (P<.01). Although C. trachomatis was detected in 36 (30%) of 121 NGU case patients and in 4 (3%) of 117 control subjects (P<.01), only 3 men with NGU were infected with both C. trachomatis and M. genitalium. U. urealyticum was not associated with NGU. By multivariate analyses, controlling for age, race, history of prior urethritis, and chlamydial infection, M. genitalium was associated with a 6.5-fold increased risk of urethritis (95% confidence interval, 2.1-19.5), which supports a role of this organism in the etiology of NGU.

Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication.

An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

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.

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.

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.

Molecular characterization of Haemophilus ducreyi strains from Jackson, Mississippi, and New Orleans, Louisiana.

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.

Haemophilus ducreyi Detection by PCR.

Genital ulcers are typically caused by one of three organisms, Haemophilus ducreyi, Treponema pallidum, or herpes simplex virus, which cause chancroid, syphilis, and genital herpes, respectively. Although traditionally these diseases have been differentiated by their clinical presentation, there is considerable overlap in their clinical manifestations (1).

An investigation of genital ulcers in Jackson, Mississippi, with use of a multiplex polymerase chain reaction assay: high prevalence of chancroid and human immunodeficiency virus infection.

In 1994, an apparent outbreak of atypical genital ulcers was noted by clinicians at the sexually transmitted disease clinic in Jackson, Mississippi. Of 143 patients with ulcers tested with a multiplex polymerase chain reaction (PCR) assay, 56 (39%) were positive for Haemophilus ducreyi, 44 (31%) for herpes simplex virus, and 27 (19%) for Treponema pallidum; 12 (8%) were positive for > 1 organism. Of 136 patients tested for human immunodeficiency virus (HIV) by serology, 14 (10%) were HIV-seropositive, compared with none of 200 patients without ulcers (P < .001). HIV-1 DNA was detected by PCR in ulcers of 6 (50%) of 12 HIV-positive patients. Multivariate analysis indicated that men with chancroid were significantly more likely than male patients without ulcers to report sex with a crack cocaine user, exchange of money or drugs for sex, and multiple sex partners. The strong association between genital ulcers and HIV infection in this population highlights the urgency of preventing genital ulcers in the southern United States.

Haemophilus ducreyi is susceptible to protegrin.

Protegrins, potent antimicrobial peptides found in porcine leukocytes, have activity against the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, and human immunodeficiency virus type 1. We tested synthetic protegrin 1 (PG-1) for activity against nine isolates of Haemophilus ducreyi, the etiologic agent of chancroid. The test organisms included CIP 542 (the type strain), 35000HP (a human-passaged variant of 35000), 35000HP-RSM2 (an isogenic D-glycero-D-manno-heptosyltransferase mutant of 35000HP), and six clinical isolates. The isolates were epidemiologically unrelated, represented three HindIII ribotypes, and had varying antimicrobial resistance patterns. In bactericidal assays, five isolates were rapidly killed by synthetic PG-1. In radial diffusion assays, all nine isolates were exquisitely sensitive to PG-1. These data highlight the potential of protegrins for development as topical agents to prevent many sexually transmitted diseases, including chancroid.

Role of the Haemophilus ducreyi Ton system in internalization of heme from hemoglobin.

By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressing hgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes was exbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.

Chancroid.

Chancroid is a genital ulcerative disease GUD). These diseases are common throughout the world and include syphilis, genital herpes, chancroid, lymphogranuloma venereum, and donovanosis. Chancroid is particularly common an Africa, Asia, and Latin America where its incidence may exceed that of syphilis as a cause of genital ulceration (1,2). However, chancroid is considered an uncommon sexually transmitted infection in the United States.

Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture.

Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, produces several factors that damage human cells. We used isogenic mutants of H. ducreyi 35000 to demonstrate that the hemolytic activity and the cytotoxic effect of H. ducreyi on human foreskin fibroblasts are due to the same toxin.

Characterization of the hemolytic activity of Haemophilus ducreyi.

H. ducreyi is the causative agent of chancroid, a genital ulcer disease most prevalent in developing countries. Chancroid enhances the heterosexual transmission of human immunodeficiency virus and is identified in focal outbreaks in the United States, but little is known about its pathogenesis. We studied the hemolysin produced by H. ducreyi because this molecule might be an important virulence factor in the pathogenesis of chancroid. Ten strains of H. ducreyi were tested on newly devised blood agar plates and were found to have hemolytic activity. We examined the hemolytic activity of H. ducreyi 35000 further and found that it was heat labile, cell associated, greatest at pH 7.0, and produced in logarithmic- but not stationary-phase cultures. Using transposons Tn916 and Tn1545-delta 3, we have isolated three classes of transposon mutants of strain 35000: those with no detectable hemolytic activity, those with reduced hemolytic activity, and those with enhanced hemolytic activity. Transposon insertions in the nonhemolytic mutants were located in a DNA sequence which hybridized to the Proteus mirabilis hemolysin gene. Analysis of clones containing overlapping sections of this region served to further localize the H. ducreyi hemolysin gene and allow its expression in Escherichia coli and complementation of the nonhemolytic defect in an H. ducreyi mutant. These experiments indicate that H. ducreyi 35000 produces a hemolysin that is related to the calcium-independent hemolysin produced by P. mirabilis. Further experiments are needed to define the similarity of the H. ducreyi hemolysin to other calcium-independent hemolysins and to determine its role in the pathogenesis of chancroid.

Haemophilus ducreyi attaches to and invades human epithelial cells in vitro.

Haemophilus ducreyi is a sexually transmitted pathogen that causes genital ulcers and inguinal adenopathy. Because chancroidal ulcers are most commonly located on the foreskins of uncircumcised males, we utilized human foreskin epithelial cells (HFECs) to investigate the initial interaction of H. ducreyi with its host. The eight different strains of H. ducreyi that were studied varied in their abilities to attach to these epithelial cells, with six strains consistently attaching to > or = 90% of HFECs and two strains attaching to < 25% of HFECs. The strains with low levels of adherence also failed to exhibit chaining in broth culture and were avirulent in the rabbit model, suggesting that virulence in this model and attachment may be linked. The most adherent strain, LA228R, was further evaluated for its ability to invade HFECs and HEp-2 cells. Scanning electron microscopy and transmission electron microscopy of HFECs after interaction with LA228R produced images consistent with attachment, ingestion into vesicles, and escape from the vesicles into the cytoplasm. In addition, the gentamicin protection assay and inhibition of invasion by cytochalasin B and D indicated that LA228R was able to invade both HFECs and HEp-2 cells. Further examination of the mechanisms involved in the adherence and invasion of H. ducreyi into epithelial cells and their correlation with virulence will provide a better understanding of the pathogenesis of the disease caused by this important pathogen.

Clear broth and plate media for culture of Haemophilus ducreyi.

Using catalase as a source of heme, we have developed both clear plate and broth media for the growth of Haemophilus ducreyi, the causative agent of chancroid. In the broth medium, the growth phase of the organism can be monitored and the organisms achieve a cell density of > 10(8) CFU/ml.