Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies.

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.

TEM-type beta-lactamase production in Haemophilus ducreyi.

A TnA-containing 6.0-megadalton plasmid (pJB1) isolated from Haemophilus ducreyi was shown to code for a beta-lactamase similar to the TEM-1-type beta-lactamase originating from the ampicillin transposon Tn2.

In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein.

A murine monoclonal antibody, which binds to an epitope on the major outer membrane protein of Chlamydia trachomatis and with species specificity in the micro-immunofluorescent assay, effectively neutralized in vitro two antigenically distinct serovars of C. trachomatis. Optimal concentrations of both organism and antibody were required to produce maximal neutralization of the organism. Neutralization was less effective and more variable at lower dilutions of antibody than at higher dilutions, suggesting a prozone phenomenon. A radiolabeled attachment assay demonstrated that attachment of elementary bodies was unaffected by earlier treatment with antibody and that neutralization occurred at a step after attachment. The epitope to which this antibody is directed, on the major outer membrane protein of C. trachomatis, may have an important role in determining infectivity of the organism.

Identification of genus-specific epitopes on the outer membrane complexes of Chlamydia trachomatis and Chlamydia psittaci immunotypes 1 and 2.

Polyclonal and monoclonal antibodies were used to study the immunogenic and antigenic characteristics of chlamydiae. We focused on the most predominant proteins in the outer membrane complex, the major outer membrane protein (MOMP) and the doublet consisting of proteins of 57 and 62 kilodaltons (57-62 kDa doublet). Immunoblot analyses were performed with chlamydial elementary bodies by using (i) immune sera from sheep which had undergone a recent episode of abortion due to the ovine abortion (OA) strain of C. psittaci, (ii) rabbit hyperimmune anti-C. psittaci (OA) and -C. trachomatis sera, and (iii) monoclonal antibodies to the MOMP of C. trachomatis. The typical pattern of response with polyclonal antisera against heterologous elementary bodies was reactivity with the 57-62 kDa doublet and lipopolysaccharide with weak and sometimes no anti-MOMP activity. Three distinct genus-specific anti-C. trachomatis MOMP monoclonal antibodies showed different patterns of reactivity with the MOMPs of the two immunotypes of C. psittaci and C. trachomatis serovars. Our data confirm the predominance of a genus-specific 57-62 kDa doublet response despite the presence of genus-specific epitopes on the MOMP.

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.

Synergistic antibacterial activity of clavulanic acid and amoxicillin against beta-lactamase-producing strains of Haemophilus ducreyi.

The susceptibility of 47 strains of Haemophilus ducreyi to amoxicillin, clavulanic acid, and the combination of both drugs was determined by agar dilution susceptibility testing. A synergistic antibacterial activity of the combination was found for beta-lactamase-producing strains.

Chlamydial heat shock proteins and trachoma.

Two chlamydial proteins (HSP-60 and HSP-70) have marked homology with bacterial and mammalian heat shock proteins. Previous studies have indicated that when inoculated into the eyes of immune animals, a Triton X-100 extract of chlamydia containing HSP-60 induces an ocular delayed-type hypersensitivity reaction. The potential for HSP-70 to induce a similar reaction was tested in six cynomolgus monkeys that had been sensitized to both antigens by previous ocular chlamydial infection. Whereas the chlamydial extract containing HSP-60 induced a marked clinical response within 24 h of inoculation, no response followed inoculation of HSP-70 in the contralateral eye. The lack of a response to HSP-70 suggests that further assessment of its potential as a trachoma vaccine is warranted.

The 75-kilodalton protein of Chlamydia trachomatis: a member of the heat shock protein 70 family?

The gene encoding a 75-kilodalton (kDa) protein of Chlamydia trachomatis was cloned, expressed, and sequenced. Genomic libraries from C. trachomatis serovar D DNA were constructed in vectors pUC18 and lambda gt11 and were screened with a panel of monoclonal antibodies against C. trachomatis antigens. The only recombinants identified were those that reacted with antibody UM-13, which has specificity for a genus-specific epitope on the 75-kDa protein. The gene was localized to a 2.9-kilobase DNA fragment and sequenced. The gene consists of a long open reading frame of 1,956 nucleotides, which translates into 652 amino acids totalling 70,558 daltons in mass. Putative promoter elements and a ribosome binding site were identified within 5'-flanking sequences, and a typical rho-independent terminator was identified within 3'-flanking sequences. Screening of the GenBank nucleic acid sequence data bank revealed extensive similarity between the chlamydial 75-kDa gene and the heat shock protein 70 (hsp70) family or proteins. In particular, 71 and 69% amino acid sequence similarities were identified with hsp70 of Escherichia coli and Bacillus megaterium, respectively. Polyclonal antibodies were produced to the recombinant antigen in rabbits and detected epitopes on elementary bodies in enzyme-linked immunosorbent and indirect microimmunofluorescence assays. Antibodies reacted with an antigen of identical molecular mass in L2 and C serovars in an immunoblot assay and neutralized these serovars in cell culture. The 75-kDa protein appears to be a chlamydial homolog of hsp70, is immunoaccessible on native elementary bodies, and is a target for neutralization.

Chlamydia trachomatis: its role in tubal infertility.

We compared the prevalence of antibody to Chlamydia trachomatis among 88 women undergoing an evaluation for infertility and 49 women attending an antenatal clinic. Demographic data regarding sexual behavior were also collected. Eighteen women had tubal infertility and 70 had infertility due to a variety of other reasons. In comparison with women who had other causes for infertility, women with tubal infertility began coitus sooner (17.7 +/- 2.2 years vs. 19.5 +/- 3.4 years, P less than .05) and had more lifetime sex partners (4.5 vs. 1.33, P less than .001). Women with tubal infertility had a higher prevalence of antibody to C. trachomatis (13 of 18) than did women with nontubal causes for infertility (6 of 70, P less than .0001) or pregnant women (11 of 49, P = .0003). This high prevalence of antibody to C. trachomatis among women with tubal infertility was independent of sexual experience. By immunoblot analysis, an antigen of approximately 57,000 Da was immunodominant in 11 of 13 seropositive subjects with tubal infertility vs. 2 of 6 seropositive subjects with nontubal infertility (P = .046) and 1 of 11 seropositive pregnant women (P = .0003). Thus, women with tubal infertility frequently have serological evidence of prior infection with C. trachomatis and have a distinctive antigen-specific humoral immune response. These results further support the etiologic role of infection with C. trachomatis in tubal infertility.

Identification of a ROB-1 beta-lactamase in Haemophilus ducreyi.

A collection of 100 clinical isolates of Haemophilus ducreyi from Thailand were all found to harbor a 5.4-kb plasmid, designated pTH126, which was shown to contain the bla ROB-1 gene. Restriction enzyme analysis and DNA-DNA hybridization studies confirmed that pTH126 was similar to the ROB-1 beta-lactamase plasmid pVM105 from Actinobacillus pleuropneumoniae. In approximately one-half of the isolates, pTH126 was found together with pHD131, which mediates TEM-1 beta-lactamase production.

Treatment of chancroid. A comparison of sulphamethoxazole and trimethoprim-sulphamethoxazole.

Since sulphonamides are no longer predictably effective in the treatment of chancroid the combination of trimethoprim-sulphamethoxazole (TMP-SMX) was evaluated to identify other effective regimens. One hundred and nine patients with genital ulcers (75 men and 34 women) seen at the Special Treatment Clinic in Nairobi, Kenya, were randomly assigned to treatment with a seven day course of either sulphamethoxazole 1000 mg twice daily or trimethoprim (160 mg)-sulphamethoxazole (800 mg) (TMP-SMX) twice daily. Haemophilus ducreyi was isolated from the ulcer in 57 patients (33 men and 24 women). 16 patients were subsequently diagnosed serologically as having syphilis. No aetiological diagnosis was made in 40 patients. Treatment with sulphamethoxazole failed in five of 21 (24%) culture positive patients who were available for evaluation after seven days, whereas all 19 of such patients who were treated with TMP-SMX responded to treatment. Of the 21 isolates available for susceptibility testing, all were susceptible to trimethoprim alone (MIC less than 0.5 mg/l) and three were resistant to sulphonamides, all three containing a 4.9 megadalton (Mdal) plasmid. Two of the three patients from whom these isolates had been obtained were treated with sulphamethoxazole and both were clinical and bacteriological failures. Five of six patients with sulphonamide-susceptible H ducreyi responded to treatment with sulphamethoxazole. Failure of sulphonamides to eradicate H ducreyi in some patients with chancroid is associated with the presence of a sulphonamide resistant plasmid. In regions where this plasmid is present in H ducreyi TMP-SMX is the preferred treatment for chancroid.

Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclines limits the use of these agents for the treatment of chancroid.

Antimicrobial therapy of chancroid: an evaluation of five treatment regimens correlated with in vitro sensitivity.

One hundred fifty-one men with genital ulcer disease were assigned randomly to treatment with one of five oral antimicrobial regimens: (1) sulfadimidine (1 g four times daily for seven days); (2) tetracycline (500 mg four times daily for seven days); (3) trimethoprim-sulfamethoxazole (TMP-SMZ; 160 mg of TMP and 800 mg of SMZ twice daily for seven days); (4) doxycycline (300 mg as a single dose); or (5) TMP-sulfametrole (640 mg of TMP and 3,200 mg of sulfametrole once as a single dose). Haemophilus ducreyi was isolated from 81 (54%) of the men, and 35 strains were available for testing of antimicrobial susceptibility. The TMP-SMZ and TMP-sulfametrole regimens were more effective than sulfadimidine, tetracycline, or single-dose doxycycline in curing ulcers. Only one of 35 strains tested was susceptible to tetracycline (less than or equal to 8 mg/liter), and only ten of 35 strains were susceptible to doxycycline (less than or equal to 4 mg/liter), whereas all were susceptible to trimethoprim (less than or equal to 2 mg/liter). The correlation between in vitro susceptibility and bacteriologic response to the antimicrobial agents requires further investigation. In particular, sulfonamide resistance did not always identify failure to respond to sulfadimidine.

Single-dose therapy of chancroid with trimethoprim-sulfametrole.

We conducted a randomized double-blind trial comparing a single dose of trimethoprim-sulfametrole (640 to 3200 mg) with five-day regimens of either trimethoprim-sulfametrole (160 to 800 mg twice daily) or trimethoprim alone (200 mg twice daily) for the treatment of men with chancroid. Of 95 patients, 78 had cultures positive for Hemophilus ducreyi. Twenty-seven, 23, and 28 patients, respectively, were assigned to the single-dose trimethoprim-sulfametrole, the five-day trimethoprim-sulfametrole, and the five-day trimethoprim treatments. The rate of ulcer and bubo resolution, the mean (+/- S.D.) healing times (10.3 +/- 5.7, 11.0 +/- 7.4, and 11.9 +/- 8.2 days, respectively), the microbiologic response, the number of treatment failures, and the number of recurrent ulcers were similar in all three treatment groups. We conclude that single-dose trimethoprim-sulfametrole is a highly effective, inexpensive therapy for chancroid in men and may prove to be an important strategy for the control of H. ducreyi infection.

Antimicrobial therapy of chancroid: effectiveness of erythromycin.

The emergence of Haemophilus ducreyi resistant to multiple antibiotics has limited the effectiveness of sulfonamides and tetracycline for the therapy of chancroid. A randomized, double-blind study compared 10-day courses of erythromycin base (500 mg) and rosaramicin (250 mg) each given four times daily for the treatment of men with chancroid in Nairobi, Kenya. Of 99 evaluable patients, 84 were positive for H ducreyi. H ducreyi-positive genital ulcers in men treated with either drug resolved with mean +/- SD healing times of 10.8 +/- 5.1 days for erythromycin and 10.7 +/- 5.5 for rosaramicin. There were no clinical or bacteriologic failures with either agent. Fifteen men with H ducreyi-negative genital ulcers for whom no other etiology could be determined also responded rapidly to treatment with either agent. Both erythromycin and rosaramicin are highly effective in the treatment of chancroid.

Cefotaxime treatment of Haemophilus ducreyi infection in Kenya.

The authors conducted a double-blind randomized clinical trial comparing single-dose cefotaxime (1 g im) plus daily placebo injections with cefotaxime (1 g im on each of three days). Each regimen was given with probenicid (1 g orally) for the treatment of chancroid. Twenty Haemophilus ducreyi culture-positive men received the single-dose cefotaxime regimen; in eight patients ulcers or buboes failed to respond to therapy. Nineteen H. ducreyi culture-positive men received cefotaxime on each of three days; H. ducreyi was eradicated from all patients, but one had a continuing ulcer and another had a bubo that failed to respond. Thus cefotaxime (1 g im daily for three days) plus probenicid (1 g orally) is effective therapy for chancroid. The lack of efficacy for chancroid of the single-dose cefotaxime regimen is surprising, given the remarkable susceptibility of H. ducreyi to cefotaxime; presumably the half-life of cefotaxime is too short for predictable eradication of H. ducreyi from the ulcer with a single-dose regimen.

Genetic diversity and mosaicism at the por locus of Neisseria gonorrhoeae.

The por genes of the predominant serovars of Neisseria gonorrhoeae circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of por gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However, por mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different por sequences may be a important strategy by which N. gonorrhoeae generates por gene diversity within core group populations. Despite extensive sequence variability, por expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among por alleles may also be finite.

The clinical diagnosis of genital ulcer disease in men in the tropics.

Since the clinical diagnosis of genital ulcers without laboratory confirmation is not reliable in developed countries, we postulated that clinical diagnosis alone would be no more reliable in developing countries. A presumptive clinical diagnosis of chancroid, genital herpes, syphilis, or lymphogranuloma venereum was made for 100 male patients at the Special Treatment Clinic in Nairobi, Kenya. This diagnosis was then compared to the final diagnosis determined by laboratory identification of the pathogen, by culture, or by serologic response. In 64 patients, a final diagnosis of either chancroid, syphilis, or genital herpes was established. The diagnostic accuracy varied from 75% for chancroid to 42% for syphilis and 43% for herpes. The overall diagnostic accuracy was 66%. The predictive values of positive clinical diagnoses were 84% for chancroid, 60% for syphilis, and 75% for herpes. Thus, clinical diagnosis of genital ulcer disease was not sufficiently reliable in this study.

Treatment of chancroid by clavulanic acid with amoxycillin in patients with beta-lactamase-positive Haemophilus ducreyi infection.

Multiresistant strains of Haemophilus ducreyi, the aetiological agent of chancroid, are prevalent in Nairobi, Kenya, where tetracyclines and sulphonamides are no longer very effective in the treatment of chancroid. The following regimens (given three times daily for seven days) were compared in a double-blind randomised trial--amoxycillin 500 mg, amoxycillin 500 mg and clavulanic acid 125 mg, and amoxycillin 500 mg and clavulanic acid 250 mg. 68 of 100 ulcers were culture-positive for H. ducreyi. All strains of H. ducreyi produced beta-lactamase. At day 7 none of the amoxycillin-treated patients had responded clinically or bacteriologically, whereas all but 2 of 56 patients treated with an amoxycillin/clavulanic-acid regimen had responded clinically and H. ducreyi had been eradicated from their ulcers. The combination of amoxycillin-clavulanic acid appears to be very effective for the treatment of chancroid. The results of this study accord with H. ducreyi as the primary pathogen of chancroid.

Clinical and microbiologic studies of genital ulcers in Kenyan women.

The etiology of genital ulcers in women in tropical regions is poorly understood. Eighty-nine women, presenting to a sexually transmitted disease clinic in Nairobi (Kenya) with a primary complaint of genital ulcers, were evaluated prospectively in a clinical and laboratory study. A final etiologic diagnosis was possible for 60 (67%) of the women. Culture for Haemophilus ducreyi was positive for 43 women, eight had secondary syphilis with ulcerated condyloma latum, three had primary syphilis, one had both chancroid and syphilis, two had moniliasis, two had herpetic ulceration, and one had a traumatic ulcer. The clinical characteristics that best distinguished chancroid from secondary syphilis were ulcer excavation and a rough ulcer base. No etiologic diagnosis was established for 29 patients. However, the clinical and epidemiologic features of these patients suggested that they were similar if not identical to the patients with H. ducreyi culture-positive chancroid. Further studies are necessary to determine the etiology of ulcers in females in whom no pathogen was identified.