Epidemiologic evidence for the development of serovar-specific immunity after gonococcal infection.

We tested the hypothesis that strain-specific immunity occurs after gonococcal infection in a longitudinal study of 227 prostitutes resident in one small community who experienced frequent gonococcal infections. Women were examined and cultured for Neisseria gonorrhoeae at 2-wk intervals. Gonococcal isolates were typed according to protein 1 serovar, auxotype, and beta-lactamase plasmid type, and classified as to serovar and strain. The hypothesis was tested by comparing the predictions of the hypothesis with the observations of the study. Over the 14-mo period of the study, major changes in the prevalence of specific serovars were observed in the gonococcal population infecting these women. Women with HIV infection experienced a higher rate of gonococcal infection (0.56 +/- 0.03 vs. 0.46 +/- 0.04, P less than 0.05, t test) compared with HIV-negative women and were more likely to experience multiple infections with the same strain. The duration of prostitution was inversely related to the frequency of gonococcal infection. Women experiencing an infection with a specific gonococcal serovar were at a 2- to 10-fold reduced risk of reinfection with the same serovar, except for the 1B-1 serovar. The results of the study were consistent with all four predictions of the hypothesis. Infection with a specific gonococcal serovar results in specific but incomplete protection against subsequent infection with the homologous serovar. The mechanism of this protection remains to be determined.

Antibodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal salpingitis.

Acute salpingitis complicating cervical gonococcal infection is a significant cause of infertility. Relatively little data are available concerning the pathophysiologic mechanisms of this disease. A cohort of 243 prostitutes residing in Nairobi were followed between March 1985 and April 1988. Gonococcal cultures were performed at each visit, and acute salpingitis was diagnosed clinically. Serum at enrollment was tested by immunoblot for antibody to gonococcal outer membrane proteins. 8.6% (146/1689) of gonococcal infections were complicated by salpingitis. Increased risk of salpingitis was associated with younger age, shorter duration of prostitution, HIV infection, number of gonococcal infections, and episodes of nongonococcal salpingitis. Rmp antibody increased the risk of salpingitis. Antibody to Opa decreased the risk of salpingitis. By logistic regression analysis, antibody to Opa was independently associated with decreased risk of gonococcal salpingitis (adjusted odds ratio [OR], 0.35; 95% confidence interval [95%CI], 0.17-0.76); HIV infection (adjusted OR, 3.5; 95% CI, 0.96-12.8) and episodes of nongonococcal salpingitis (adjusted OR, 3.4; 95% CI, 1.8-6.4) were independently associated with an increased risk of salpingitis. Antibody to Opa appears to protect against ascending gonococcal infection, perhaps by interfering with Opa mediated adherence and endocytosis. The demonstration of natural immunity that protects against upper genital tract infection in women suggests that a vaccine to prevent gonococcal salpingitis is possible.

Antibody to Rmp (outer membrane protein 3) increases susceptibility to gonococcal infection.

The severe adverse effects of gonococcal infection on human fertility suggests that Neisseria gonorrhoeae would exert powerful selection for the development of a protective immune response in humans. N. gonorrhoeae is an obligate human pathogen and must persist in humans to survive. Since it is an ecologically successful organism, it must have evolved strategies to evade any human immune response it elicits. In a longitudinal study among 243 women working as prostitutes and experiencing frequent gonococcal infection, younger women, women with HIV infection, and women with antibody to the gonococcal outer membrane protein 3 (Rmp) were at increased risk of infection (adjusted odds ratio 3.4, CI95% 1.1-10.4, P < 0.05). Rmp is highly conserved in N. gonorrhoeae and the blocking of mucosal defences may be one of its functions. As similar proteins occur in many gram negative mucosal pathogens, the enhancing effect of such proteins may be a general strategy whereby bacteria evade human immune responses.

PIP: Between March 1985 and July 1986 researchers enrolled 243 female prostitutes in Pumwani community of Nairobi, Kenya, in a longitudinal study to examine the relationship between the antibody to the gonococcal outer membrane protein 3 (Rmp Ab) and gonococcal mucosal infection. Few women used condoms. 69% were HIV-1 seropositive. Just 9.5% (23) of the women had not had any gonococcal infections, despite probable exposure to them, indicating the possibility of some acquired protective immunity to Neisseria gonorrhoea. 90.5% had had at least 1 gonococcal infection. Women with Rmp Ab faced a greater risk of gonococcal infection than those who were Rmp Ab negative (OR = 3.4;l p .05), denoting that Rmp Ab increases susceptibility to gonococcal mucosal infections. Women older than 29 years were at lower risk of gonococcal infection than those younger than 29 years (odds ratio [OR] = 0.3; p .03). Women who used oral contraceptives (OCs) were also likely to be infected with N. gonorrhoea (OR = 3; p = .062). Further, 31% of OC users had cervical ectopy compared to just 14% of nonusers (OR = 2.8; p .005), suggesting that the effect of OCs on the cervix make it more susceptible to gonococcal infection. Rmp Ab also exists in many other gram-negative mucosal pathogens, often playing the same role as it does in N. gonorrhoea infection. Thus, Rmp Ab may be a common scheme bacteria used to elude human immune responses. These findings provide more understanding as to why N. gonorrhoea is an ecologically successful human pathogen.

Nasopharyngeal pneumococcal colonization among Kenyan children: antibiotic resistance, strain types and associations with human immunodeficiency virus type 1 infection.

OBJECTIVES: To compare pneumococcal nasopharyngeal colonization rates among HIV-1-infected children with those of uninfected children born to seropositive mothers and those of seronegative controls. To determine the predominant serotypes and antimicrobial susceptibility among pneumococcal isolates in Kenya. METHODS: Nasopharyngeal pneumococcal colonization was examined in 207 children recruited from the Perinatal HIV-1 Transmission Study conducted in Nairobi, Kenya. Colonization was compared among HIV-1-infected children, uninfected children born to seropositive mothers and control seronegative children. Isolates were serotyped and tested for antibiotic susceptibility to penicillin, tetracycline, erythromycin, chloramphenicol, clindamycin and rifampin. RESULTS: Colonization was higher among HIV-1-infected and uninfected children than among controls only when associated with respiratory illnesses (86% of 7 and 60% of 20 vs. 29% of 31, P = 0.004). No differences were observed when children were asymptomatic (20% of 35, 35% of 94 and 22% of 101). Intermediate penicillin resistance was found in 60% of 94 isolates, 28% were resistant to tetracycline and all isolates were susceptible to the other antibiotics tested. Sixteen serotypes were identified, with 13, 15, 14, 6B and 19F comprising 73% of isolates. Serotype 13 was found in 31% of colonized children. This serotype and 2 others isolated are not found in the current 23-valent polysaccharide vaccine. Overall 41% of colonized children harbored nonvaccine strains. CONCLUSIONS: Although nasopharyngeal pneumococcal colonization was high among children with respiratory illness born to HIV-1-seropositive mothers, increased asymptomatic colonization did not explain the increased risk of invasive pneumococcal disease associated with HIV-1 infection. Intermediate penicillin resistance was common but high level penicillin and multiple antibiotic resistance were not seen. The prevalence of the unique strains circulating in this region will need to be considered in the design of effective pneumococcal vaccines for use in East Africa.

Identification of highly conserved and species-specific polypeptides of Haemophilus ducreyi.

Chancroid is a sexually transmitted diseased caused by Haemophilus ducreyi. The pathological manifestations of chancroid are unique among Haemophilus species and the virulence factors of H. ducreyi that account for these features have not been identified. Some of these virulence factors may be unique components of H. ducreyi, but attempts to identify H. ducreyi-specific components have been unsuccessful. Four polypeptides--A, B, C and D of 83, 77, 56 and 28 kDa, respectively--were identified with a panel of nine H. ducreyi-specific monoclonal antibodies (MAbs). Polypeptide C was one of the five major proteins in H. ducreyi and demonstrated micro-heterogeneity in SDS-PAGE. Polypeptides A, B and D were present in only small amounts in whole-cell lysates of H. ducreyi. The relative amounts of A and B varied, suggesting that they may be precursor molecules. The unique polypeptides C and D were not exposed on the surface. Polypeptide C was highly soluble and did not appear to be membrane-bound, whereas polypeptide D appeared to partition with the cytoplasmic membrane and was soluble in Sarkosyl. All four polypeptides appeared to be unique to H. ducreyi since MAbs directed against them did not cross-react with H. influenzae, H. parainfluenzae or Neisseria gonorrhoeae. The mol. wts of all of these polypeptides were conserved throughout 35 clinical isolates collected from 15 cities in eight countries and one reference strain of H. ducreyi that were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Voluntary euthanasia in Northern Ireland: general practitioners' beliefs, experiences, and actions.

BACKGROUND: There has been much recent interest in the press and among the profession on the subject of euthanasia and physician-assisted suicide. The BMA recently conducted a 'consensus conference' over the internet to collect views on physician-assisted suicide. Any surveys to date have addressed a variety of specialties; however, no recent surveys have looked at general practitioner (GP) attitudes and experiences. AIM: To explore the attitudes of GPs in Northern Ireland towards the issue of patient requests for euthanasia, their nature, and doctors' experiences of such requests. METHOD: An anonymous, confidential postal survey of all (1053) GP principals in Northern Ireland. RESULTS: Seventy per cent of responders believe that passive euthanasia is both morally and ethically acceptable. Fewer (49%) would be prepared to take part in passive euthanasia. However, over 70% of physicians responding consider physician-assisted suicide and voluntary active euthanasia to be wrong. Thirty per cent of responders have received requests from patients for euthanasia in the past five years. One hundred and seven doctors gave information about these requests. Thirty-nine out of 54 patient requests for passive euthanasia had been complied with, as had one of 19 requests for physician-assisted suicide and four out of 38 patient requests for active euthanasia. Doctors perceived the main reasons why patients sought euthanasia was because of fear of loss of dignity and fear of being a burden to others. CONCLUSIONS: While the majority of GPs support passive euthanasia, they, in common with those who approve of assisted suicide and active euthanasia, often express a reluctance to take part in such actions. This may reflect the moral, legal, and emotional dilemmas doctors encounter when facing end-of-life decisions.

In-vitro activity of azithromycin, erythromycin, ciprofloxacin and norfloxacin against Neisseria gonorrhoeae, Haemophilus ducreyi, and Chlamydia trachomatis.

The minimum inhibitory concentrations (MICs) of azithromycin, erythromycin, ciprofloxacin and norfloxacin for 300 strains of Neisseria gonorrhoeae, 100 strains of Haemophilus ducreyi and six strains of Chlamydia trachomatis were determined. The two quinolones were more active against gonococcal strains than were the two macrolides. Azithromycin was approximately eight-fold more active against N. gonorrhoeae than was erythromycin (MIC90: 0.25 mg/l azithromycin, 2.0 mg/l erythromycin). The Mtr phenotype of gonococci increased azithromycin MICs approximately four fold. Azithromycin was less active than erythromycin against C. trachomatis. Azithromycin had considerable activity against H. ducreyi and was ten-fold more active than was erythromycin (MIC90: 0.004 mg/l azithromycin, 0.03 mg/l erythromycin). Clinical trials of azithromycin in the treatment of chlamydial infection and genital ulcer disease are indicated.

In-vitro activity of meropenem against Neisseria gonorrhoeae, Haemophilus influenzae and H. ducreyi from Canada and Kenya.

Meropenem, a new parenteral carbapenem demonstrated increased activity as compared to imipenem against 336 strains of Neisseria gonorrhoeae, 119 strains of Haemophilus influenzae, and 110 strains of H. ducreyi. Neither carbapenem was affected by the beta-lactamase activity of the organisms tested. Ceftriaxone and ciprofloxacin demonstrated activity superior to that of both carbapenems while the activity of ceftazidime was similar to that of meropenem.

Biochemical characteristics of Haemophilus influenzae in relationship to source of isolation and antibiotic resistance.

Based on a limited number of biochemical properties, a system for biotyping Haemophilus influenzae (M. Kilian, Acta Pathol. Microbiol, Scand. Sect. B82:835-842, 1976) was used to analyze the relationship of biotype to source of infection and antibiotic resistance for 600 clinical strains. The distribution of biotypes from bacteremic patients was significantly different (P less than 0.001) from the distribution of biotypes from nonbacteremic patients. Although there appeared to be a correlation between biotype and source of isolation, no single biotype correlated with a specific clinical syndrome in bacteremic patients. The frequency of resistance to antibiotics (ampicillin, tetracycline, chloramphenicol, and kanamycin), which was known to be at least in part plasmid mediated, was determined. Of the 600 isolates, 43 were resistant to at least one antibiotic (30 were ampicillin resistant, 11 were tetracycline resistant, 1 was ampicillin-tetracycline resistant, and 1 was tetracycline-chloramphenicol resistant). Of these 43 resistant isolates, 42 were either biotype I or II. This distribution of biotypes among antibiotic-resustant isolates was significantly different from the overall distribution of biotypes (P is less than 0.001).

Transfer of Haemophilus influenzae chromosomal genes by cell-to-cell contact.

A low-frequency exchange of chromosomal markers was observed in matings of Haemophilus influenzae. Transfer did not appear to be due to classical transformation or to be plasmid mediated, and chromosomal gene transfer differed in several respects from plasmid transfer by conjugation.

Correlation of auxotype and protein I type with expression of disease due to Neisseria gonorrhoeae.

To explore the correlation of outer membrane protein I (PI) type and auxotype to clinical expression of infection with Neisseria gonorrhoeae, we characterized nutritional requirements by auxotyping and PI serovars by coagglutination assay with monoclonal antibodies of 325 consecutive clinical isolates of N. gonorrhoeae. The clinical status of each infected individual was determined by chart review. The predominant auxotype required proline, citrulline, and uracil with or without hypoxanthine (Pro-Cit-Ura-[Hyx-]) and accounted for 22% of all gonococcal strains. Pro-Cit-Ura-(Hyx-) strains were recovered from seven of 15 men with asymptomatic urethral infections (P less than .01). Of the 325 strains, 33% were PIA and 67% were PIB serovars. All five isolates in disseminated gonococcal infection were PIA serovars (P less than .01). Three percent of 168 urethral infections in men were complicated by epididymitis, and 17% of 135 cervical infections in women were complicated by pelvic inflammatory disease (P less than .0005). Neither of these complications was associated with a particular auxotype or PI serovar.

Trimethoprim sulphamoxole in the treatment of chancroid. Comparison of two single dose treatment regimens with a five day regimen.

In a prospective blinded study, 135 men with genital ulcers culture positive for Haemophilus ducreyi, were randomized to one of three regimens. Two single dose regimens, either the combination of sulphamoxole 3200 mg/trimethoprim 640 mg or trimethoprim 700 mg alone were compared to a five day regimen of sulphamoxole 800 mg/trimethoprim 160 mg twice daily. All 31 treated with a five day regimen of trimethoprim sulphamoxole healed without further treatment. Of 27 patients treated with the single dose sulphamoxole/trimethoprim regimen, only 21 were cured and of 34 treated with trimethoprim alone, 25 responded. Antibacterial susceptibilities were performed on 31 H. ducreyi isolates. The laboratory susceptibility of these strains to trimethoprim correlated with the clinical response to the single agent. Trimethoprim alone in a dose of 700 mg or the combination of sulphamoxole (3200 mg) and trimethoprim (640 mg) is not satisfactory for the single dose treatment of genital ulcer disease. However, when prescribed for five days, sulphamoxole/trimethoprim is effective and compares favourably with other treatment regimens.

Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi.

Plasmid pLS88 from a clinical isolate of Haemophilus ducreyi encoded resistance determinants for sulfonamides and streptomycin related to those of RSF1010 and for kanamycin related to Tn903 but lacked the inverted repeats of the transposon. Its host range included Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Escherichia coli; and it was compatible with pDM2 and RSF1010.

Plasmid-mediated sulfonamide resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi from patients with chancroid were shown to have one or more 4.9- to 7.0-megadalton non-self-transferable plasmids and to have in vitro resistance to sulfonamides. Transformation of Escherichia coli to sulfonamide resistance was associated with the acquisition of a 4.9-megadalton plasmid, which did not confer linked resistance to streptomycin. The guanine-plus-cytosine content of this plasmid was found to be 57%. Filter-blot hybridization and restriction endonuclease digestion studies suggested a relationship of this plasmid to RSF1010. Electron microscope heteroduplex analysis confirmed this relationship. The identification in H. ducreyi of a plasmid closely related to plasmids found in enteric species, rather than transposition of a resistance determinant to an indigenous plasmid, suggests that further dissemination of the enteric plasmid pool to this genus is possible since plasmid transfer between certain Haemophilus species is readily demonstrated.

Molecular epidemiology of beta-lactamase-specifying plasmids of Haemophilus ducreyi.

We have studied the genetic basis of beta-lactamase production in eight strains of Haemophilus ducreyi isolated in diverse areas of the world. Beta-lactamase production in all strains was mediated by plasmids having a molecular mass of either 5.7 or 7.0 megadaltons. Plasmids of 5.7 megadaltons were shown to carry the entire sequence of pFA7, the beta-lactamase specifying plasmid found in isolates of Neisseria gonorrhoeae epidemiologically linked to West Africa. Plasmids of 7.0 megadaltons were shown to carry the entire sequence of pFA3, the beta-lactamase specifying plasmid found in Far Eastern isolates of N. gonorrhoeae. Both groups of H. ducreyi plasmids were shown to carry physically complete and functional TnA sequences. Thus we have identified two types of H. ducreyi beta-lactamase plasmid which are identical to the two types of N. gonorrhoeae beta-lactamase plasmid, except that they carry complete TnA sequences.

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.

Haemophilus influenzae: comparison of respiratory tract isolates with genitourinary tract isolates.

Haemophilus influenzae isolates recovered from the genitourinary (GU) tract were shown to have a significantly different biotype distribution compared with respiratory tract isolates. Biotype IV strains were recovered more commonly from the GU tract, and most strains were non-serotypable. Antibiotic-susceptible strains isolated from the GU tract more frequently harbored plasmids of less than 10 megadaltons than did antibiotic-susceptible respiratory tract strains. One 2.8-megadalton plasmid resident in a GU tract isolate and one 1.8-megadalton plasmid resident in a respiratory tract isolate were shown to be related to the small ampicillin resistance plasmids previously described in H. influenzae, Haemophilus parainfluenzae, Haemophilus ducreyi, and Neisseria gonorrhoeae. This supports the suggestion that these ampicillin resistance plasmids originated by transposition or recombination of the ampicillin transposon (TnA) with cryptic endogenous Haemophilus plasmids.

Antimicrobial susceptibility testing and phenotyping of Neisseria gonorrhoeae isolated from patients with ophthalmia neonatorum in Nairobi, Kenya.

Antimicrobial susceptibility testing, auxotyping-serotyping, and plasmid analysis were performed on 41 ocular isolates, 7 nasopharyngeal isolates, and 18 cervical isolates of Neisseria gonorrhoeae obtained during a recent treatment trial of gonococcal ophthalmia neonatorum in Nairobi, Kenya. Fourteen distinct serovar-auxotype patterns were observed with IB-1/Pro-strains which accounted for 59% of the isolates. Infection with multiple types of gonococci appeared to occur in 22% of the mothers since 4 of 18 paired maternal cervical and neonatal ocular isolates had mismatched serovar-auxotype patterns. Among 10 treatment failure isolates only 1 had a mismatched serovar-auxotype pattern. Six (15%) of the ocular isolates were penicillinase-producing N. gonorrhoeae (PPNG). Five had the 4.4-megadalton (Md) beta-lactamase plasmid and one had the 3.2-Md beta-lactamase plasmid. The 24.5-Md plasmid was found in 5 of 6 PPNG strains and in 8 of 35 non-PPNG strains (P less than 0.02). For most antimicrobial agents, PPNG and non-PPNG strains showed similar patterns of susceptibility. Ceftriaxone was the most active of the antibiotics tested, with all strains having an MIC less than or equal to 0.06 mg/liter. Among non-PPNG strains, 15 (43%) had a penicillin MIC greater than or equal to 2 mg/liter and were considered intrinsically resistant to penicillin. Overall, non-PPNG intrinsically resistant strains had greater resistance to other antibiotics than did non-intrinsically resistant strains (P less than or equal to 0.006). The Mtr phenotype was found in 53% of these strains.

Use of an adsorption enzyme immunoassay to evaluate the Haemophilus ducreyi specific and cross-reactive humoral immune response of humans.

Serodiagnosis of chancroid is limited by the cross-reactivity of Haemophilus ducreyi with Haemophilus influenzae and Haemophilus parainfluenzae. This research describes an adsorption enzyme immunoassay (EIA) that assesses the humoral immune response of North Americans and Africans to H. ducreyi. Adsorption effectively removed anti-H. influenzae and anti-H. parainfluenzae antibodies, revealing that North American control sera had no residual anti-H. ducreyi reactivity. However, African control sera still had a residual anti-H. ducreyi response. Assessment of the duration of the humoral immune response in sera from African patients with chancroid showed that the humoral antibodies persisted for up to 8 months after the diagnosis. This may explain the lack of specificity of the adsorption EIA in areas where chancroid is endemic. The detection of the humoral immune response was affected by the strain of H. ducreyi used, with indigent strains being most useful. Using H. ducreyi 35000 for Canadian sera, the sensitivity of the adsorption EIA was 100% and the specificity was 88%. For African sera, H. ducreyi strain R018 was used, and the adsorption EIA had a sensitivity of 81% and a specificity of only 23%. These data reveal that the existing humoral response in a country where chancroid is endemic differs from that in a country where it is not, and that care must be used interpreting unadsorbed humoral immune responses. The adsorption EIA approach may prove useful as an epidemiologic tool for definition of existing (past and present) levels of exposure to H. ducreyi.

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.