Diversity of the transferrin-binding protein Tbp2 of Neisseria meningitidis

In order to investigate the genetic basis for the observed polymorphism amongst meningococcal transferrin-binding proteins, Tbp2, the corresponding genes of different Neisseria meningitidis strains were cloned and sequenced. Comparison of the deduced amino acid (aa) sequences indicated that the Tbp2 were 76.6 to 81.2% homologous. Several stretches of aa have been found repeated both in the N- and C-terminal halves of the molecule.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Correlates of gonococcal infection and of antimicrobial-resistant Neisseria gonorrhoeae among female sex workers, Republic of the Philippines, 1996-1997

From 1994 to 1997, the proportion of Neisseria gonorrhoeae highly resistant to ciprofloxacin (MIC >/=4 microg/mL) increased substantially among female sex workers (FSWs) in the Philippines. Among 1499 Filipina FSWs, we evaluated factors associated with gonococcal infection and with gonococcal antimicrobial resistance. By multivariate analysis, gonococcal infection was associated with sex with a new client, self-prescribed prophylactic antimicrobial use, work in a brothel, and inconsistent condom use and was negatively associated with registration status and vaginal hygiene practices. Factors associated with ciprofloxacin-resistant gonococci included: marital status, living alone, duration of sex work, and clinic site. Further, gonococci highly resistant to ciprofloxacin were isolated from 10 (11.5%) of 87 FSWs reporting self-prescribed antimicrobial use versus 44 (3.4%) of 1295 reporting no antimicrobial use (P<.001). Self-prescribed prophylactic antimicrobial use and inconsistent condom use could be important factors in the continued emergence of gonococcal antimicrobial resistance in the Philippines.     

Prevalence and horizontal propagation of gonococcal infections among Nigerian children

The prevalence and mode of spread of gonococcal infections was studied among prepubertal children in Nigeria. Of 16 children with symptoms suggestive of sexually transmissible diseases (STD), 9 (56%) had gonorrhoea, while no causative organism was found in 7. The majority (7; 78%) of the gonococcal isolates produced penicillinase. Three of the cases were by child-to-child transmission, with female peers as the initiators. Prepubertal children should no longer be ignored as propagators of STD.     

Direct DNA probe assay for Neisseria gonorrhoeae in pharyngeal and rectal specimens

The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal (234) and pharyngeal (608) swab specimens were obtained from 249 men and 372 women attending sexually transmitted disease clinics in Las Vegas and Reno, Nevada. The prevalence of gonococcal infection by culture at the pharyngeal and rectal sites was 2.9% (16 of 548 specimens) in women and 2.7% (8 of 294 specimens) in men. No false-positive reactions were observed among the 234 rectal specimens tested. Two probe-positive, culture-negative specimens were detected among the 361 pharyngeal specimens obtained from women. Both of these samples were confirmed as Neisseria gonorrhoeae by a probe competition assay. The overall correlation of the DNA probe test with pharyngeal and rectal cultures was 99.4% (837 of 842 cultures), with a sensitivity of 87.5% (21 of 24 cultures) and specificity of 99.7% (816 of 818 cultures). The positive and negative predictive values of the DNA assay were 91.3 and 99.8%, respectively. The direct DNA probe assay provides an alternative to culture screening for rectal and/or pharyngeal gonococcal infections.     

Preparation and characterization of Neisseria meningitidis mutants deficient in production of the human lactoferrin-binding proteins LbpA and LbpB

Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285-297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.     

Studies on Neisseria gonorrhoeae strains from test-of-cure specimens. Correlation between the in vitro susceptibility to penicillin and the sensitivity to the complement-dependent bactericidal activity of normal and convalescent human serum

Sixty-seven out of 88 Neisseria gonorrhoeae strains isolated from test-of-cure (TOC) specimens during a five-months' period were included in the study. For 62 patients sufficient information was obtained in order to distinguish between relapse (34 ptt) and re-infection (28 ptt). For comparison with strains from these two groups of patients, 63 urogenital and 21 pharyngeal gonococcal strains isolated during the same period of time were randomly selected. The distributions according to susceptibility to penicillin for TOC strains and control strains corresponded to those found for the total number of TOC strains (275) and other strains (3,345) tested in 1979, respectively. The TOC strains did not differ from the control strains in sensitivity to the complement-dependent. The TOC strains did not differ from the control strains in sensitivity to the complement-dependent bactericidal activity of normal human serum. However, gonococcal strains less susceptible to penicillin in vitro (MIC values within the range 0.1-2.0 microgram/ml) were significantly more sensitive to the complement-dependent activity of normal human serum (P less than 0.01) than strains fully susceptible to penicillin (MIC less than 0.01 microgram/ml.) Penicillin-resistant strains (MIC greater than 2.0 microgram/ml) did not differ from strains susceptible to less than 0.1 microgram penicillin/ml and were slightly more serum-resistant than the less susceptible strains (P less than 0.05). No difference in serum-sensitivity of urogenital and pharyngeal isolates could be demonstrated. The level of bactericidal activity of homologous convalescent serum was unrelated to the presence of antibodies either to gonococcal pili or crude gonococcal antigen preparations. The sensitivity to normal human serum of a certain strain was not correlated with sensitivity to homologous convalescent serum.     

Emergence of in vitro resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Japan

To investigate emerging fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Japan, we compared the in vitro antimicrobial susceptibilities of 79 gonococcal isolates from 1992 through 1993 to 14 fluoroquinolones and 14 other antibiotics with those of 27 isolates from between 1981 and 1984. The MICs at which 90% of the isolates were inhibited by nine fluroquinolones, including norfloxacin, enoxacin, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, fleroxacin, levofloxacin, and sparfloxacin, for isolates from 1992 to 1993 were 8- or 16-fold higher than those for isolates from 1981 to 1984. Furthermore, the MICs at which 90% of the isolates were inhibited by five fluroquinolones, including OPC-17116, T-3761, DU-6859a, AM-1155, and Q-35, that have recently been synthesized but have not yet been introduced for clinical use in Japan for isolates from 1992 to 1993 were also 2- to 16-fold higher than those for isolates from 1981 to 1984. The gonococcal isolates from 1992 to 1993 showed no significant decreases in susceptibility to beta-lactams, tetracyclines, macrolides, and spectinomycin, compared with those for isolates from 1981 to 1984. Our data indicate that the incidence of gonococcal strains with decreased susceptibilities to fluoroquinolones is increasing in Japan.     

An evaluation of the IFA-GC procedure in a low risk population

An automated indirect fluorescent antibody (IFA) test for gonococcal antibody was evaluated in 600 low risk females; 392 were prenatal patients from public and private clinics and 208 were apparently healthy females state employees in a multiphasic screening program. Clinical histories, physical examinations, and cervical cultures were obtained on all patients. The results of the cultures and serologic test were compared. Although the number of culture positive patients in the study was low, the lack of sensitivity of the IFA test in detecting gonococcal infection in these patients was disturbing. This observation and the number of apparent false positive IFA tests lead us to conclude that the automated IFA test for detection of gonorrhea shows promise, but we could not consider adopting the test in its present stage of development.     

Comparing the efficacy of pefloxacin and ciprofloxacin in the treatment of acute uncomplicated gonococcal urethritis in males

The aim of this study was to compare the efficacy of single-dose pefloxacin 400 mg and ciprofloxacin 250 mg in the treatment of acute uncomplicated gonococcal urethritis in males. One hundred and twenty male patients with uncomplicated gonococcal urethritis were assigned alternately to receive single oral doses of either pefloxacin 400 mg or ciprofloxacin 250 mg. Forty-one out of 43 patients (95.3%) of the pefloxacin group and 46 of 47 (97.9%) of the ciprofloxacin group were cured of gonorrhoeae. The rates of post-gonococcal urethritis were 57.7% and 53.3% in the pefloxacin and ciprofloxacin groups respectively. There was a high incidence of penicillinase-producing gonococci (34.2%). High level resistance to pefloxacin (minimum inhibitory concentration [MIC] >1.0 mg/l) resulting in clinical failure on 400 mg stat dose was noted in 1 isolate. It also showed decreased susceptibility to ciprofloxacin (MIC 0.25 mg/l). Another isolate showed high-level resistance (MIC 0.06 mg/l) to ciprofloxacin 250 mg stat dose with concomitant decreased susceptibility to pefloxacin (MIC >1.0 mg/l). Ciprofloxacin 250 mg stat dose is still useful for the treatment of uncomplicated gonococcal urethritis in males. The cure rate of 95.3% with pefloxacin at 400 mg stat dose is acceptable, but needs to be monitored with caution. The emergence of a more resistant strain of Neisseria gonorrhoeae to fluoroquinolones calls for vigilance in the monitoring of antimicrobial susceptibility.     

Collaborative study of a semiautomated method for the analysis of prednisolone and prednisone tablets

A simple, adequately evaluated serologic test is not now available for use in gonorrhea screening programs. Some of the factors responsible for the relatively low sensitivity and specificity of serologic tests for gonorrhea as compared to cultural techniques are discussed. These include the time required for development of an immunologic response after infection, cross-reactivity with other antigens, and the persistence of antibody after therapy. At present, gonococcal serologic tests may be useful to the clinician in establishing the diagnosis of disseminated gonococcal infection, especially if paired sera demonstrate a change in antibody titer. Current research on the antigenic structure of the gonococcus may lead to the development of improved serologic tests for use in screening programs. However, the performance of any test may vary according to sex of the patient, presence of symptoms, history of prior gonococcal infection, and duration of the current infection. The physician must understand these factors when considering the use of any serologic test for gonorrhea.     

An outbreak of non-sexually transmitted gonococcal conjunctivitis in Central Australia and the Kimberley region

From 13 February to 27 June 1997, 447 cases of gonococcal conjunctivitis were identified by Communicable Disease and Public Health Centres and Community Clinics in the Northern Territory, Western Australia and South Australia. The outbreak involved Aboriginal communities predominantly in Central Australia and the Kimberley region in Western Australia. This was the first outbreak recorded in the Kimberley region. It is not yet known whether the Kimberley cases were part of the larger Central Australian outbreak or whether they represented a separate and unrelated outbreak. Environmental factors associated with this outbreak were similar to those seen in previous outbreaks. Control measures were based on early recognition and treatment of index cases and identifying and treating contacts. Until sexually transmitted Neisseria gonorrhoeae is controlled in communities gonococcal conjunctivitis is likely to appear again. The role of oropharyngeal carriage of N. gonorrhoeae needs to be evaluated further.     

The use of tampons for identifying asymptomatic N. gonorrhoeae infections

Female contacts of males with gonococcal urethritis were screened for asymptomatic infection using self-inserted tampons. Results of cultures were compared with specimens collected with cervical swabs during a pelvic examination. The sequence of collection of specimens was randomized, and specimens were promptly incubated. Among 297 patients, 40.4% had positive cervical specimens and 35.4% had positive tampon specimens. A difference in rates of recovery by method of collection was 5.0% +/- 1.7%. A better correlation in results was noted among patients using oral contraceptives and among patients whose tampon specimen was collected after a pelvic examination. A relatively small difference in results by method of collection suggests that tampons may serve as an inexpensive screening device for sexually active women, especially in settings where pelvic examinations are not routinely performed, but where immediate processing of specimens is possible.     

Asymptomatic sexually transmitted disease prevalence in four military populations: application of DNA amplification assays for Chlamydia and gonorrhea screening

The prevalence of asymptomatic chlamydial and gonococcal infections in male and female military populations was determined using urine-based ligase chain reaction DNA amplification assays (DAAs). Cross-sectional surveys in four military settings revealed an overall prevalence of asymptomatic chlamydial infection of 4.2% (56/1338). This included 3.4% (21/618) of Western Pacific shipboard US Marine Corps enlisted men; 5.2% (21/406) of male marines shore-based in Okinawa, Japan; 2.7% (5/183) of female enlisted US Navy subtender personnel in dry dock; and 6.9% (9/131) of shore-based female naval personnel in San Diego. No gonococcal infections were detected. All subjects were treated within 2 weeks of screening; none of them had progressed to symptomatic disease. General population-based screening for asymptomatic sexually transmitted diseases, and in particular chlamydial infection, can be successfully implemented using urine-based DAA tests. Benefits are maximized in a population in which compliance for follow-up therapy is high.     

Transrectal ultrasound in male urethritis

OBJECTIVE: To assess the prevalence of prostatic abnormalities in men with gonococcal and non-gonococcal urethritis using trans-rectal ultrasonic markers. DESIGN: A case control study of patients attending a department of genitourinary medicine with symptoms of urethritis. SETTING: Department of Genitourinary Medicine and Department of Radiology in Manchester Royal Infirmary. RESULTS: A total of 42 patients were recruited to the study: 26 with urethritis and 16 controls. Of the 26 study patients, six were gonococcal, four chlamydial, two mixed gonococcal and chlamydia and nine non specific (no organisms detected). Of the 26 study patients, 16 had abnormal scans (61.5%), eight from the chlamydia group and eight from the non specific group. No abnormalities were found in the gonococcal and mixed group. Of the 16 control patients, five had abnormal scans (31.25%), three of these have had a past history of chlamydial urethritis. CONCLUSION: The prevalence of prostatic abnormalities in patients with non-gonococcal urethritis was significantly higher when compared with controls. The cause of these abnormalities is unclear, but is compatible with inflammatory changes within the gland.     

Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae

In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected.     

Analysis of quinolone resistance mechanisms in a sparfloxacin-resistant clinical isolate of Neisseria gonorrhoeae

BACKGROUND AND OBJECTIVES: Recently, a reduction in the susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including sparfloxacin in vitro has been recognized in Japan. The quinolone resistance mechanisms in gonococcal isolates from a patient with clinical failure of sparfloxacin treatment was investigated. GOAL: To report a man with gonococcal urethritis in whom clinical failure of sparfloxacin treatment occurred and to examine the quinolone resistance mechanisms in gonococcal isolates from the patient. STUDY DESIGN: A man with gonococcal urethritis was treated with oral 100 mg sparfloxacin three times daily for 5 days. However, clinical failure of the sparfloxacin treatment was observed. The antimicrobial susceptibilities of pretreatment and posttreatment isolates to sparfloxacin and other agents were measured. To analyze quinolone resistance mechanisms in the set of isolates, DNA sequencing of the genes corresponding to the quinolone resistance-determining regions within the GyrA and ParC proteins was performed. We also assayed the intracellular sparfloxacin accumulation level in these gonococcal cells. Moreover, we performed pulsed-field gel electrophoresis analysis to determine whether the pretreatment and posttreatment isolates were isogenic. RESULTS: The minimum inhibitory concentration of sparfloxacin for the posttreatment isolate (4 micrograms/ml) was 16 times higher than that for the pretreatment isolate (0.25 microgram/ml). The pretreatment isolate contained three mutations, including a Ser-91 to Phe mutation and an Asp-95 to Asn mutation in GyrA and a Ser-88 to Pro mutation in ParC. The posttreatment isolate had four mutations, including the same three mutations and an additional Glu-91 to Gly mutation in ParC. The sparfloxacin accumulation level within 30 minutes in the posttreatment isolate was four times less than that in the pretreatment isolate. There were no differences in the pulsed-field gel electrophoresis patterns between the pretreatment and posttreatment isolates from the patient. CONCLUSIONS: The emergence of a fluoroquinolone-resistant N. gonorrhoeae isolate with multiple mutations involving GyrA and ParC reduced the response to sparfloxacin treatment. Multiple dosing and long-term treatment with sparfloxacin seems to induce a mutation in ParC and an alteration leading to reduced drug accumulation that contribute to increasing the fluoroquinolone resistance level.     

Gonorrhoea in the asymptomatic patient: presentation and the role of contact tracing for heterosexual men and women and for homosexual men

Clinically silent gonorrhoea is the major problem in the control of the disease. Only 12 per cent of infected women reported in 1974 because of symptoms, compared with 97 per cent of infected heterosexual men and only 35 per cent of homosexual men with gonococcal proctitis alone. Homosexual men, compared with heterosexual men, had twice as many subsequent sexual contacts after infection and had a higher incidence of early syphilis. Eighty-four per cent had experienced passive anorectal intercourse. Ninety-seven per cent of men with gonococcal urethritis reported because of symptoms, but occasionally (particularly after unsuccessful treatment) urethral gonorrhoea in men may be clinically silent and even require tests of the overnight urethral secretion for diagnosis. For women, and for homosexual men who have had passive anorectal (or oral) intercourse, the indication for attendance for tests for gonorrhoea should be having run the risk, and not the presence of symptoms. Routine tests of the anorectum for gonorrhoea are essential in cases of 80 women at risk, and for most homosexual men since over 80 per cent of these men will have had passive anorectal intercourse. Because gonococcal infections following treatment-failure are often clinically silent in both women and men, symptoms cannot be relied upon to indicate such failure. Follow-up smears and cultures are always essential.     

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from Tucumán, Argentina

At present, most Neisseria gonorrhoeae testing is done with beta-lactamase and agar dilution tests using common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N. gonorrhoeae is based on beta-lactamase determination and agar dilution method using common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produces results similar to the reference agar dilution method for antibiotic susceptibility of N. gonorrhoeae. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the minimum inhibitory concentration (MIC's) and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods, with a sensitivity of 89% and specificity of 91%.     

ATP radionuclide ventriculography; its usefulness for the evaluation of ischemic heart disease

The susceptibility of 45 strains of Neisseria gonorrhoea was studied measuring minimal inhibitory concentrations of five antimicrobials. Results show that 66.7% of strains are resistant to tetracycline and 11.2% to penicillin. All strains were susceptible to ciprofloxacin, ceftriaxone and cefotaxime. This results should prompt to perform surveillance in different areas of Santiago and to establish new therapeutic norms for the treatment of gonococcal infections.