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Rapid Sequence-Based Identification of Gonococcal Transmission Clusters in a Large Metropolitan Area

In large metropolitan areas, which typically have the highest rates of gonorrhea, the identification of chains of transmission by use of partner notification is problematic, and there is an increasing interest in applying molecular approaches, which would require new discriminatory high-throughput procedures for recognizing clusters of indistinguishable gonococci, procedures that identify local chains of transmission. Sequencing of internal fragments of 2 highly polymorphic loci, from 436 isolates recovered in London during a 3-month period, identified clusters of antibiotic-resistant and antibiotic-susceptible isolates with indistinguishable genotypes, the vast majority of which were also identical or closely related by other methods, and defined groups of individuals who typically had similar demographic characteristics. This discriminatory sequence-based approach produces unambiguous data that easily can be compared via the Internet and appears to be suitable for the identification of linked cases of gonorrhea and the timely identification of transmission of antibiotic-resistant strains, even within large cities.

NEISSERIA GONORRHOEAE ACQUIRES MUTATIONS IN ANALOGOUS REGIONS OF GYRA AND PARC IN FLUOROQUINOLONE-RESISTANT ISOLATES

Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the O‐terminus of the predicted open reading frame. A series of ciprofloxacin‐resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10000‐fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high‐level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are invoived in the establishment of extreme levels of ciprofloxacin resistance.

Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics

Objectives: To validate a simplified grading scheme for Gram stained smears of vaginal fluid for the diagnosis of bacterial vaginosis (BV) against the accepted “gold” standard of Amsel’s composite criteria. Methods: Women attending genitourinary medicine (GUM) clinics, as part of a multicentre study, were diagnosed as having BV if three or more of the following criteria were present; homogeneous discharge, elevated vaginal pH, production of amines, and presence of “clue” cells. Women with less than three of the criteria were considered as normal. Simultaneously, smears were made of vaginal fluid and Gram stained and then assessed qualitatively as normal (grade I), intermediate (grade II), or consistent with BV (grade III). Two new grades were used, grade 0, epithelial cells only with no bacteria, and grade IV, Gram positive cocci only. Results: BV was diagnosed in 83/162 patient visits using the composite criteria, the remainder being regarded as normal. The majority of patients with BV had a smear assessed as grade III (80/83, 96%) and the majority of normal women had a smear assessed as grade I (normal, 48/79, 61%), giving a high sensitivity (97.5%), specificity (96%), and predictive value for a positive (94.1%) and negative (96%) test, kappa index = 0.91. Smears assessed as grade II were found predominantly (12/13) among patients diagnosed as normal, with less than three of the composite criteria. Grades 0 and IV were both only found among normal women. Conclusion: This simplified assessment of Gram stained smears can be used as an alternative to Amsel’s criteria and is more applicable for use in busy GUM clinics.

Opa‐typing: a high‐resolution tool for studying the epidemiology of gonorrhoea

A single gonococcus possesses a family of 11 distinct and highly variable opa genes. The extensive variation and rapid evolution of the opa gene repertoire has been exploited to provide a high‐resolution typing method for studies of the short‐term transmission of gonorrhoea. The 11 opa genes are amplified with a single pair of primers by the polymerase chain reaction, digested with frequently‐cutting restriction enzymes, and the fragments are fractionated on polyacrylamide to provide an opa‐type. The method appeared to be highly discriminatory as the opa‐types of gonococci, isolated world‐wide over the last 30 years, were all different. Opa‐typing discriminated between isolates of the same auxotype/serovar class. Similarly, there were 41 opa‐types among 43 consecutive isolates from a sexually transmitted disease (STD) clinic. The two pairs of isolates from this clinic that gave the same opa‐types were identical by other criteria and may have been from unsuspected sexual contacts. With one minor exception, identical opa‐types were obtained from gonococci recovered from known sexual contacts. These results suggest that variation in the family of 11 opa genes evolves so rapidly that the opa‐types of gonococci are distinguishable, unless the isolates are from sexual contacts or a short chain of disease transmission. The identification of gonococci with identical opa‐types is therefore believed to be a good indicator that the individuals from which they were recovered were sexual partners, or part of a short chain of disease transmission.

Ciprofloxacin resistance in Neisseria gonorrhoeae in England and Wales in 2002

The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) monitors trends in antimicrobial resistance in consecutive gonococcal isolates from 26 genitourinary medicine clinics in England and Wales. In 2002, 2204 gonococcal isolates were tested, and the overall prevalence of ciprofloxacin resistance (minimum inhibitory concentration > or =1 mg/L) was 9.8%, compared with 3.1% in 2001 and 2.1% in 2000. Between 2001 and 2002, prevalence of ciprofloxacin resistance increased two to three-fold, irrespective of recent sexual contact overseas, sex, or residence within or outside of London. These findings suggest that national and local treatment guidelines need to be reviewed urgently.

A prospective social and molecular investigation of gonococcal transmission

BACKGROUND Gonorrhoea is a common infectious disease, poorly controlled despite effective treatments. Tracing chains of transmission is difficult, because sexual partners are commonly difficult or impossible to identify. We assess the use of gonococcal opa-typing in identifying transmission links not revealed through interview. METHODS Epidemiological data and gonococcal isolates were collected prospectively from patients at two UK clinics in London and Sheffield. Social and epidemiological data were combined with molecular typing of gonococcal isolates by a new methodology based on the polymorphisms of the opa gene. FINDINGS In London, interview data and opa-typing on samples from 215 cases showed a diverse population with few links. In Sheffield, interview data identified links between 51 (43%) of 120 cases, whereas opa-typing suggested a more connected population: 95 (79%) of cases had shared profiles. There was a highly significant correlation between the two distributions with epidemiological clusters appearing as a subset of the opa clusters. Two large opa clusters, of 18 and 43 cases, accounted for 50% of local cases of gonorrhoea. Discordance between epidemiological and opa-typing data was observed at highly connected points in the sexual network. INTERPRETATION Opa-typing is a more powerful tool for epidemiological investigation of gonorrhoea transmission than earlier methods. Opa-typing can link infections that would otherwise remain unlinked, and may aid interventions to control endemic disease.

Whole blood model of meningococcal bacteraemia—a method for exploring host-bacterial interactions

An ex vivo whole blood model of meningococcal bacteraemia was developed to examine the total bactericidal activity of blood. Using a single defined donor and strains belonging to serogroups A, B and C and an unencapsulated strain, we demonstrated that the bactericidal mechanisms operating in whole blood varied with anticoagulant, serogroup and bacterial growth conditions. The choice of anticoagulant had a major effect on the survival of the serogroup A strain with 94% (SEM 7.6) survival in citrated blood compared to 19.7% (SEM 19.6) survival in heparinised blood after 60 min incubation. The serogroup C strain showed enhanced survival when grown in liquid medium compared to growth on solid medium (73.5%, SEM 7.5, and 8.2%, SEM 3.1, respectively, in citrated blood after 60 min). The pattern of survival of serogroup B and the unencapsulated strain were largely unaffected by these variables. Comparison with cell free conditions allowed the contribution of cellular components in meningococcal killing to be determined. Secreted levels of tumour necrosis factor and neutrophil elastase secreted during whole blood assays did not correlate with bacterial growth or viability indicating a lack of relationship between killing and activation of phagocytes.

Antimicrobial agents and gonorrhoea: therapeutic choice, resistance and susceptibility testing.

INTRODUCTION: Neisseria gonorrhoeae, the causative agent of gonorrhoea is a particularly well adapted pathogen that has continued to evolve mechanisms to evade treatment with antimicrobial agents. THERAPEUTIC CHOICE: The choice of antibiotic for use in the first-line treatment of gonorrhoea should be made with knowledge of the susceptibility of the isolates of N gonorrhoeae to be encountered. RESISTANCE: High-level resistance to penicillin and tetracycline in N gonorrhoeae is plasmid-mediated and a major therapeutic problem. Penicillinase-producing N gonorrhoeae, first described in 1976, have now spread worldwide and tetracycline-resistant N gonorrhoeae, described in 1985, are becoming increasingly prevalent. Chromosomal resistance to penicillin is low-level and affects a range of antibiotics. High-level resistance to spectinomycin has been sporadic and has not limited its use whereas the emergence of resistance to ciprofloxacin will have a significant impact on its use for gonorrhoea. SUSCEPTIBILITY TESTING: A variety of methods are available including disc diffusion, breakpoint agar dilution technique, E-test and determination of the minimum inhibitory concentration (MIC). The choice of methodology will depend on the number and type of isolates and the facilities available for testing. DISCUSSION: Surveillance programmes to monitor levels of antibiotic resistant isolates are essential to ensure therapeutic success.

Neutrophil Response to Neisseria meningitidis: Inhibition of Adhesion Molecule Expression and Phagocytosis by Recombinant Bactericidal/Permeability-Increasing Protein (rBPI21)

Polymorphonuclear neutrophil (PMNL) activation enhances microbial clearance but also contributes to the vascular damage and multiorgan failure associated with severe meningococcal sepsis. By use of a whole blood model of meningococcal bacteremia, loss of PMNL L-selectin and up-regulation of CD11b was observed in response to Neisseria meningitidis serogroups B and C, which is followed by opsonophagocytosis. PMNL priming with either Escherichia coli lipopolysaccharide (LPS) or FMLP prior to meningococcal challenge resulted in enhancement of both PMNL L-selectin shedding (1.5- to 4-fold) and phagocytosis (2- to 3-fold). Blockade of meningococcal LPS lipid A with recombinant bactericidal/permeability-increasing protein (rBPI21) resulted in partial inhibition of the PMNL activation and phagocytosis response to N. meningitidis. The effect of rBPI21 was reversed by excess E. coli LPS or FMLP. It is proposed that PMNL priming by N. meningitidis results in an exaggerated activation and phagocytosis response to the organism.

Male carriage of Gardnerella vaginalis

The prevalence of Gardnerella vaginalis in the urethra of 430 men attending a clinic for sexually transmitted disease was 11·4%; it was significantly higher in heterosexuals (14·5%) than in homosexuals (4·5%). There was no evidence of rectal or subpreputial carriage of G vaginalis, and urethral carriage was not associated with symptoms of urethritis.