Characteristics of patients infected with common Neisseria gonorrhoeae NG-MAST sequence type strains presenting at the Edinburgh genitourinary medicine clinic.

OBJECTIVE: Characterisation of population groups infected with common Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence types, presenting at the Edinburgh genitourinary medicine clinic. METHODS: All patients with gonococcal infection attending over a 2-year period were reviewed. Patients infected with unique, paired and clustered gonococcal sequence types were compared. The characteristics of patients infected with common sequence types were analysed. The concordance of gonococcal strains between sexual partners was examined. RESULTS: There were 78 unique, 17 paired and 34 clustered sequence types: the three groups varied significantly in relation to patient gender and origin/location of recent sexual contacts. There were nine large sequence type clusters (containing 11-24 isolates each) and these varied in terms of patient gender, sexual orientation and HIV prevalence. There was high concordance (94%) of sequence types between sexual contacts. CONCLUSION: There was a trend towards significance when comparing the risks of carriage/contact with HIV between different sequence type clusters. Further research is therefore warranted to determine if NG-MAST data can be used to help identify high-risk sexual networks.

Molecular epidemiology of syphilis in Scotland.

OBJECTIVE: To examine the molecular epidemiology of syphilis in Scotland. METHODS: Ulcer specimens were collected from 85 patients with infectious syphilis. Typing of Treponema pallidum was performed using a method that examines variation in two loci; the number of 60-basepair repeats within the arp gene and sequence variation in the tpr genes. RESULTS: Patients were predominately white men who have sex with men (MSM). Treponemal DNA was detected in 75 specimens and a total of six subtypes were identified from 58 typeable specimens (77%). The most common subtypes were 14d (44/58, 76%), followed by 14e (7/58, 12%), 14j (3/58, 5%), 14b (2/58, 3%), 14p and 14k (1/58, 2%). CONCLUSIONS: This study shows that subtype 14d is the predominant subtype circulating in Scotland and there is a surprising level of genetic diversity within the Scottish MSM community.

To what extent does Neisseria gonorrhoeae multiantigen sequence typing of gonococcal isolates support information derived from patient interviews?

Gonococcal isolates from genitourinary (GU) medicine clinic attendees in Glasgow, Scotland were typed using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST). Correlation between named partners (contacts) and NG-MAST type was sought and associations between specific NG-MAST types, and the social, epidemiological and geographical data were explored. We found NG-MAST typing to be a supportive and confirmatory tool for contact tracing. Specific NG-MAST types were found to be associated with distinct characteristics such as sexuality or chlamydial co-infection. An increased number of gonococcal infections were reported from those resident in deprived areas of Glasgow than from those resident in more affluent areas. However, there was no clear geographic clustering of specific NG-MAST types found within the city. Routinely observing the spread of common strains of gonorrhoea is likely best done from a larger geographical perspective unless a specific outbreak occurs.

Prediction of antibiotic resistance using Neisseria gonorrhoeae multi-antigen sequence typing.

OBJECTIVES: To establish whether antibiotic resistance in Neisseria gonorrhoeae is uniform within a given sequence type as determined by N gonorrhoeae multi-antigen sequence typing (NG-MAST). METHODS: Antibiotic susceptibility testing and typing was performed on all N gonorrhoeae isolated in Scotland over a 2-year period. Antibiotic susceptibility to seven antibiotics was determined using the agar dilution method and NG-MAST was performed. RESULTS: Isolates from 1762 episodes of infection were tested, of which 8.0% were penicillinase-producing N gonorrhoeae, 8.4% were tetracycline-resistant N gonorrhoeae, 2.7% had chromosomal penicillin resistance, 30.5% had chromosomal tetracycline resistance, 2.0% had decreased susceptibility to azithromycin and 25.3% were ciprofloxacin resistant (including 1.7% with intermediate resistance). Resistance to spectinomycin or decreased susceptibility to ceftriaxone or cefixime was not observed. Of 405 sequence types, 169 contained two to 85 isolates accounting for 1526 isolates. The overall concordance between sequence type and antibiotic susceptibility category was 98.1% (95% CI 97.8 to 98.3). The concordance for penicillin (chromosomal and plasmid-mediated resistance) was 97.1% (95% CI 96.1 to 97.8), for ciprofloxacin it was 99.5% (95% CI 99.1 to 99.8), for azithromycin it was 97.8% (95% CI 96.9 to 98.5) and for tetracycline (chromosomal and plasmid-mediated resistance) it was 92.0% (95% CI 90.5 to 93.3). CONCLUSIONS: Antibiotic resistance in N gonorrhoeae was usually uniform within a given sequence type. Therefore the sequence type of an isolate allows the presence of antibiotic resistance to be predicted with a high degree of accuracy. Further studies on the geographical variation and temporal stability of antibiotic susceptibility patterns within sequence types are required.

Opa-typing can identify epidemiologically distinct subgroups within Neisseria gonorrhoeae multi-antigen sequence type (NG-MAST) clusters.

A collection of 106 Neisseria gonorrhoeae ciprofloxacin-resistant isolates were typed using Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). Opa-typing was performed on 74 isolates which had non-unique sequence types to determine if further discrimination could be achieved and if so whether this had any epidemiological basis. The 74 isolates were separated into 12 sequence types and 20 opa-types (OT). Seven opa-type clusters were congruent with the sequence types and five sequence types could be subdivided by opa-typing. These results demonstrate that opa-typing can add a further level of discrimination compared with NG-MAST. The surveillance data for isolates in the largest sequence type cluster (ST 147) indicated that two major subdivisions OT 1 and OT 2 differed epidemiologically by patients' sexual preference and geographical location. ST 147 is a common strain that has been isolated in several countries since 1999; our results suggest that it has diverged into at least two epidemiologically discrete forms.

Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae strains: implications for changes in diagnostic strategies.

BACKGROUND: Species confirmation of Neisseria gonorrhoeae is commonly performed with biochemical kits, rely on the activity of the enzyme prolyliminopeptidase (PIP). This enzyme has previously been considered to be almost universally present in N gonorrhoeae. However, increasing numbers of N gonorrhoeae isolates lacking PIP activity have been identified. OBJECTIVES: To investigate the possibility of a widespread transmission of one or several N gonorrhoeae PIP-negative strains among several countries worldwide. METHODS: PIP-negative N gonorrhoeae isolates cultured from 2001 to 2004 in Australia, New Zealand and Scotland were comprehensively characterised and compared with previous data from England and Denmark. All isolates were characterised by antibiotic susceptibility testing, serovar determination, pulsed-field gel electrophoresis (PFGE), opa-typing, sequencing of the entire porB gene and N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Most (83%) of the viable Australian isolates, and all the New Zealand and Scottish isolates were assigned serovar IB-4, with similar antibiograms, nearly identical porB1b gene sequences, identical (ST210) or highly related (ST292, ST1259) NG-MAST STs, and indistinguishable or related PFGE fingerprints as well as opa-types. The isolates showed characteristics indistinguishable or highly related to the previously described English and Danish outbreak strain. CONCLUSIONS: A comprehensive characterisation indicates a widespread dissemination, mainly among men who have sex with men (MSM), of indistinguishable and highly related genotypes that have evolved from a single N gonorrhoeae PIP-negative serovar IB-4 strain among several countries worldwide. An increased awareness of PIP-negative N gonorrhoeae strains is crucial and changes in the diagnostic strategies may need to be considered.

Extended surveillance of gonorrhoea in Scotland 2003.

In 2003, a national surveillance of demographic, behavioural, clinical and laboratory data on gonorrhoea at genitourinary (GU) medicine clinics in Scotland was undertaken. The data-set represented 77% of all gonorrhoea cases. Findings were compared with data reported from England and Wales. Young women (16-19 years) and young men (20-24. years) represented the greatest proportion of heterosexual infections in Scotland (36 and 30%, respectively) and in England and Wales (37 and 32%, respectively). In Scotland (relative to England and Wales), men who have sex with men (MSM) accounted for more of the total gonorrhoea; there were more heterosexuals aged 45+ years; fewer belonged to ethnic minorities; fewer had had gonorrhoea previously; more heterosexual men had a sexual partner abroad; ciprofloxacin resistance was higher. During the year, first-line therapy changed from ciprofloxacin to a third-generation cephalosporin. Extended surveillance for gonorrhoea is vital in guiding appropriately targeted interventions as the epidemiology of gonorrhoea may differ in neighbouring countries.

Dramatic increase in a single genotype of TRNG ciprofloxacin-resistant Neisseria gonorrhoeae isolates in men who have sex with men.

In 2003, episodes of gonorrhoea caused by ciprofloxacin-resistant strains increased to 15.3% from 11% in 2002. This was coincident with a marked increase in strains characterized as serogroup WI, ciprofloxacin-resistant bearing the tetracycline resistance plasmid. Molecular typing of these strains, using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) revealed 71% (34/48) were of the same sequence type, ST338, accounting for 4.1% (34/824) of all strains in 2003. Epidemiological data demonstrated that transmission of ST338 was associated with men who have sex with men (MSM; 23/27), acquisition within the UK (22/26) and having two or more partners in the previous three-month period (18/27). The combined use of highly discriminatory typing and epidemiological surveillance helps to identify successful transmission networks.

The epidemiology of ciprofloxacin resistant isolates of Neisseria gonorrhoeae in Scotland 2002: a comparison of phenotypic and genotypic analysis.

OBJECTIVES: To characterise all isolates with reduced susceptibility or resistance to ciprofloxacin received by the Scottish Neisseria gonorrhoeae Reference Laboratory (SNGRL) in 2002 using N gonorrhoeae multi-antigen sequence typing (NG-MAST); to compare NG-MAST with conventional typing and to describe the epidemiology of ciprofloxacin resistant gonorrhoea in Scotland in 2002. METHODS: Isolates were characterised on receipt by auxotyping and serotyping (A/S typing), and antibiotic susceptibility testing, and retrospectively by NG-MAST. Epidemiological data were requested for all isolates in the study. RESULTS: The 106 isolates were separated into more sequence types (ST) than A/S classes (44 versus 17). All isolates within a sequence type had the same serotype, were homogeneous with respect to ciprofloxacin resistance category, but were sometimes heterogeneous with respect to auxotype or plasmid borne resistance to penicillin. Combined NG-MAST and epidemiological data revealed sustained transmission of several gonococcal strains predominantly within Greater Glasgow and Lothian. Clusters of isolates were associated with transmission within the United Kingdom, whereas isolates with unique STs were associated with foreign travel (p < 0.0001). CONCLUSIONS: NG-MAST is more discriminatory than A/S typing. Ciprofloxacin resistant gonococcal isolates in Scotland are heterogeneous, with endemic spread of some strains occurring predominantly in Greater Glasgow and Lothian.

Phenotypic and genotypic characterization of prolyliminopeptidase-negative Neisseria gonorrhoeae isolates in Denmark.

In the study presented here 26 recent Danish clinical isolates of prolyliminopeptidase (PIP)-negative Neisseria gonorrhoeae were phenotypically and genotypically characterized to investigate whether one or more PIP-negative strains are circulating in the Danish community. The profiles of these isolates were compared with those of three isolates from a recent outbreak of PIP-negative N. gonorrhoeae infection in the UK. Twenty-five of the Danish isolates and all three UK isolates had similar antibiograms and were designated serovar IB-4. Genotypic characterization by pulsed-field gel electrophoresis, porB1b gene sequencing, and opa-typing revealed that these isolates were indistinguishable or closely related. The results indicate that at least one PIP-negative N. gonorrhoeae strain is currently circulating in the Danish community, and this strain is indistinguishable from the one that caused an outbreak in the UK.

Use of PCR in the diagnosis of early syphilis in the United Kingdom.

OBJECTIVES: To evaluate a Treponema pallidum polymerase chain reaction (PCR) test in the laboratory diagnosis of early syphilis in the United Kingdom. SUBJECTS AND SETTING: Men and women attending genitourinary medicine clinics in England. METHODS: A trial PCR service was offered for the analysis of swabs of ano-genital or oral ulcers suspected to be syphilitic in origin. Clinical details, results of treponemal serology, and other relevant laboratory tests carried out by the sending laboratories were obtained retrospectively by questionnaire. RESULTS: Data from 98 patients, representing 100 episodes of ulceration, were analysed. The majority of patients (70) attended clinics in the Greater Manchester area. Eighty six patients were male and 58 were men who have sex with men (MSM), of whom 24 were HIV positive. PCR results agreed with the clinical diagnosis for 95 patients; samples from 26 patients were PCR positive and serologically diagnosed as primary (18) or secondary (8) syphilis, whereas 70 patients had PCR negative samples and were not diagnosed as having active syphilis. These data include two HIV positive patients who were PCR positive 12 and 21 days before their treponemal seroconversion. One positive PCR result was not supported by positive treponemal serology (this patient coincidentally received a 10 day course of co-amoxiclav 1 week after sampling). Three patients had negative PCR results but positive syphilis serology. The sensitivity, specificity, positive and negative predictive value for primary syphilis were 94.7%, 98.6%, 94.7%, and 98.6%, respectively, and for secondary syphilis these were 80.0%, 98.6%, 88.9%, and 97.2%, respectively. CONCLUSION: PCR is a sensitive and specific test for T pallidum, and an important adjunct to dark ground microscopy and treponemal serology in diagnosing infectious syphilis in the United Kingdom.

Evaluation of the specificities of five DNA amplification methods for the detection of Neisseria gonorrhoeae.

The intragenus specificities of five molecular diagnostic methods for Neisseria gonorrhoeae were determined. Three assays were considered suboptimal. Molecular detection of N. gonorrhoeae from sites where other Neisseria spp. commonly occur or from any site in low-prevalence settings should be confirmed by a test targeting a different genetic locus.

Investigation of an outbreak of ciprofloxacin-resistant Neisseria gonorrhoeae using a simplified opa-typing method.

Ciprofloxacin-resistant gonococci have been isolated from patients in the United Kingdom since 1993. Until recently, evidence has suggested that the majority of infections are not endemic but have been acquired abroad. In October 1999, increasing numbers of ciprofloxacin resistant isolates of the non-requiring auxotype were reported in Oldham and Rochdale (Northwest England). These and similar isolates from elsewhere in England and Wales were genetically characterized using a simplified opa-typing method (a non-radioactive PCR-RFLP method targeting the opa family of genes). Of 73 isolates studied, 24 had unique opa-types (10 from infections acquired abroad), whilst the remaining 49 were indistinguishable (none were known to be acquired abroad). This cluster included 31 isolates from Oldham and Rochdale, 16 from elsewhere in the north of England, and 2 from Southern England and South Wales with known epidemiological links to cases from Manchester and Rochdale respectively. This study illustrates the potential for spread of an antibiotic resistant clone of N. gonorrhoeae both locally and nationally and demonstrates that endemic acquisition of ciprofloxacin-resistant gonococci is now a significant problem in the United Kingdom.

Genotyping Neisseria gonorrhoeae using fluorescent amplified fragment length polymorphism analysis.

An evaluation of fluorescent amplified fragment length polymorphism (FAFLP) for typing Neisseria gonorrhoeae using 52 isolates revealed that its discriminatory power approached that of Opa-typing in identifying epidemiologically linked isolates. Automated, accurate sizing of FAFLP amplified fragments permits objective data analysis and storage, making it an attractive method for large surveillance projects.

A multiplex polymerase chain reaction to differentiate beta-lactamase plasmids of neisseria gonorrhoeae.

In penicillinase-producing Neisseria gonorrhoeae (PPNG), resistance to penicillin may be mediated by one of several related plasmids of different sizes. These include the Asian, African and Rio/Toronto plasmids. Identification of these plasmids provides useful epidemiological information, but has necessitated plasmid purification and gel analysis. We have developed a rapid, simple multiplex polymerase chain reaction (PCR) which discriminates between the beta-lactamase resistance plasmids that are frequently found in strains of N. gonorrhoeae. Amplicons of 1191, 958 and 650 bp were produced from strains containing the African, Asian and Rio/Toronto plasmids, respectively, whilst no products resulted from non-PPNG strains harbouring the cryptic, conjugative or tetracycline resistance plasmids. PCR analysis of 123 strains of PPNG identified 60 strains with African, 16 strains with Asian and 47 strains with Rio/Toronto plasmids and showed complete agreement with the standard plasmid analysis.

Neisseria meningitidis expressing transferrin binding proteins of Actinobacillus pleuropneumoniae can utilize porcine transferrin for growth.

Homologous recombination was used to generate a number of mutants of serogroup B Neisseria meningitidis B16B6 with the following characteristics: (i) an inability to bind human or porcine transferrin because of loss of both transferrin binding proteins (Tbp) A and B [strain B16B6(Str(r))/tbpA(-)B(-)] and (ii) an ability to bind porcine transferrin but not human transferrin [strain B16B6(Str(r))/tbpA(ap)B(ap)] due to replacement of the meningococcal Tbp with the Tbp of Actinobacillus pleuropneumoniae. During construction of the B16B6(Str(r))/tbpA(ap)B(ap) strain, transformants expressing only TbpA or TbpB of A. pleuropneumoniae were isolated [strains B16B6(Str(r))/tbpA(ap)B(-) and B16B6(Str(r))/tbpA(-)B(ap)]. Expression of the A. pleuropneumoniae Tbp in N. meningitidis B16B6 was iron regulated and expressed under the control of the meningococcal promoter. The relative abilities of the meningococcal transformants to bind porcine transferrin were in the order B16B6(Str(r))/tbpA(ap)B(ap) > B16B6(Str(r))/tbpA(ap)B(-) > B16B6(Str(r))/tbpA(-)B(ap). Of these transformants, only B16B6(Str(r))/tbpA(ap)B(ap) could grow in the presence of porcine transferrin as the sole iron source, achieving a growth rate similar to that of the B16B6 parent strain in the presence of human transferrin.

Diagnosis of invasive aspergillosis in bone marrow transplant recipients by polymerase chain reaction.

A nested polymerase chain reaction (PCR) test targeting Aspergillus spp. large ribosomal subunit genes was evaluated retrospectively on 175 serum samples from 37 bone marrow transplant recipients, 70% of whom received grafts from unrelated donors. Six patients had proven infection, seven had probable infection, and three had possible infection, using the revised EORTC case definitions. These 16 patients were all PCR positive (57 out of 93 samples tested). Two additional patients who did not fulfil current diagnostic criteria, but in whom invasive aspergillosis (IA) was thought clinically probable, were also PCR positive (five out of nine samples). Invasive aspergillosis was unlikely in the remaining 19 patients, four of whom were PCR positive on a single occasion (four out of 70 samples). Three samples were inhibitory to PCR. Sensitivity of PCR in diagnosing patients with IA was 100%, specificity was 79% and positive predictive value was 80%, using the criterion of a single positive result. If two positive results were required, these values were 81%, 100% and 100% respectively. The median duration of infection documented by PCR was 36 days (range 3-248 days) in 17 out of 18 patients (94%) who did not survive. Positive PCR results predated the institution of antifungal therapy in two-thirds of patients. Four patients became PCR positive during pretransplant conditioning therapy.

Differential binding of apo and holo human transferrin to meningococci and co-localisation of the transferrin-binding proteins (TbpA and TbpB).

Apo-transferrin (apo-hTf) and holo-transferrin (holo-hTf) were separately conjugated to 15-nm colloidal gold. Iron-restricted Neisseria meningitidis strain SD (B:15:P1.16) bound up to three-fold more holo-hTf than apo-hTf (p <0.001). The ability of meningococcal mutants lacking either transferrin-binding protein A (TbpA) or TbpB to discriminate between apo-hTf and holo-hTf was also investigated. There was no significant difference between the amount of gold-labelled apo-transferrin bound by the isogenic TbpA mutant (expressing TbpB) and the parent strain, whereas an isogenic TbpB mutant (expressing TbpA) bound significantly less gold-labelled apo-hTf. The isogenic TbpA and TbpB mutants and the parent strain all bound significantly more holo-hTf than apo-hTf, whereas the double 'knock-out' mutant failed to bind hTf irrespective of the iron-loading. In the isogenic mutants, TbpB was more effective in binding either apo- or holo-hTf than TbpA. Monoclonal antibodies against TbpA and TbpB were used to co-localise the transferrin-binding proteins on strain SD. The ratio of TbpA:TbpB was approximately 1:1. TbpA and TbpB were occasionally observed in close proximity to each other, but the two proteins were generally quite separate, which may indicate that they do not usually form a complex to act as a transferrin receptor.

Self-medication and memory in an elderly Canadian sample.

The ability of elderly people to self-medicate is a critical function for successful independent living. The current research investigated the predictive value of three aspects of memory potentially related to success or failure in a self-medication program. Results show that a combination of memory measures successfully discriminated between those subjects who advanced in the program and those who did not. The results of the present study provide information that will aid in improving the selection process for admission to self-medication programs.