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.

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.

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.

Radioimmunoassay of plasma vasopressin in physiological and pathological states in man.

A radioimmunoassay method for the measurement of arginine-vasopressin (AVP) in human plasma has been developed which requires 5 ml of plasma and has a lower limit of detection of 1-8 pg/ml plasma. Arginine-vasopressin was found to be stable in whole blood for up to 1 h at room temperature and for at least 4 h at 4 degrees C, while in plasma stored at -20 degrees C no loss was seen over 10 days. Dehydration and rehydration in normal subjects produced appropriate changes in AVP concentration but there was considerable variability in the levels attained by individual subjects and no obvious correlation with plasma osmolality. No consistent increase in plasma AVP concentration was seen on change of posture from the recumbent to the upright position. Vigorous exercise produced a marked rise in plasma AVP concentrations in most subjects which could not be attributed simply to an increase in plasma osmolality. In fusion studies with Pitressin in normal subjects showed a mean half-life of 6-4 min with an overall plasma clearance rate of 8-5 ml/min/kg body weight and a mean volume of distribution of 5-33 l. In patients with a biochemical picture suggestive of inappropriate antidiuretic hormone secretion, markedly raised plasma AVP concentrations were found only in patients with bronchial carcinoma.

Detection of Chlamydia trachomatis by the polymerase chain reaction in swabs and urine from men with non-gonococcal urethritis.

A polymerase chain reaction (PCR) was developed for Chlamydia trachomatis in which a 380 base pair DNA fragment was amplified. Amplification occurred with the DNA from the 15 serovars but not with that from other Chlamydia spp or with DNA from a variety of other organisms. Chlamydial DNA (10(-16) g) could be detected and the PCR seemed to be able to detect single organisms. Urethral swabs were obtained from 37 men with acute non-gonococcal urethritis (NGU), 18 (49%) of whom were positive for C trachomatis by MicroTrak. As a result of clinical re-examinations 65 urethral swabs were available for analysis by the PCR. In comparison with MicroTrak, PCR had a sensitivity of 95%, a specificity of 94%, a positive predictive value of 86% and a negative predictive value of 98%. The PCR was apparently less sensitive (82%) in tests on urine samples. Overall, however, values of sensitivity and specificity of the PCR compared favourably with those of MicroTrak. The PCR for C trachomatis is likely to be a valuable technique for research, but problems of DNA contamination suggest that it should not be recommended for routine diagnosis.

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: twin N-terminal PCR.

The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae.

Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium.

The polymerase chain reaction (PCR) was developed to detect Mycoplasma genitalium. Oligonucleotide primers were used to amplify a 374 bp region of the attachment protein of the mycoplasma. DNA from three strains of M. genitalium tested gave a characteristic PCR product which was not seen with DNA from any other source. As little as 10(-15) g of M. genitalium DNA could be detected and it was found in the vagina of progesterone-treated BALB/c mice inoculated with M. genitalium organisms later than they could be cultured from this site, but not in mice that never became colonised vaginally.

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin.

A gene library of Neisseria meningitidis B15 P1.16 DNA was established in lambda Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of tbp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae, and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.

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.

Acute intravascular hemolysis with minimal renal impairment in Clostridium perfringens infection.

We report here a case of post-abortal clostridium prefringens infection in which there was severe intravascular hemolysis with black urine, but only minor abnormalities of the clotting mechanism and mild renal failure. The patient recovered following supportive therapy only.

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.

Detection of Mycoplasma genitalium in the genitourinary tract of women by the polymerase chain reaction.

A polymerase chain reaction (PCR) was used to demonstrate the presence or absence of Mycoplasma genitalium in the lower genital tract of 57 women who attended a sexually transmitted diseases clinic. The mycoplasma was detected in the cervix of 10 (17.5%) women and also in the vagina of 4 (16%) and the urethra of 6 (24%) of 25 women from whom multiple samples were obtained. Chlamydia trachomatis was detected also by a PCR in 9 (16%) of the women, but only 3 were chlamydia-positive and mycoplasma-positive. M. genitalium was detected occasionally in women with vaginal disease (for example, bacterial vaginosis), whereas C. trachomatis was not, but whether there is any causal relationship between the mycoplasma and vaginal or cervical disease requires further study.

A reappraisal of chlamydial and nonchlamydial acute non-gonococcal urethritis.

A cohort of 112 men presenting with acute non-gonococcal urethritis (NGU) was investigated for the presence of Chlamydia trachomatis. Men with 3 or more episodes of NGU in the preceding 12 months, or who had received treatment for NGU in the preceding 3 months were excluded. C. trachomatis was sought by examination of urethral smears by direct immunofluorescence, and by examination of the centrifuged deposit from a first pass urine (FPU) sample by direct immunofluorescence, IDEIA, and the polymerase chain reaction. Urethral samples from 48 men were positive for CT, and the FPU samples from an additional 7 men were positive by at least 2 assays. With such intensive investigation it is likely that those men identified as chlamydia-negative were genuinely free from the infection. The clinical history and response to treatment of those men who were chlamydia-positive were compared with those of the chlamydia-negative men. They differed in that a larger proportion of the chlamydia-positive men reported having had intercourse with more than one partner in the previous 3 months, and having had fewer previous episodes of NGU. Moreover, in contrast to some previous studies, after one week of treatment with doxycycline, a larger proportion (65%) of the chlamydia-negative men than the chlamydia-positive men (40%) was cured, although the difference was not sustained following later treatment.