Molecular epidemiology of gonorrhoea in Wales (UK).

OBJECTIVES: After a trend of increasing incidence of gonorrhoea in the 1990s, by 2004 the incidence was declining in England, but continuing to increase in Wales. This prompted an investigation of the epidemiology of gonorrhoea in Wales to inform future prevention and control measures. METHODS: As an extension to Gonococcal Resistance to Antimicrobials Surveillance Programme, between May 2005 and September 2006, 540 consecutive gonococcal isolates were collected from three microbiology laboratories in South Wales. Isolates were typed using Neisseria gonorrhoeae Multi Antigen Sequence Typing tested for susceptibility to therapeutic agents and demographic and behavioural data were collected retrospectively from patient notes. RESULTS: 163 sequence types (STs) were identified in 475 N gonorrhoeae isolates from 502 patient episodes. The most frequently observed STs (>20 isolates) were: 2, 752, 471, 249 and 8, all of which were susceptible to the antimicrobial agents tested. A significant association between ST and sexual orientation was identified, the most frequently observed STs occurring in young (median age <25 years) heterosexuals. STs 147, 4, 1634 and 64 predominated in men who have sex with men. CONCLUSIONS: We confirm the existence of common STs across the UK, as well as identify a number of types that were novel to Wales. Discrete sexual networks were identified, the most localised being in young heterosexuals. Molecular typing provides a method for identifying local clusters of gonorrhoea, and could assist in the implementation and evaluation of targeted interventions.

A comparison of two methods for the diagnosis of lymphogranuloma venereum.

A recent outbreak of lymphogranuloma venereum (LGV) within the men who have sex with men (MSM) community and their requirement for extended therapy has highlighted the need for laboratory tests that differentiate LGV- from non-LGV-associated serovars of Chlamydia trachomatis. Two previously described methods were evaluated against 495 clinical specimens referred to the Sexually Transmitted Bacteria Reference Laboratory (London, UK): (i) PCR amplification of a 1.1 kb region of the ompI gene followed by restriction enzyme digestion (ompI RFLP-PCR); and (ii) real-time PCR targeting a 36 bp deletion present within the polymorphic membrane protein H gene of LGV-associated serovars (pmpH real-time PCR). For specimens that could be categorized using both methods, a 94.7 % (390/412) concordance was achieved. Eighty-three specimens were found to be untypeable by ompI RFLP-PCR due to a failure to amplify the 1.1 kb fragment. Of these 83 untypeable specimens, 19 were determined to be an LGV-associated serovar by pmpH real-time PCR. Despite the high level of concordance, there were differences found in the technical complexity of the two methods. The pmpH real-time PCR exhibited greater sensitivity, a more rapid turnaround time and a lower technical requirement. Whilst the ompI RFLP-PCR was not as robust as a laboratory diagnostic method, it did enable serovar-level identification. LGV infection remains an important threat to the health of high-risk MSM in Europe. In conclusion, the two methods for the detection of LGV from clinical samples were found not only to have a high concordance (94.7 %) but also to be complementary, and could be used in an integrated way to aid LGV detection.

Concordance between Neisseria gonorrhoeae genotypes recovered from known sexual contacts.

Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) is a highly discriminatory molecular typing procedure that provides precise and unambiguous strain characterization. Since molecular typing can complement contact tracing for reconstructing gonorrhea sexual networks, the concordance between the NG-MAST genotypes of pairs of N. gonorrhoeae isolates from recent sexual contacts was examined. Among 72 pairs of gonococci from recent sexual contacts, the genotypes of each pair were concordant in 65 cases (90.3%). In two further pairs, the isolates from sexual contacts differed by only a single nonsynonymous substitution in the porin gene, and in both of these pairs, the isolates were the same by opa typing. The other five nonconcordant pairs of isolates were clearly different strains. opa typing data were available for 51 of the pairs of isolates from sexual contacts, and concordant opa types were obtained in 38 cases (74.5%). NG-MAST should therefore be better than opa typing at identifying recent sexual contacts and has the important advantage over opa typing of being a more precise method of strain characterization.

Confirming the Chlamydia trachomatis status of referred rectal specimens.

OBJECTIVES: To assess the reliability of different laboratory methods for the detection of Chlamydia trachomatis in rectal specimens METHODS: 1782 rectal specimens confirmed as C trachomatis positive using a standard laboratory method, were forwarded to the Sexually Transmitted Bacteria Reference Laboratory (STBRL). All specimens were retested using a C trachomatis specific independent in-house real time polymerase chain reaction (PCR). If this test was negative, a second test (Artus Real-Art PCR Kit) was employed as a confirmation. A correlation between real time PCR results obtained at the reference centre (STBRL), and the method of C trachomatis detection used in the primary laboratory was undertaken. RESULTS: The percentage of specimens that could be confirmed as positive, compared with primary method of detection was as follows: C trachomatis culture 87.5%, strand displacement assay (SDA: Becton Dickinson) 93.4%, Cobas Amplicor (Roche) 89.2%, Aptima Combo Two assay (Genprobe) 83.3%, and enzyme immunoassays (EIA) 35.4%. CONCLUSIONS: High rates of confirmation can be achieved using an independent real time PCR assay to examine rectal specimens which had initially tested C trachomatis positive using nucleic acid amplification tests and chlamydia tissue culture. This is not possible for specimens that had been screened using EIA tests, which reflects the low specificity of this test when used for rectal specimens. Laboratories currently using EIA based assays to test rectal specimens should review this approach.

Non-cultural detection and molecular genotyping of Neisseria gonorrhoeae from a piece of clothing.

Isolation of Neisseria gonorrhoeae is currently the gold standard for the definitive diagnosis of gonorrhoea and for use in medico-legal cases in the UK. Molecular detection methods are used increasingly but are untested as evidence of infection in a court of law. An isolate of N. gonorrhoeae was obtained from a child and an article of clothing from an adult male who was suspected of sexual abuse of the child. Biochemical and immunological tests were used to confirm the isolate as N. gonorrhoeae. Amplification by PCR using two targets, cppB and ompIII, was used both as further confirmation of the isolate and to detect the presence of gonococcal-specific DNA from the clothing. The relationship of the gonococcal DNA from the child and the adult was investigated using genotyping (N. gonorrhoeae multi-antigen sequence typing; NG-MAST), including a nested PCR for the por gene. Both samples were indistinguishable by NG-MAST and shared the same sequence type, 403. This is the first report of molecular detection and genotyping of N. gonorrhoeae on an article of clothing, which resulted in conviction of the man for sexual assault.

Lymphogranuloma venereum in the United kingdom.

BACKGROUND: Over the past 2 years, lymphogranuloma venereum (LGV), caused by L serovars of Chlamydia trachomatis, has emerged as a significant problem among men who have sex with men (MSM). We report on, to our knowledge, the largest case series of LGV to date, with detailed epidemiological and clinical characteristics of the epidemic in the United Kingdom. METHODS: A national diagnostic service and surveillance system was established in October 2004. Cases were confirmed by the presence of C. trachomatis and an LGV serovar (L1, L2, or L3) from genotyping. For confirmed cases, an enhanced surveillance questionnaire was sent to the clinician. RESULTS: Through February 2006, a total of 327 cases of LGV were confirmed. Cases were diagnosed across the United Kingdom, with the majority from London (71%) and Brighton (13%). Case reports were received for 282 MSM. The majority (96%) had proctitis, many with severe local and systemic symptoms. There was a high level of coinfection with human immunodeficiency virus (76%), hepatitis C (19%), and other sexually transmitted infections (39%). Nine cases of human immunodeficiency virus infection were diagnosed around the same time as LGV. Most cases were acquired within the United Kingdom, although patients with early cases were more likely to report contacts in The Netherlands. CONCLUSIONS: We found a significant burden of this once-rare sexually transmitted infection among MSM in the United Kingdom. LGV may be contributing to the epidemic of human immunodeficiency virus infection by facilitating transmission. Further control efforts are required, including awareness campaigns, continued detailed surveillance, and expanded chlamydia testing among MSM.

The molecular diagnosis of lymphogranuloma venereum: evaluation of a real-time multiplex polymerase chain reaction test using rectal and urethral specimens.

OBJECTIVES: The objectives of this study were to evaluate the use of a real-time multiplex polymerase chain reaction (M-PCR) assay to differentiate between trachoma and lymphogranuloma venereum (LGV) biovars of Chlamydia trachomatis and to validate its performance with the conventional genotyping method. STUDY: Swab specimens from 115 patients with anorectal symptoms or syndromes associated with LGV were tested by a real-time M-PCR assay and the results compared with the PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1). RESULTS: A high agreement of 96.5% (111 of 115 specimens) was found between the real-time M-PCR testing and the standard genotyping method for the detection of C. trachomatis DNA (kappa value, 0.945, P <0.00001). Both methods identified 53 LGV, 32 non-LGV C. trachomatis, and 26 negative specimens. CONCLUSIONS: The real-time M-PCR assay simultaneously detects and differentiates LGV from non-LGV strains using swab specimens. This assay offers a relatively rapid and sensitive alternative for the diagnosis of LGV infection and is a useful tool for screening and for outbreak investigations.

Lymphogranuloma venereum in Australia.

Lymphogranuloma venereum (LGV), caused by C. trachomatis serovars L1, L2 and L3, is an invasive disease capable of causing tissue destruction with many patients experiencing complex, severe symptoms. LGV, endemic to areas of Africa, Asia, South America and the Caribbean, has emerged as a cause of significant morbidity among men who have sex with men (MSM) in more affluent nations. The high prevalence of HIV in LGV cases could suggest either that LGV is confined to a dense sexual network, or that clinicians are selectively testing HIV-positive MSM for LGV The increase in reported LGV cases highlights the need to improve sexual health overall among MSM; experience from the recent syphilis outbreaks suggests that control could prove difficult.

Molecular epidemiology of recently emergent ciprofloxacin-resistant Neisseria gonorrhoeae in South Africa.

OBJECTIVES: Syndromic management guidelines for male urethritis syndrome and female discharge syndrome (nonpregnant) in South Africa advocate the use of ciprofloxacin for potential infection with Neisseria gonorrhoeae. In 2003, reports of clinical failure of gonorrhea following ciprofloxacin treatment prompted a clinic-based surveillance to detect the presence of resistant isolates. STUDY: Urethral samples for the isolation of N gonorrhoeae were obtained from consecutive male patients with urethral discharge attending the largest sexually transmitted disease clinic in KwaZuluNatal. Molecular typing of isolates was performed by means of N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Of 139 isolates, 31 (22%) were resistant to ciprofloxacin (minimum inhibitory concentration >or=1 mg/l). NG-MAST revealed novel, as well as previously described, sequence types (ST). The largest cluster of our isolates belonged to ST 217. This ST has been reported among ciprofloxacin-resistant isolates from Europe. CONCLUSION: : The results underscore the urgency of review of the current treatment guidelines for discharge disease in KwaZuluNatal.

Epidemiological correlates of asymptomatic gonorrhea.

OBJECTIVES: To assess correlates of asymptomatic gonorrhea among patients attending Genitourinary Medicine Clinics participating in the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England for 2001-2003. STUDY DESIGN: GRASP is a sentinel surveillance program that monitors antimicrobial resistance to Neisseria gonorrhoeae. Data collection occurs annually in June to August each year. RESULTS: Women with previously diagnosed gonorrhea had decreased odds of asymptomatic gonococcal infection, as did women diagnosed with other sexually transmitted infections (all except chlamydia, syphilis, herpes, and warts). Heterosexual men, but not women, coinfected with chlamydia had significantly higher likelihood of being diagnosed with asymptomatic gonorrhea, as did homosexual men coinfected with syphilis and warts. CONCLUSION: The heterogeneity in correlates of asymptomatic gonorrhea has implications for screening in clinical settings. Such findings also depend on the extent of testing on sexually transmitted infections from different sites of infection, which has particular relevance in homosexual men and would thus need to be investigated in other studies.

Changing epidemiologic profile of quinolone-resistant Neisseria gonorrhoeae in London.

The percentage of quinolone-resistant Neisseria gonorrhoeae isolated in London increased between 2000 and 2003, from 0.9% to 7.9% of total isolates. This increase was investigated by genotyping resistant isolates and comparing demographic and behavioral data. In 2000, resistant isolates predominantly had unique sequence types (STs) that were associated with imported infection, whereas, in 2002 and 2003, large ST clusters of indistinguishable isolates were associated with endemic acquisition. Resistant isolates that belonged to these large clusters were typically from patients who had similar epidemiological characteristics (such as ethnicity and sexual orientation) and behavioral characteristics (such as multiple sex partners and previous gonorrhea). In London, quinolone resistance is no longer associated with importation from areas of high prevalence and is spreading endemically in high-risk groups.

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.

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.

Typing of Neisseria gonorrhoeae reveals rapid reinfection in rural South Africa.

A recent study afforded us the opportunity to collect pre- and post-treatment isolates of Neisseria gonorrhoeae from women who supposedly failed to eradicate the organism when tested 8 to 10 days following treatment with a single, directly observed 250-mg dose of ciprofloxacin. In an attempt to differentiate true treatment failure from reinfection, we determined the ciprofloxacin MICs and performed auxotyping, serotyping, and opa typing of the pre- and post-treatment isolates. Paired isolates of N. gonorrhoeae were obtained from seven different women, despite susceptibility of the initial isolates to ciprofloxacin. Six of seven patients were infected with gonococcal isolates that differed significantly from their primary isolate. These most probably represent reinfection with a different strain, which could originate from the same partner infected with multiple strains or reinfected with a new strain or from a different partner. The susceptibility to ciprofloxacin of all isolates makes the possibility of multiple strains in the patient unlikely. The diversity of the isolates within the pairs therefore suggests rapid reinfection within the partnerships.