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Molecular detection ofNeisseria gonorrhoeae causes the second most prevalent bacterial sexually transmitted infection (STI) in men and women globally (42). As clinical signs of gonococcal infection may overlap with those of other STIs, laboratory testing is crucial for appropriate diagnosis and subsequent adequate treatment. The accurate diagnosis of gonorrhea relies on laboratory tests that are sensitive, specific, reproducible, and robust because of nonspecific clinical manifestations or lack of symptoms in women as well as men (43). Nucleic acid amplification tests (NAATs) have a number of recognized advantages over other diagnostic assays, which include increased sensitivity of detection and ease of sample collection (including use of self-collected samples) and transport, compared to culture-based methods (13,26,27,31,32). A number of commercial NAAT systems and assays developed "in-house" are currently in use for the detection of urogenital gonococcal infection, and their use has seen many of the anticipated benefits of NAATs. However, with increasing use of commercial and "in-house" NAAT systems over time and in different geographical settings, the need for both a considered approach to the application of NAATs and for an awareness of their limitations has arisen. These considerations include the sensitivity and specificity of the primary NAAT screening test, and where used, supplementary "confirmatory" tests as well as the prevalence of infection in various population groups (18). A number of assays have been shown to cross-react with other Neisseria species (10,20,23,29,36). The Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia) and the Australian Public Health Laboratory Network have proposed a number of testing algorithms for confirmation of STIs by NAATs, which require the use of additional or supplementary assays (12,28,43). There is increasing evidence for the rise in infection in extragenital sites, which include the rectum and oropharynx, particularly in population groups of men who have sex with men (38). Commercial NAAT systems currently on the market have not been cleared by the FDA for diagnosis of specimens from the rectum, pharynx, or conjunctiva; however, as detection of N. gonorrhoeae by NAAT is more sensitive than culture (43), laboratories continue to offer these tests to diagnose extragenital gonorrhea (4). Extragenital sites carry a number of commensal Neisseria species and commonly Neisseria meningitidis, which due to having a high nucleic acid homology to N. gonorrhoeae may cross-react in the NAAT assay utilized (9). As crossreactions occur in screening assays, steps to include at least one

Sundsfjord A, Simonsen GS, Haldorsen BC, Haaheim H, Hjelmevoll SO, Littauer P, Dahl KH. Genetic methods for detection of antimicrobial resistance. APMIS 2004;112:815-37.Accurate and rapid diagnostic methods are needed to guide antimicrobial therapy and infection control interventions. Advances in real-time PCR have provided a user-friendly, rapid and reproducible testing platform catalysing an increased use of genetic assays as part of a wider strategy to minimize the development and spread of antimicrobial-resistant bacteria. In this review we outline the principal features of genetic assays in the detection of antimicrobial resistance, their advantages and limitations, and discuss specific applications in the detection of methicillin-resistant Staphylococcus aureus, glycopeptide-resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad-spectrum resistance to b-lactam antibiotics in gram-negative bacteria, as well as genetic elements involved in the assembly and spread of antimicrobial resistance.

Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n ‫؍‬ 34) and N. gonorrhoeae clinical isolates (n ‫؍‬ 176) but not isolates of the 13 different nongonococcal Neisseria species (n ‫؍‬ 68) that we tested. Furthermore, a panel of gram-negative bacterial (n ‫؍‬ 18), gram-positive bacterial (n ‫؍‬ 23), fungal (n ‫؍‬ 1), and viral (n ‫؍‬ 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/ PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.

Gonorrhea may become untreatable, and new treatment options are essential. Verified resistance to spectinomycin is exceedingly rare. However, we describe a high-level spectinomycin-resistant (MIC, >1,024 g/ml) Neisseria gonorrhoeae strain from Norway with a novel resistance mechanism. The resistance determinant was a deletion of codon 27 (valine) and a K28E alteration in the ribosomal protein 5S. The traditional spectinomycin resistance gene (16S rRNA) was wild type. Despite this exceedingly rare finding, spectinomycin available for treatment of ceftriaxone-resistant urogenital gonorrhea would be very valuable.

Background  The prevalence of Mycoplasma genitalium and Ureaplasma genitalium in populations outside sexually transmitted infection clinics in Norway is unknown. Objective  To assess the prevalence of potential sexually transmitted organisms in a non‐clinical setting, among college students in Northern Norway. Methods  In total 655 students, 449 men and 206 women, were tested for Chlamydia trachomatis, M. genitalium, and U. urealyticum by nucleic acid amplification testing of urine samples. All subjects completed questionnaires. Results  Among the included men, the prevalences of C. trachomatis, M. genitalium, and U. urealyticum were 4.2%, 1.1% and 8.9%, respectively. Prevalence among included women was 1.9%, 1% and 8.2%, respectively. In men, the number of sexual partners in the preceding 6 months was associated with prevalence of U. urealyticum and C. trachomatis. Conclusions  U. urealyticum appeared more prevalent than C. trachomatis and increased number of sexual partners was associated with increased risk of a positive test. M. genitalium had a low prevalence.

The present porA pseudogene real-time PCR comprises a valuable supplement to the traditional culture techniques for diagnosis of N. gonorrheae, especially for samples from extragenital sites such as pharynx and rectum.

BackgroundThere has been an increasing number of diagnosed cases of Chlamydia trachomatis in many countries, in particular among young people. The present study was based on a growing request to examine urine as a supplementary or primary specimen in screening for Chlamydia trachomatis in women, with the Becton Dickinson ProbeTec (BDPT) Strand Displacement Assay (SDA). Urine samples may be particularly important in screening young people who are asymptomatic.MethodsA total of 603 women aged 15 and older were enrolled from the Sexually Transmitted Infection (STI) clinic at Haukeland University Hospital, Norway, in 2007. Only 31 women were older than 35 years. Cervical swabs and urine samples were tested with BDPT for all participants. In cases of discrepant test results from a given patient, both samples were retested by Cobas TaqManCT and a Polymerase Chain Reaction (PCR)-method (in-house). Prevalence of C. trachomatis, sensitivity, and specificity were estimated by latent class analysis using all test results available. Bootstrap BC confidence intervals (10 000 computations) were estimated for sensitivity and specificity, and their differences in cervix vs. urine tests.ResultsA total of 1809 specimens were collected from 603 patients. 80 women (13.4%) were positive for C. trachomatis. Among these, BDPT identified 72 and 73 as positive in cervix and urine samples, respectively. Of the 523 C. trachomatis negative women, BDPT identified 519 as negative based on cervical swabs, and 514 based on urine samples. Sensitivity for cervical swabs and urine samples with the BDPT were 89.0% (95% CI 78.8, 98.6) and 90.2% (95% CI 78.1, 95.5), respectively. The corresponding values for specificity were 99.2% (95% CI 98.3, 100) and 98.3% (95% CI 96.4, 100).ConclusionsThis study indicates that urine specimens are adequate for screening high-risk groups for C. trachomatis by the SDA method (BDPT). Such an approach may facilitate early detection and treatment of the target groups for screening, and be cost-effective for patients and the health services.