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- Hjelmevoll, Stig Ove, et al.
(author)
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A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
- 2006
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In: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 8:5, s. 574-581
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
- Hjelmevoll, Stig Ove, et al.
(author)
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Appropriate Time for Test-of-Cure when Diagnosing Gonorrhoea with a Nucleic Acid Amplification Test
- 2012
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In: Acta Dermato-Venereologica. - Uppsala : Acta Dermato-Venereologica. - 0001-5555 .- 1651-2057. ; 92:3, s. 316-319
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Journal article (peer-reviewed)abstract
- Culture is commonly regarded as the gold standard for diagnosis of Neisseria gonorrhoeae. However, nucleic acid amplification tests (NAATs) have rapidly replaced culture for diagnostics in many settings. The aim of the present study was to investigate the appropriate time for test-of-cure (TOC) when NAATs are used for diagnosis of gonorrhoea. In total, 30 patients (28 men and 2 women) provided urethral, cervical, rectal or pharyngeal specimens for TOC. All included patients, except one who did not return for second TOC before day 19, tested negative within 2 weeks after treatment with cefixime 400 mg x 1. Antimicrobial susceptibility testing showed that 68% of the culture-positive strains were resistant to ciprofloxacin. Thus, the recommended empirical treatment with ciprofloxacin in Norway should be changed immediately. TOC can be performed 2 weeks after treatment when NAATs are used for diagnosis of gonorrhoea.
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| 3. |
- Hjelmevoll, Stig Ove, et al.
(author)
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Clinical validation of a real-time polymerase chain reaction detection of Neisseria gonorrheae porA pseudogene versus culture techniques
- 2008
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In: Sexually Transmitted Diseases. - Philadelphia : American Venereal Disease Association. - 0148-5717 .- 1537-4521. ; 35:5, s. 517-520
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Journal article (peer-reviewed)abstract
- Background: Diagnosing Neisseria gonorrheae using nucleic acid amplification tests (NAATs) might increase the sensitivity, compared to cultivation. However, using NAATs has also been problematic mainly due to the close genetic relationships between different Neisseria species, resulting in false positive diagnoses. This study was conducted to clinically validate a previously published real-time polymerase chain reaction (PCR) method targeting the porA pseudogene in N. gonorrheae in comparison to culture techniques.Methods: In total, 360 samples, urethra (n = 109), rectum (n = 84), pharynx (n = 119), and cervix (n = 48) from 185 males and 57 females, were analyzed using porA pseudogene PCR and cultivation. Sequencing of the entire porA pseudogene and the 16S rRNA gene were used to resolve discrepant results.Results: Of the 360 samples, 37 were positive by both culture and PCR, however, the PCR identified 15 additional confirmed positive samples. The PCR method showed a sensitivity, specificity, positive predictive value, and negative predictive value of 100% in a preselected population. The preselected population had a true gonorrhea prevalence of 17.4%.Conclusions: 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.
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| 4. |
- Unemo, Magnus, et al.
(author)
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Neisseria gonorrhoeae Strain with High-Level Resistance to Spectinomycin Due to a Novel Resistance Mechanism (Mutated Ribosomal Protein S5) Verified in Norway
- 2013
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 57:2, s. 1057-1061
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Journal article (peer-reviewed)abstract
- 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 mu 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.
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