A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene. Eur J Clin Microbiol Infect Dis

Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Clinical Medical Virology Centre, University of Queensland, Herston, Brisbane, Australia.
European Journal of Clinical Microbiology (Impact Factor: 2.67). 10/2004; 23(9):705-10. DOI: 10.1007/s10096-004-1170-0
Source: PubMed


The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition, testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor.

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    • "In case of a CT positive result, molecular genotyping, based on omp1 gene semi-nested PCR, followed by RFLP analysis was performed as previously described [5-7]. GC reactive results were confirmed by in-house PCR assay targeting porA pseudogene [8]. "
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    ABSTRACT: We evaluated LGV prevalence and predictors in a high risk population attending a STI Outpatients Clinic in the North of Italy. A total of 108 patients (99 MSM and 9 women), with a history of unsafe anal sexual intercourses, were enrolled. Anorectal swabs and urine samples were tested for Chlamydia trachomatis (CT) DNA detection by Versant CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Terrytown, USA). RFLP analysis was used for CT molecular typing. L2 CT genotype was identified in 13/108 (12%) rectal swabs. All LGV cases were from MSM, declaring high-risk sexual behaviour and complaining anorectal symptoms. Patients first attending the STI Outpatient Clinic received a significant earlier LGV diagnosis than those first seeking care from general practitioners or gastroenterologists (P = 0.0046).LGV prevalence and characteristics found in our population are in agreement with international reports. Statistical analysis showed that LGV positive patients were older (P = 0.0008) and presented more STIs (P = 0.0023) than LGV negative ones, in particular due to syphilis (P < 0.001), HIV (P < 0.001) and HBV (P = 0.001).Multivariate logistic regression analysis revealed that HIV and syphilis infections are strong risk factors for LGV presence (respectively, P = 0.001 and P = 0.010). Even if our results do not provide sufficient evidence to recommend routine screening of anorectal swabs in high-risk population, they strongly suggest to perform CT NAAT tests and genotyping on rectal specimens in presence of ulcerative proctitis in HIV and/or syphilis-positive MSM. In this context, CT DNA detection by Versant CT/GC DNA 1.0 Assay, followed by RFLP analysis for molecular typing demonstrated to be an excellent diagnostic algorithm for LGV identification.
    BMC Research Notes 04/2014; 7(1):225. DOI:10.1186/1756-0500-7-225
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    • "Recent reports have shown that other Neisseria species cross react with the targets used in the respective assays, including the opaA gene of the Abbott m2000 (Maze et al., 2011), the confirmatory porA gene assay (Whiley et al., 2004) resulting in one case out of 8 positive cases where the opaA gene was positive and the porA gene was negative and some variable has false positive results in the VERSANT® CT/GC DNA 1.0 Assay (kPCR) (Bongaerts et al., 2011). The multiplex PCR method developed in Whiley's laboratory (Goire et al., 2008) detects both the opaA and porA genes and has been used in the present study. "
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    ABSTRACT: A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.
    Journal of microbiological methods 03/2012; 89(2):148-52. DOI:10.1016/j.mimet.2012.03.002 · 2.03 Impact Factor
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    ABSTRACT: Gonorrhea is a sexually transmitted disease caused by bacteria called gonococci. The disease was described in ancient times, but until the late nineteenth century it was mixed up with syphilis. Eventually microscopy could differ between the organisms and give a reliable diagnosis. Human is the only known reservoir for gonorrhea. It is transferred during all acts of sexual activity, and an infection cause characteristic symptoms if established. Reliable diagnostic tools are important not only because gonorrhea is a severe disease but also because the means of spreading will always have a negative social impact on people given the diagnosis. Gonococci are fastidious bacteria, and survive poorly outside its human host. Thus diagnostic methods have been challenged by the problem of retrieving living bacteria for phenotypic tests. There have been several attempts to identify gonococci by specific characteristic in its genome, but these have been less successful. Due to its fluctuating genome it has been difficult to find a specific target for the gonococci. Here I present an attempt to establish a reliable diagnostic tool for reproducible detection and specific identification of gonococci in samples from different body sites.
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