A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the porA pseudogene. Eur J Clin Microbiol Infect Dis
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.
[Show abstract] [Hide abstract] ABSTRACT: Screening for extra-genital Chlamydia trachomatis and Neisseria gonorrhoeae infections is a crucial component for sexually transmitted diseases management, even if at present days no commercial methods have been approved for use on pharyngeal and rectal specimens by the US FDA or have received the conformity CE marking. Here we report the analytical sensitivities of the Versant CT/GC 1.0 assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) on rectal and pharyngeal swabs, and an evaluation about the suitability for this assay with two widely used swab collection devices (E-Swab and eNAT, Copan, Brescia, Italy). The limits of detection for rectal and pharyngeal specimens with the Versant assay were 10 copies/ml and 1.0 copies/ml, for C. trachomatis and N. gonorrhoeae, respectively. False positive results due to the presence of non-gonococcal Neisseria species were excluded when clinical rectal and pharyngeal samples containing organisms identified as N. meningitidis, N. sicca, N. flavescens and N. subflava were tested. Due to its sensitivity and specificity, the Versant assay represents a good choice for the diagnosis of chlamydial and/or gonococcal infections not only in genito-urinary samples, but also on rectal and pharyngeal swabs.0Comments 0Citations
- "A PCR assay targeting a region of 132 bp of porA pseudogene was performed for the detection of N. gonorrhoeae, using papF (5'-CGGTTTCCGTGCGTTACGA-3') and papR (5'- CTGGTTTCATCTGATTACTTTCCA-3') as primer pair [18,19]. The amplified products were visualized after electrophoresis in agarose gel by ethidium bromide staining. "
[Show abstract] [Hide abstract] 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.0Comments 3Citations
- "In case of a CT positive result, molecular genotyping, based on omp1 gene semi-nested PCR, followed by RFLP analysis was performed as previously described567. GC reactive results were confirmed by in-house PCR assay targeting porA pseudogene . "
[Show abstract] [Hide abstract] 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.0Comments 2Citations
- "These problems do not appear to be localized to one specific test but appear to be a general problem; for example the Roche Cobas Amplicor GC assay (Roche Molecular Systems, Brandenburg, NJ, USA) cross reacts with strains of N. flavescens, N. lactamica, N. sicca and N. subflava, the ProbeTec SDA GC assay (Becton Dickinson, Sparks, MD, USA) cross reacts with strains of N. subflava and N. cinerea (Whiley et al., 2008) and in-house assays using the piv ng gene (cross reacts with N. flavescens, N. lactamica, N. subflava, N. cinerea (Palmer et al., 2003)) and the cppB gene (cross reacts with N. cinerea (Farrell, 1999)). 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. "