Last data update: May 13, 2024. (Total: 46773 publications since 2009)
Records 1-3 (of 3 Records) |
Query Trace: Onyekwuluje JM[original query] |
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A real-time PCR assay for HPV52 detection and viral load quantification.
Onyekwuluje JM , Steinau M , Swan DC , Unger ER . Clin Lab 2012 58 61-6 BACKGROUND: Human papillomavirus (HPV) 52 is one of the most frequent high risk HPV types found in cervical and anal infections. Reliable and well characterized methods for HPV 52 detection are therefore of great importance. Unfortunately one of the most widely used commercially available HPV typing assays, the Roche Linear Array (LA), detects type 52 only through its XR probe which cross-reacts with types 33, 35 and 58 and fails to give unambiguous detection of HPV 52. METHODS: To address this problem a real-time TaqMan PCR assay for HPV 52 targeting the E6/E7 region was developed and validated, which can be applied as robust duplex assay simultaneously detecting beta-globin as genomic control and reference or as highly sensitive single target detection assay. RESULTS: Optimized primer and probe concentrations produced linear PCR amplifications over seven logs of targets (10(1) - 10(7)). The detection limit for HPV 52 was reproducibly at 10 copies per reaction for the duplex assay format and 5 copies for the single-plex format. The assay was very type-specific and no amplification signal was observed with 10(7) copies of the related HPV33, 35, and 58 DNA. Of 89 samples that tested unambiguously positive for HPV 52 in the LA, 75 were confirmed in the duplex format and 88 in the single-plex format. An additional 100 samples negative for HPV 52 in LA were all negative in both HPV 52 real-time PCR assay formats. CONCLUSIONS: These results indicate 92.6% and 99.5% accuracy relative to LA for the duplex and single-plex formats, respectively. In ongoing testing of 18,484 from various studies, 10.8% required the HPV52 TaqMan assay to unequivocally determine the status. Including the HPV 52 duplex assay provides the ability to monitor variations in the cell yield in various methods of sample collection and processing. This additional information is useful in QC monitoring of epidemiologic studies. |
Performance of commercial reverse line blot assays for human papillomavirus genotyping.
Steinau M , Onyekwuluje JM , Scarbrough MZ , Unger ER , Dillner J , Zhou T . J Clin Microbiol 2012 50 (5) 1539-44 The performance of three line blot assays (LBA) - Linear Array HPV genotyping assay (LA; Roche Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA; Innogenetics) and the Reverse Hybridization assay (RH; Qiagen) was evaluated using quantitated whole genomic HPV plasmids (types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, 68b) as well as epidemiologic samples. In a plasmid titration series LiPA and RH did not detect 50 international units (IU) of HPV 18 in the presence of 5 x 10(4) or more IU HPV16. HPV DNA (1-6 types) in the plasmid challenges at 50 (IU) or genome equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA and 95.4% by RH with positive reproducibility of 99.8% (Kappa=0.992), 88.2% (Kappa=0.928) and 88.1% (Kappa=0.926) respectively. Two instances of mis-typing occured with LiPA. Of the 120 epidemiologic samples 76 were positive for high-risk types by LA, 90 by LiPA and 69 by RH with a positive reproducibility of 87.3% (Kappa=0.925), 83.9% (Kappa=0.899) and 90.2% (Kappa=0.942) respectively. Although all the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that the LA assay has an advantage for internationally comparable genotyping studies. |
Differences and changes of HPV16 variant status in HIV-positive adults are not uncommon
Steinau M , Swan DC , Onyekwuluje JM , Brooks JT , Vellozzi C , Unger ER , Sun Study Investigators . J Gen Virol 2010 91 2068-2072 HPV16 genotype variants have been the subject of several investigation, but study participants were rarely sampled more than ones. Among a cohort of Human immunodeficiency virus (HIV)-infected adults, we investigated HPV16 variants in samples collected concurrently from anus and cervix, as well as in serial samples collected from the same anatomical site at twelve-month intervals. We determined HPV16 variants in stored extracts of cervical and anal samples from subjects with multiple visits and at least one sample positive for HPV16. Seven polymorphic nucleotide positions within the E6 region were analyzed by pyrosequencing to determine genotype variants. Of 364 samples examined, 176 anal and 39 cervical swabs from 84 different subjects yielded unequivocal sequences of eight major HPV16 variants. Eight samples contained probable novel HPV16 variants and in 1 sample two variants were detected. In eight of 29 (27.6%) anal-cervical sample pairs positive for HPV 16, discordant variants were found. From 57 anal and 9 cervical sample series of HPV 16 positive samples a change in HPV16 variant status over time was seen in nine (15.8%) instances (7 anal, 2 cervical) from eight different participants. Changes of HPV 16 variants in HIV-infected adults was most frequently seen when different anatomic sites were sampled, but was also observed over time. |
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