高危型肛门人乳头瘤病毒（HPV）基因分型：离子激流下一代测序法与线性阵列法的比较
Genotyping of high-risk anal human papillomavirus (HPV): ion torrent-next generation sequencing vs. linear array.
作者信息
Nowak Rebecca G, Ambulos Nicholas P, Schumaker Lisa M, Mathias Trevor J, White Ruth A, Troyer Jennifer, Wells David, Charurat Manhattan E, Bentzen Søren M, Cullen Kevin J
机构信息
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA.
University of Maryland Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, 22 S. Green St. N9E17, Baltimore, MD, 21201, USA.
出版信息
Virol J. 2017 Jun 13;14(1):112. doi: 10.1186/s12985-017-0771-z.
BACKGROUND
Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it's diagnostic performance has not been tested in cases with multiple concurrent HPV infections.
METHODS
We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections.
RESULTS
In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2-91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5-96.2; sensitivity: 93.1%, 95% CI: 83.3-98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1-87.7; sensitivity: 61.1, 95% CI: 43.5-76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4-95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114).
CONCLUSIONS
The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.
背景
我们基于下一代测序（NGS）的人乳头瘤病毒（HPV）基因分型检测方法，在头颈部和宫颈癌样本中，与罗氏线性阵列（LA）检测方法显示出高度一致性，福尔马林固定石蜡包埋来源的基因组DNA用量低至1.25 ng。这种灵敏的基因分型检测方法使用适用于人群研究的带条形码的HPV PCR广谱通用引物5+/6+（BSGP）5+/6+，但其诊断性能尚未在多重并发HPV感染病例中得到测试。
方法
我们进行了一项横断面研究，以比较NGS检测方法与LA检测方法在检测HPV基因型感染方面的阳性和阴性预测值（PPV和NPV）、敏感性和特异性。DNA先前从参与TRUST/RV368队列研究的尼日利亚男男性行为者的10份肛门拭子样本中提取。使用双样本比例检验来检查NGS检测方法在检测高危与低危HPV型特异性感染方面诊断性能的差异。
结果
在10个样本中共检测到94种型特异性感染，每个样本中位数为9.5种，范围为（9至10）种。以LA作为金标准，在NGS检测方法中检测到的相同肛门型特异性感染中，84.4%（95%CI：75.2 - 91.2）也被LA检测到。与低危HPV相比，高危HPV的PPV和敏感性差异显著（PPV：90%，95%CI：79.5 - 96.2；敏感性：93.1%，95%CI：83.3 - 98.1）（低危HPV：PPV：73%，95%CI：54.1 - 87.7；敏感性：61.1，95%CI：43.5 - 76.9）（所有p < 0.05）。所有类型的NPV为92.5%（95%CI：88.4 - 95.4）。高危和低危HPV的NPV和特异性相似（所有p > 0.05）。NGS检测方法检测到10种HPV基因型，这些基因型不在LA检测到的37种基因型（30、32、43、44、74、86、87、90、91、114）之中。
结论
NGS检测方法能够在样本量有限的个体临床标本中准确检测多重HPV感染，并且与LA相比具有更广泛的覆盖范围。
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