多重PCR法和HPV分型基因芯片法在高危型人乳头瘤病毒感染检测中的应用

目的评价高危型人乳头瘤病毒（HPV）多重核酸扩增荧光检测法和HPV分型基因芯片检测法在HPV感染女性患者标本基因分型的临床应用效果。方法应用13种高危型HPV多重PCR荧光检测法对在深圳市人民医院进行宫颈癌筛查的653例疑似HPV感染女患者的宫颈细胞样本进行检测,并与HPV分型基因芯片检测法的检测结果进行比较,2种方法检测13种高危型HPV不一致的样本经序列分析方法进一步验证。结果13种高危型HPV多重PCR荧光检测法检测HPV阳性样本,阳性检出率为21．5％（140／653）;用HPV分型基因芯片检测法验证,与13种高危型HPV多重PCR荧光检测法一致的阳性样本占20．4％（133／653）,总一致率为98．2％。2种方法的检测结果具有高度一致性（kappa值一o．945）;用HPV分型基因芯片检测法检出：HPV单一型别感染占59．4％（79／133）,主要的高危HPV型别为HPV16、52、39、68、33和59型,6种高危型占总数的87．3％（69／79）,其中HPV16和HPV52为主要感染,占44．9％。结论高危型HPV多重PCR荧光检测法和HPV分型基因芯片检测法在13种高危型HPV的检结果具有高度一致性;多重PCR荧光检测法覆盖主要的13种高危型HPV,分型基因芯片检测法可进行具体的单一型别分型。2种检测方法的联合应用,对宫颈癌筛查和预防具有较高的临床应用价值,同时可为HPV分子流行病学和HPV疫苗的应用研究提供依据。     

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen''s kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.Infection with a carcinogenic type of human papillomavirus (HPV) is necessary for development of cervical cancer.^1,2 There are 13 types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) confirmed to be of high or moderate risk for cervical cancer and four types (26, 73, 66 and 82) that are probable high-risk. Other HPV types are low-risk and may be associated with genital warts, such as HPV 6 and 11. Molecular-based screening for the most oncogenic types of HPV is being used more frequently to diagnose risk for cervical cancer because it is easier and more reliable than cytological screening.³ For example, the digene HC2 HPV DNA Test (HC2) is widely used for screening⁴ because it has exceptional clinical performance for a cancer and high-grade disease end point (ie, negative predictive value). Molecular screening, however, is not designed to determine which HPV type is present in an infection. The type of HPV in an infection is important to monitor because the risk of cancer and high-grade lesions increases for those whose HPV infection type is the same over time, especially for HPV 16 and 18.⁵Because of the numerous HPV types, genotyping assays require a high level of multiplex. The development of PCR-based assays for HPV genotyping is useful to identify infections with carcinogenic HPV-types, but they have some technical limitations. Many genotyping assays involve PCR with consensus primers that amplify conserved regions of the targeted HPV types.^6,7 The efficiency of consensus-PCR is limited because primers have a variable number of nucleotide matches to the various HPV targets. An alternate approach of using multiple primer sets in a single tube is also limited due to interference among multiple primer sets. Multiplex real-time PCRs in a single reaction is also limited by the number of dyes that may be discriminated by the PCR detector instrument. Furthermore, currently described PCRs amplify a relatively small region of HPV, most in the L1 region, so they may miss HPV that has been disrupted due to viral integration.⁸ Besides integration, many point mutations in the HPV genome have been described, which may decrease the efficiency of any specific primer or probe set.⁹ The efficiency of HPV genotyping assays may also be reduced if a multitude of human DNA and nontargeted DNA is included in the reaction. This occurs for DNA isolation methods that are not specific to the targeted HPV sequences.Our HPV genotyping assay was developed to permit rapid and accurate genotyping with a high level of multiplex, while avoiding or mitigating the stated drawbacks of PCR. The assay is called Hybrid Capture (HC)-whole genome amplification (WGA)-Luminex (LMX) and is described as follows. First, there is a sequence-specific sample enrichment process to isolate single-stranded HPV DNAs while removing much of the nonspecific genomic DNA that may interfere with some assays. This sample preparation was devised from HC technology used in the HC2 Test^10,11 but requires less time and captures the RNA:DNA hybrids on magnetic beads instead of an assay plate.¹² The second, WGA step amplifies the enriched single-stranded DNA using isothermal amplification with φ-29 DNA polymerase. This polymerase is highly processive and has strand-displacement activity, which creates multiple, long linear strands of target in the presence of short random primers or where nicks occur in the DNA.^13,14 This step amplifies all sequences present, which permits a high level of multiplex. The long amplicon size allows use of several probes for simultaneous detection of multiple regions of the same HPV target. The final, multiplex detection step utilizes Luminex XMAP technology¹⁵ combined with HC signal amplification. LMX technology consists of capturing HPV target amplicons onto oligonucleotides that are fixed to fluorescent beads. For this assay, there are two specific oligonucleotide sequences per bead per HPV target, and each bead has a distinct fluorescent emission. Long complementary HPV RNA probes are bound to the denatured captured DNA creating hybrids on the LMX bead complex. The hybrids are detected using a fluorescent HC antibody (reporter). Multiple reporter antibodies are bound to each hybrid, which provides signal amplification and improves the analytical sensitivity of the assay. The bead and the reporter emissions are analyzed by a LMX flow cytometer instrument. The assay was designed to detect the 17 most common high-risk types detected in cervical cancers and the two most common low-risk types found in genital warts. The assay was tested on samples of HPV DNA plasmids and with clinical specimens. Results were compared with the widely-used clinical diagnostic assay, HC2 test, and with a PCR-based genotyping assay.     

Evaluation of the cobas Liat detection test for SARS-CoV-2 and influenza viruses following the emergence of the SARS-CoV-2 Omicron variant

ObjectivesThis study assessed the clinical performance of the cobas Liat SARS?CoV?2 & Influenza A/B assay (LiatCOVID/flu) for the detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses during the SARS-CoV-2 Omicron outbreak.MethodsResidual nasopharyngeal swab samples (NPS) previously tested with cobas SARS-CoV-2 & Influenza A/B for SARS-CoV-2 and with the Allplex Respiratory Panel 1 for influenza viruses were collected. All samples were submitted to the LiatCOVID/flu assay.ResultsA total of 1147 samples were collected comprising 167 SARS-CoV-2-positive, 556 SARS-CoV-2-negative, 224 influenza-positive, and 200 influenza-negative cases. The positive percent agreement (PPA)/negative percent agreement (NPA) of LiatCOVID/flu for SARS-CoV-2 and influenza viruses compared to the previously tested methods were 100% of 100% and 99.6% of 100%, respectively.ConclusionsThe LiatCOVID/flu assay shows an acceptable performance in the detection of SARS-CoV-2 and influenza viruses using NPS samples.     

A profile of the cobas® CT/NG assay on the cobas® 6800/8800 system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

ABSTRACT

Introduction: Chlamydia trachomatis and Neisseria gonorrhoeae infection rates continue to rise worldwide. Increasing screening of the largely asymptomatic infections is critical for timely and effective disease control. Laboratory solutions that can handle increasing volumes and generate highly accurate test results are needed in reference labs that provide the majority of chlamydia/gonorrhea testing.

Areas covered: This review will describe the Roche cobas CT/NG assay performed on the cobas 6800/8800 system. The instrument features will be described as will the performance of the assay.

Expert opinion: The high throughput cobas 6800/8800 can be integrated with clinical chemistry, hematology and immunology systems which will increase reference lab efficiency. The broad menu, ease of expansion with lab developed tests, in combination with features that support lab efficiencies and cost management while assuring high sensitivity and specificity, make this a uniquely effective diagnostic tool.     

Comparison of the analytical and clinical performances of Abbott RealTime High Risk HPV,Hybrid Capture 2, and DNA Chip assays in gynecology patients

The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18.     

A profile on cobas® EGFR Mutation Test v2 as companion diagnostic for first-line treatment of patients with non-small cell lung cancer

ABSTRACT

Introduction

Among non-small cell lung cancer (NSCLC) patients, there is one molecularly defined subgroup harboring activating mutations in the epidermal growth factor receptor gene (EGFR), which results in constitutive activation of its intrinsic kinase activity. Consistent data have demonstrated that these patients have a better outcome when treated with specific tyrosine-kinase inhibitors (EGFR-TKIs). Therefore, analysis of EGFR mutational status for treatment guidance is mandatory in this context.     

2种方法学检测对女性宫颈病变诊断的临床价值

目的研究沈阳地区女性宫颈感染高危型人乳头瘤病毒(hrHPV)状况及年龄分布情况,以及与宫颈活检病理诊断的相关性。方法该院491例18~75岁女性行第2代基因杂交捕获技术(hc2HPV DNA)和薄层液基细胞学检查(TCT)2种方法的联合检测,并对有必要进行电子阴道镜检查的患者进行病理活检,且以病理诊断为宫颈病变诊断的金标准。结果 491例女性hrHPV DNA检测阳性率为48.9%,TCT阳性率为61.9%,两者比较差异有统计学意义(P0.01)。TCT异常女性中,浸润性宫颈癌(ICC)组中位年龄(55.6岁)与不典型鳞状上皮细胞(ASC)/未明确诊断意义的不典型鳞状上皮细胞(ASCUS)、低度鳞状上皮细胞内瘤变(LSIL)、高度鳞状上皮细胞内瘤变(HSIL)组中位年龄(43.0、38.6、44.2岁)差异有统计学意义(P0.05),提示该地区ICC的高发年龄为(55.6±12.3)岁。宫颈病变中,39~49岁组(33.6%)与≤29岁组(13.8%)、49岁组(25.6%)患病率比较,差异有统计学意义(P0.05),与29~39组(27.0%)比较差异无统计学意义(P0.05)。宫颈活检病理异常者有70.1%感染hrHPV,其中慢性宫颈炎、宫颈上皮内瘤样变(CIN)Ⅰ、CINⅡ、CINⅢ、ICC患者的hrHPV感染率随着宫颈病变程度的加重而增加,分别为53.0%、77.8%、90.0%、97.6%、100.0%;hrHPV DNA病毒相对荧光值检测(RLU/CO)中位数分别为1.89、10.01、23.81、77.93、216.76,其中CINⅡ、CINⅢ、ICC组的hrHPV病毒相对荧光值与慢性炎性组的病毒相对荧光值比较,差异有统计学意义(均P0.05),与CINⅠ组比较差异无统计学意义(P0.05)。结论 hc2HPV DNA与TCT检测并结合宫颈病理学是筛查宫颈癌及癌前病变的必要手段,具有重要的临床意义。     

Comparison of 2 line blot assays for defining HPV genotypes in oral and oropharyngeal squamous cell carcinomas

Patients with invasive oral and oropharyngeal squamous cell carcinomas infected with human papillomaviruses (HPV) demonstrate improved survival. HPV detection in tumors may assist in risk stratification of patients and in guiding optimum treatment. Two reverse line blot assays [Linear Array (LA) and INNO-LiPA (LiPA)] were evaluated for detection of HPV genotypes in paraffin-embedded biopsies. Overall, 82.4% of 131 biopsies were HPV+ by LiPA versus 61.1% by LA (κ = 0.32). Completely concordant results were observed in 52.7% of cases: 18 negative and 51 with exactly the same genotype(s). An additional 13 cases had partial agreement. These 82 completely or partially concordant cases revealed a high rate of HPV positivity (78.0%), primarily involving HPV16 (90.6%). HPV+ tumors occurred preferentially in the oropharynx, especially tonsils, with trends for male patients and poor differentiation. Significant differences in these associations were found when LA and LiPA results were analyzed independently. No relationships were found between tumor HPV status and tobacco or alcohol use.     

第二代杂交捕获法与多重荧光PCR在检测高危型人乳头瘤病毒中的应用比较

目的探讨第2代杂交捕获法（HC2）和多重荧光聚合酶链反应（PCR）在女性生殖道高危型人乳头瘤病毒（HPV）中的检测性能。方法随机选择267例宫颈疾病患者，采集宫颈部位分泌物，运用美国食品药品监督管理局（FDA）认证的HC2和多重荧光PCR分别检测标本中的HPV，并将2种方法检测结果不一致的标本进行测序分析。结果HC2和多重荧光PCR检测高危型HPV的阳性率分别为33．33％和31．46％；HC2和多重荧光PCR对高危型HPV感染的诊断敏感性分别为85．88％和90．32％，特异性分别为91．21％和99．43％。结论与HC2相比，多重荧光PCR检测高危型HPV的敏感性和特异性均有提高。     

HPV病毒检测联合液基细胞学和鳞状细胞癌抗原检测在筛查宫颈癌中的价值

目的探讨HPV病毒阳性患者联合应用液基细胞学检测(TCT)、鳞状细胞癌抗原检测(SCCAg)检测在筛查宫颈癌中的价值。方法 1125例常规门诊体检HPV病毒阳性的患者,按照1:1:1比例随机分为三组(TCT组、SCC-Ag组和TCT+SCC-Ag组),所有患者行阴道镜检查并取活检,以病理诊断为金标准,计算各组筛查宫颈癌的灵敏度、特异度、阳性预测值和阴性预测者,评价各方法的诊断价值。结果 TCT联合SCC-Ag筛查宫颈癌的敏感度、特异度、阳性预测值、阴性预测者分别为0.68、0.96、0.89和0.85,与单独TCT筛查宫颈癌相比,有明显优越性,差异有统计学意义(P0.05)。结论 HPV病毒检测联合TCT和SCC-Ag明显降低筛查宫颈癌的假阳性率,提高特异度和诊断符合率,在宫颈癌筛查中有推广价值。     

高危型HPV联合TCT检测在宫颈病变Leep术后的应用价值

目的探讨高危型人乳头瘤病毒（high-risk human papilloma virus,HR-HPV）联合液基薄层细胞学检测（liquid-based thinprep cytology test,TCT）在宫颈上皮内瘤样病变（cervical intraepithelial neoplasia,CIN）行宫颈环形电切术（loop electrosurgical excision procedure,Leep）后的临床应用价值。方法回顾性对比分析528例患者,经术前HR-HPV、TCT检测并宫颈活检病理确诊≥CINⅠ,Leep术后接受HR-HPV联合TCT检测随访12-36个月。结果术前所有病例TCT≥ASCUS,术后3月、6月、12月、18月、24月、30月和36月随访TCT异常率分别为0.12%（1/528）、2.84%（15/528）、1.33%（7/528）、1.17%（6/528）、0.76%（4/528）、0.38%（2/528）、0%;HR-HPV阳性率分别为0%（未查）、26.89%（142/528）、19.32%（102/528）、12.12%（64/528）、6.06%（32/528）、3.60%（19/528）、2.46%（13/528）;术后病变持续或复发的23例,HR-HPV联合TCT双项检测病理诊断符合率为91.30%。结论 Leep术后HR-HPV联合TCT检测对评判手术效果及术后随访、预防宫颈病变复发具有较高的临床应用价值。     

A profile of the cobas® TV/ MG test for the detection of Trichomonas vaginalis and Mycoplasma genitalium

ABSTRACT

Introduction: Trichomonas vaginalis and Mycoplasma genitalium are highly prevalent sexually transmitted pathogens that may be asymptomatic or may cause cervicitis and pelvic inflammatory disease in women and urethritis in men. Our limited understanding of the epidemiology of these infections has been hampered by a lack of diagnostic capacity, but the new cobas® TV/MG assay runs on the cobas® 6800/8800 platform offers a solution to this gap in our current diagnostic capacity.

Areas covered: This article will describe what we know about the epidemiology and impact of untreated infections with these organisms as well as current recommendations for testing. The features and performance of the cobas 6800/8800 and the TV/MG assay will be described based on the emerging data related to this assay.

Expert commentary: Molecular diagnostics for trichomonas and mycoplasma that can be performed on a high-throughput system with the flexibility to order only those tests required are needed in order to reduce the burden of disease and of consequences of undiagnosed infections caused by these pathogens. As a result of the complexities in the needs for testing in different populations, sample-specific flexibility in test ordering is an absolute need in the molecular laboratory.     

健康知识护理宣教对宫颈癌早期筛查中高危型 HPV 检测及 TCT 检查结果的影响

目的 探究健康知识护理宣教对宫颈癌早期筛查中高危型 HPV 检测及 TCT 检查结果的影响。方法 选取 2019 年 6 月至 2021 年 6 月收治的在我院开展宫颈癌早期筛查的 60 例宫颈炎患者作为研究对象。采取随机数字表法将患者分为对照组和观察 组,每组各 30 例。对照组实施常规护理干预;观察组在对照组的基础上实施健康知识护理宣教。比较两组的心理状态、筛查 恐惧情绪、筛查知晓程度。结果 护理干预前,两组的 SAS、SDS 评分差异较小,无统计学意义（P>0.05）。护理干预后,两 组的 SAS、SDS 评分均显著下降（P<0.05）;且观察组的 SAS、SDS 评分均优于对照组（P<0.05）。护理干预前,两组的 FAVS 评分及筛查知晓程度无统计学差异（P>0.05）;护理干预后,两组的 FAVS 评分均显著下降（P<0.05）;且观察组的 FAVS 评 分显著低于对照组（P<0.05）。护理后,两组的筛查知晓程度均显著提高（P<0.05）;且观察组的筛查知晓程度显著高于对照 组（P<0.05）。结论 通过健康知识护理教育,可以有效降低早期宫颈癌筛查患者在检查过程中的强烈反应,缓解患者的不良情 绪,提高筛查知晓程度。     

TCT联合HPV及肿瘤标志物检测在宫颈癌中的意义

目的研究薄层液基细胞学(TCT)联合高危型人乳头瘤病毒(HPV)、鳞状细胞癌抗原(SCCA)、糖类抗原(CA125)、CA19-9检测在宫颈癌中的意义。方法将86例患者分两组,宫颈上皮内瘤变(CIN)28例,58例病理结果阳性(CINⅠ、CINⅡ、CINⅢ)、子宫颈原位癌(CIS)的患者,同时进行TCT检查及HPV DNA、SCCA、CA125、CA19-9检测。对不同临床分期、病理类型TCT、HPV DNA及肿瘤标志物阳性率进行比较。结果高度病变CINⅡ、CINⅢ及CIS HPV阳性率为94.8%,与TCT的高度病变的阳性率81.1%相比,差异有统计学意义(P<0.05)。宫颈腺癌CA125、CA19-9阳性检出率高于鳞癌,差异有统计学意义(P<0.05)。宫颈鳞癌SCCA阳性检出率高于鳞癌,差异有统计学意义(P<0.05)。结论宫颈细胞学检查和HPV检测相互结合以提高检出率,肿瘤标志物及HPV DNA阳性对患者的辅助诊断、治疗及预后具有临床意义。     

液基细胞学结合HPV高危分型检测对宫颈病变应用价值的评估

目的：评估宫颈液基细胞学结合高危性HPV DNA检测在宫颈病变中的诊断价值。方法收集本院2009-2012年细胞室及HPV检测室的资料进行评估分析。结果液基细胞学阳性检出率6.01%,HPVDNA高危型分型检出率20.34%,两种方法联合检测的评估指标：阳性检测率33%,敏感性、特异性、阳性预测值、阴性预测值88.58%。结论单独应用上述两种筛查方法都有一定的局限性,但有较高敏感性,阴性预测值及阳性检出率。联合应用检测后不但能够优势互补,而且其敏感性、阴性预测值、阳性检出率更高,但其特异性反而降低。具体原因不详,有待于在今后工作中进一步深入探讨。对已婚妇女在进行细胞学及HPV检测的同时,对HPV检测阳性病例如能采用HPV疫苗治疗,对于HPV检测阴性病例如能早期接种HPV疫苗预防,对于宫颈癌的防治效果会更加科学与完善。     

惠州地区高危型HPVE6/E7 mRNA检测在宫颈癌筛查中的临床意义

目的探讨与液基细胞学(TCT)检测结果比较,分析高危型人乳头瘤病毒(HPV)E6/E7 mRNA检测在宫颈癌筛查中的临床价值。方法选取该院妇科接受TCT检查的140例患者,根据检查结果分为5个组:炎性/良性组(32例),宫颈上皮内瘤变(CIN)Ⅰ组(44例),INⅡ组(38例),CINⅢ组(17例),宫颈癌组(9例)。比较各组HPV E6/E7mRNA检测阳性率和表达水平。结果 CIN异型程度增高,HPV E6/E7 mRNA阳性率和表达水平均呈增加趋势,不同CIN分级患者的阳性率和表达水平比较,差异均有统计学意义(P0.05);与TCT病理检查结果有较高的一致性。结论高危型HPV E6/E7 mRNA可以作为惠州地区宫颈癌筛查手段之一,联合HPV E6/E7 mRNA及TCT检测可提高其诊断准确性。