双色荧光定量PCR快速分型、定量高危型人乳头状瘤病毒

目的针对高危型人乳头状瘤病毒（humanpapillomavims，HPV）亚型的多样性以及感染病毒载量的高低，旨在建立高危型HPV的定量与分型的快速检测方法，为HPV筛查与治疗提供依据。方法利用2种荧光染料分别标记HPV-16、HPV-33／52／58／67和HPV-18／45、HPV-31探针，同时以B球蛋白基因作为内对照，对120份疑为HPV感染的宫颈脱落细胞样本进行I{I＇V分型与定量，定量范围为5×10^1-5×10^7拷贝/ml，以HC-2杂交捕获法作为“金标准”，评价该方法的特异性与灵敏度。结果荧光定量PCR阳性检出率为52．5％（631120），其中HPV-16、HPV-18／45、HPV-31和m．V-33，52，58，67阳性率分别为36．51％（23／63）、11．11％（7／63）、12．70％（8／63）、58．73％（37／63）；HPV感染的平均病毒载量为1．08×10^7拷贝/ml。荧光定量PCR的特异性为100％，灵敏度为81．82％。结论双色荧光定量PCR能快速分型、定量高危型HPV，可用于HPV感染的筛查与宫颈病变程度预测以及疗效观察。     

浙江沿海地区人群TTV和HPV混合感染研究

目的了解浙江沿海地区人群TTV与HPV混合感染状况,探讨TTV传播途径。方法建立巢式聚合酶链反应方法（nPCR）,对健康体检和患宫颈疾病妇女150例宫颈病变细胞学标本及其平行97例血清标本进行TTVDNA及TTV病毒滴度的nPCR检测;应用导流杂交方法检测95例宫颈病变细胞学标本的HPV基因型。结果TTVDNA在55例健康妇女宫颈细胞标本中检出率52．7％（29／55）,与其平行42例血清样本中检出率50．0％（21／42）。在患有疾病妇女宫颈细胞中TTVDNA检出率（74．7％）高于健康体检对照组（P=0．005）。TTVDNA在患者血清样本中检出率51％（28／55）。在宫颈细胞及其平行血清中均检测出TTV基因亚型G1b。TTV病毒滴度在宫颈细胞中高于在其平行血清10～1000倍。HPV在患者组中检出率98．9％（94／95）,在健康组中检出率27．3％（15／55）。HPV基因型是高危型HPV16、18、33和低危型HPV6。HPV阳性标本TTVDNA检测率明显高于HPV阴性标本（P=0．02）。结论TTV在宫颈细胞中具有高检出率,在宫颈细胞中TTV病毒滴度高于其平行血清。TTV与HPV随性传播感染人群,并在女性生殖道内繁殖。TTV与HPV协同作用有待研究。     

不同型别人乳头瘤病毒感染不孕患者治疗方法的研究

目的 探讨不孕症患者感染不同型别高危型人乳头瘤病毒（HPV）的最佳治疗方法.方法 按宫颈病理学、宫颈状况和不孕患者生育欲望,将144例感染高危型人乳头瘤病毒的门诊不孕患者分为治疗组和不治疗组,以6个月为一复查周期,采用双色实时荧光定量PCR法,检测患者宫颈脱落细胞内的HPV-DNA,观察其阴转情况及受孕情况,并进行比较分析.结果 ①感染不同型别高危型HPV,治疗组的HPV阴转率（56.67%）较高;②次要高危型不治疗组的妊娠率（50.00%）较高,主要高危型治疗组的妊娠率（31.67%）较高,显著高于不治疗组（4.00%）;③主要高危型手术治疗后HPV阴转率和妊娠率（40.00%）均高于药物治疗（6.67%）,手术治疗二种方法中以Leep＋药物最佳.妊娠率为41.18%,高于药物治疗组的6.67%.结论 不孕患者应常规进行宫颈HPV筛查,主要高危型HPV感染是不孕的宫颈性病因,主要高危型HPV感染伴宫颈病变者以LEEP＋药物治疗为最佳.     

HPV高危型别检测联合细胞学检查对宫颈病变筛查的研究

目的 研究人乳头瘤病毒HPV高危型别检测联合液基薄层细胞学检查（TCT）及阴道镜检查对宫颈癌及癌前病变筛查的诊断价值。方法 对1375例宫颈组织细胞样本进行HPV高危型别检测,对其中阳性样本进行TCT检查,有宫颈上皮内瘤变者（CIN）行阴道镜下活检病理组织学确诊。HPV高危型别检测采用双色荧光定量PCR方法进行8种高危型HPV DNA（主要高危型：HPV16,18,45,31）和次要高危型（HPV33,52,58,67）分型及病毒载量检测。结果 1375例样本高危型HPV DNA检测结果为阳性256例,阳性率为18.62％;TCT结果为WNL的样本高危型HPV的感染率为16.41％ （42/256）;TCT结果为ASCUS以上的样本高危型HPV的感染率为83.59％（ 214/256）。HPV各型别的病毒载量在TCT结果为WNL、ASCUS及LSIL/HSIL/SCC之间差异无统计学意义（P＞0.5）。TCT与阴道镜的阳性符合率分别为WNL-正常或炎症92.86％（ 39/42）,LISL-CIN I 81.36％（48/59）,HSIL-CIN Ⅱ＆Ⅲ 85.19％ （23/27）,SCC-宫颈癌9/10。结论 HPV高危型别检测联合TCT技术及阴道镜检查能显著提高宫颈病变的阳性检出率,可作为宫颈癌及宫颈上皮内瘤变（ CIN）筛查的可靠早期诊断方法,具有重要临床应用价值。     

芯片电泳快速检测高危型人类乳头状瘤病毒

目的建立人类乳头状瘤病毒（humanpa pillomavims,HPV）52和58基因型高危型的芯片电泳检测方法。方法选取114例宫颈细胞检测患者,提取宫颈脱落细胞DNA。PCR扩增HPV高危型52和58基因型特异性DNA序列,应用优化的芯片电泳分析方法对扩增产物进行分离检测。根据细胞学诊断结果将研究对象分为4组：正常组、不典型鳞状细胞组、低度鳞状上皮内病变组和高度鳞状上皮内病变组,比较各组的HPV52、58基因型感染情况。结果114份标本中,HPV52基因型检出率为8.8%（10/114）,HPV58基因型检出率为33.3%（38/114）。与正常对照组相比较,各病变组HPV52基因型检出率差异有统计学意义（P〈0.05）;而HPV58基因型检出率在病变组和正常对照组之间差异无统计学意义（P〉0.05）。结论芯片电泳法检测快速、成本低,可用于HPV52、58基因型感染的筛查。     

人乳头瘤病毒检测在宫颈病变中的应用价值

目的了解宫颈细胞学异常患者中人乳头瘤“毒（HPV）的感染状况,评估HPV检测在宫颈病变筛查中的价值。方法随机选取宫颈薄层液基细胞学检测异常的101例患者进行了HPV检测,同时行组织病理学检查。结果（1）101例宫颈细胞学异常患者中,细胞学为ASCUS、LSIL、HSIL、鳞状细胞癌时高危型HPV阳性率分别为84．2％、88．6％、100．0％和2／2;（2）10l例细胞学异常患者中20例为CINI,81例为CINⅡ及以上级别,高危型HPV阳性率存CINI、CINⅡ及以上级别分别为60％、97．5％;（3）ASCUS组中,高危型HPV阳性患者中CINⅡ及以上病变的发生率为87．5％,HPV阴性患者中CINⅡ及以上病变的发生率为16．7％;（4）高危型HPV型别分布由高到低分别为HPVl6型39．6％（40／101）,HPV58型17．8％（18／101）,HPV52型16．8％（17／101）,HPVl8型9．9％（10／101）以及HPV33型9．9％（10／101）。结论高危型HPV感染率与宫颈病变级别呈正相关;HPV检测可作为ASCUS患者的有效分流手段。宫颈病变患者中高危型HPV感染以16、58、52、18、33型为主。     

宫颈癌与人乳头状瘤病毒16/18型的关系探讨

目的　应用荧光定量聚合酶链反应 (FQ -PCR)检测宫颈癌患者人乳头状瘤病毒 (HPV) 16 / 18型感染率 ,探讨HPV 16 / 18型与宫颈癌的关系。方法　应用荧光探针标记引物的荧光定量聚合酶链反应对 88例宫颈癌患者的宫颈分泌物进行了HPV 16 / 18型检测。结果　 88例宫颈癌患者宫颈分泌物FQ -PCR阳性率为 78% ,阳性样品定量对数平均值 (ml-1)为 5 .33× 10 6,定量测值范围 (ml-1)为 1.2 0× 10 3 ～ 2 .4 1× 10 7。对照组 85例全部阴性。结论人乳头状瘤病毒 (HPV) 16 / 18型感染与宫颈癌的发生发展关系密切。     

三明地区人乳头瘤病毒（HPV）感染FQ-PCR检测与宫颈病变的关系

目的通过对三明地区妇女感染HPV情况的调查,为三明地区妇女感染HPV的预防提供科学依据。方法采用聚合酶多重核酸扩增荧光检测方法检测女性宫颈口脱落细胞进行13种高危型,5种低危型人乳头瘤状病毒（HPV）检测和分型。结果在129例正常组女性宫颈口脱落细胞标本中,HPV高危型和低危型阳性总检出率为29.5%（38/129）,其中高危型检出率为31%,低危型为69%。421例各种不同宫颈病变总检出率为89.3%（376/421）。其中宫颈炎症高危型检出率52%,低危型为48%;CINⅠ高危型为74%,低危型为26%;CIN Ⅱ高危型为84%,低危型为16%;CIN Ⅲ高危型为92%,低危型为8%;宫颈鳞癌高危型为100%;宫颈腺癌高危型为100%;宫颈湿疣高危型为94%,低危型为6%。结论随着宫颈病变的加重,CIN级别的增加,HPV感染率上升,在较高级别的宫颈病变中,大多为高危型HPV感染。     

荧光定量聚合酶链反应检测人乳头瘤病毒6/11型

目的 了解女性阴道炎患者人乳头瘤病毒6／11型（HPV6／11）的感染情况。方法 用荧光定量聚合酶链反应（FQ－PCR）技术检测129例门诊患者阴道分泌物中HPV6／11的病毒拷贝数。结果 共检出HPV6／11阳笥者47例,阳性率为36．43％;拷贝数在10^5以上者39例,占阳性者中的82．98％;40岁以上年龄组阳性率高且病毒复制量均在10^5以上。结论 中老年阴道炎患者就诊晚,感染重。FQ－PCR检测HPV敏感、快速、准确,特别是其定量特点对临床很有意义。     

安岳地区女性高危型HPV基因分型结果分析

目的 分析安岳地区女性高危型人乳头瘤病毒的感染以及各亚型的分布情况,为本地区宫颈癌防治及流行病学研究提供依据。方法 收集2016年1月~2017年12月年安岳地区自愿进行15种高危型HPV检测的女性宫颈脱落细胞标本2078例,应用实时荧光定量PCR进行包括16、18、31、33、35、39、45、51、52、53、56、58、59、66、68在内的15种HR-HPV检测,分析HR-HPV在人群中的总体和不同年龄段感染情况以及各亚型的分布特点。结果 2078例女性宫颈脱落细胞标本总阳性检出率为22.81%,其中单一感染占17.37%,混合感染占5.44%;在各年龄组中＜20岁组与＞60岁年龄组HR-HPV感染率最高,差异具有统计学意义（P＜0.05）;15种高危型中检出前五位的依次为:52型、16型、31型、53型、58型。结论 本地区女性人群的高危型HPV检出率较高,对本地区相关人群进行高危型HPV筛查非常必要。     

Determination of HPV type 16 and 18 viral load in cervical smears of women referred to colposcopy

It has been recognized that human papillomavirus infection is the major causal factor for high-grade cervical lesions. The aim of the study was to evaluate the relationship between HPV 16 and 18 viral loads and cervical status in different age strata. A duplex real time PCR method was devised to determine HPV 16 and 18 viral load per million of human cells using an in house plasmidic construct as a standard of quantification. The 151 cervical scrapes were collected before colposcopic examination from either abnormal cervico-vaginal smear (group 1, 97 patients) or from post treatment clinical follow-up (group 2, 54 patients). In women aged 30-40, the HPV16 viral loads were significantly higher in high-grade squamous intraepithelial lesion than in low-grade squamous intraepithelial lesion in both groups and HPV18 in group 1. In women aged 20-30 of group 1, high HPV viral load was associated in few cases with high-grade squamous intraepithelial lesion or low-grade squamous intraepithelial lesion, and surprisingly in some patients with normal cervix. HPV 16 and 18 viral loads are related to the severity of cervical lesion, and may be useful in the clinical management of cervical lesions. A specific follow-up may be useful for those with high viral load despite normal cervix.     

Evaluation of type-specific HPV persistence and high-risk HPV viral load quantitation in HPV positive women under 30 with normal cervical cytology

The persistence of high-risk HPV (HR-HPV) infection is necessary for the development of cervical intraepithelial neoplasia. The aim of this study was to evaluate if HR-HPV typing and HPV16, 18, 31, and 33 quantitation are predictive for type-specific infection persistence and/or the development of CIN in women under 30 with normal cervical cytology. Young women (under 30) attending a family planning clinic who were HPV positive with normal cervical cytology were included. HPV genotyping was assessed by MY09/MY11 PCR, sequencing, phylogenetic analysis, and cloning when necessary. HR-HPV viral load was quantified using duplex real-time PCR. Study patients were offered for a second smear and HR-HPV detection and quantitation after 12 months. HR-HPV was identified in 43 (21.9%) of the 199 included women. Of these, 39 patients had a second cervical sample taken within a mean interval of 11.7 months (8.8-18.3 months). The mean HR-HPV 16, 18, 31, and 33 initial viral load was 1.9 × 10(6) copies/million cells. The level of viral load did not reveal any significant association with type-specific HR-HPV persistence or the subsequent development of cervical intraepithelial neoplasia. Only HPV16 infection was significantly more likely to persist (91.7% vs. 33.1%, P=0.001) and to develop CIN (33.3% vs. 3.7%, P=0.025). In women under 30 with normal cytology, HR-HPV viral load is common and is not predictive of HPV persistence or the development of cervical intraepithelial neoplasia. HPV16 positive women are significantly more likely to have persistent infection and to develop cervical intraepithelial neoplasia.     

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89-9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories. Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.     

Dynamics of HPV16 DNA load reflect the natural history of cervical HPV-associated lesions.

BACKGROUND: High burden of high risk human papillomavirus (HR HPV) has been shown to be predictive for the development of high grade cervical lesions and invasive cancers. However, low viral load cannot inevitably exclude progression towards cervical diseases. Moreover, few studies addressed whether viral load could predict infection clearance. OBJECTIVES: We carried out a retrospective study to analyze the variations of HPV16 load over time as a predictive marker of clinical outcome. STUDY DESIGN: The population consisted of 38 women who were found HR HPV positive by HCII test at study entry. Among them, 13 had developed a CIN2/3 (cases) and 25 had a negative HCII test and a normal cytology (controls) at study exit. The HPV16 DNA loads were quantified in 132 longitudinal cervical samples using quantitative real-time PCR. RESULTS: At study entry, the median of HPV16 load was not statistically different between controls and cases. However, when using a cut-off value of 200 copies/10(3) cells, the rate of cumulative incidence of CIN2/3 at 18 months increased from 14% in women with a load200 copies/10(3) cells. The longitudinal analysis performed on follow-up samples showed that in cases the progression to CIN2/3 was linked to HPV16 burden increasing over time, whereas in controls a decrease of at least 1 log HPV16 DNA load was observed over>or=2 time points. CONCLUSIONS: These results show that kinetics of HPV load, rather than a single HPV detection, might be more reliable to estimate whether a HPV infection will progress or be cleared.     

Associations of high-risk HPV types and viral load with cervical cancer in China.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. Infection with some genotypes of human papillomavirus is the most important risk factor associated with cervical cancer. OBJECTIVE: The aim of this study was to determine the prevalence and genotypes of HPV in China, and to evaluate the correlation between viral load of high risk HPV and cervical cancer and its precursors. STUDY DESIGN: A cross-sectional study was carried out, wherein cervical samples were collected from 541 patients with cervical cancer, 262 with CIN, 139 with cervicitis and 68 age-matched healthy controls. Hybrid Capture 2 was employed to detect HPV DNA. Specimens from HPV DNA positive cervical cancer were tested for HPV types by using type specific PCR and general primer PCR with sequence-based typing (GP PCR-SBT). RESULTS: Overall high risk HPV prevalence was 68.8% in CIN1, 80.3% in CIN2, 90.2% in CIN3, 90.9% in cervical cancer in situ, 89.9% in invasive cervical cancer and 25% in healthy controls from China. The most common HPV DNA type found in patients with cervical cancer was HPV16 (79.6%), followed by HPV58 (5.92%), HPV33 (3.29%), HPV18 (1.97%), HPV6 (1.97%), HPV31 (1.31%), HPV39 (1.31%), HPV68 (1.31%) and other HPV types (3.3%). It was found that there was a significantly increased risk of increasing CIN stage with high viral load. Frequency of low viral load found in the controls was 13.2% and 22.9% of CIN1, obtaining an OR of 4.2 (1.5-12.0). Associations (OR) among low viral load and CIN2/3, CIS, and CC were 6.7 (2.9-15.6), 9.4 (2.7-32.3) and 8.3 (3.7-18.4), respectively. While high viral loads were found in 5.9% of controls, 27.1% of CIN1, 42.1% of CIN2/3 and 48.5% of CIS, demonstrating increasing odds ratios with severity of disease (OR for CIS=68.0, 95% CI=17.8-259.7). CONCLUSIONS: HPV16 was the most common genotype in central China. The developing cervical cancer precursors were associated with elevated high-risk HPV viral load.     

高危型HPV阳性患者宫颈支原体和衣原体感染与宫颈组织学改变的关系

目的 探讨高危型HPV阳性不孕患者宫颈支原体和衣原体感染与宫颈组织病变的关系.方法 以HPV阴性不孕患者为对照,回顾分析不同高危型别HPV阳性不孕患者宫颈支原体和衣原体感染与宫颈组织病理分级、炎症分级之间的关系.结果 支原体阳性者HPV感染的发生率与支原体阴性者差异有统计学意义（P＜0.01）,衣原体阳性者HPV感染的发生率与衣原体阴性者差异无统计学意义（P＞0.05）.与HPV阴性组比较,HPV阳性者CIN和宫颈糜烂发生率以及CIN分级均较高（P＜0.01）,HPV阳性者宫颈糜烂程度无差异（P＞0.05）.与单纯HPV阳性组比较,HPV和支原体混合感染者CIN发生率及重度宫颈糜烂无差异（P＞0.05）.结论 支原体感染增加了高危型HPV的感染概率,高危型HPV感染增加了宫颈组织的病理损害程度,支原体感染可能是高危型HPV持续性感染的因素,宫颈组织病理程度是宫颈性不孕不可忽视的因素.     

High risk HPV load estimated by Hybrid Capture II correlates with HPV16 load measured by real-time PCR in cervical smears of HPV16-infected women.

BACKGROUND: High risk human papillomavirus (HR-HPV) load determined by quantitative methods has already been considered as highly predictive of future development of high grade cervical lesions. Some studies also demonstrated that Hybrid Capture II (HCII) results can be considered as a reflection of HPV DNA load, while others did not. HCI assay, well suited for routine HR-HPV screening, is not especially dedicated for quantitative use. However, we have recently shown that women with high viral loads assessed by HCII were at increased risk of cervical precancer. OBJECTIVES: The aim of the study was to determine if the values given by the HCII assay can be considered as quantitative. STUDY DESIGN: We used a real-time PCR allowing precise quantification of both HPV16 genome and albumin gene to normalize the measuring HPV16 load in cervical cells and to compare the data with those obtained by HCIIin a series of 40 HR-HPV positive samples. RESULTS: Reproducibility of the HPV16 real-time PCR, assessed from nine independent experiments of serial dilutions of SiHa cell DNA, was reflected in coefficients of variation for standard curves of crossing point (Cp) values below 5%. The HPV16 loads with a broad individual variability were significantly related to the cumulative load estimated by HCII and did not depend on the cellularity of samples. CONCLUSIONS: We assume that the HCII values can be used as a quantitative measure of HR-HPV DNA, so long as cervical specimens are collected using standardized protocols.     

Nr-CWS对TC-1细胞致瘤作用的影响及抗女性下生殖道人乳头状瘤病毒感染的研究

目的探讨NvCWS对含HPV16型病毒基因的TC-1细胞荷瘤鼠肿瘤生长的影响及对女性下生殖道HPV感染的清除效果。方法SCID小鼠分别注射代．1细胞形成肿瘤模型后随机分成3组，分别注射生理盐水、60μg/ml和120μg/ml Nr-CWS，观察肿瘤生长的情况。临床选取45例单纯HPV感染者使用NvCWS1个月、3个月后再次检测HPV，分析NvCWS对生殖道HPV感染的清除效果。结果动物实验表明两组用药组的肿瘤体重明显低于对照组，抑瘤率分别为59．1％和84．2％：三组瘤重用秩和检验分析P〈0．05，差异有统计学意义；临床实验结果显示用药后3个月HPV检测转阴23例（51．1％），型别减少9例（20％），无效13例（28、9％）；治愈率与自然转阴率31％相比较，x^2检验P=0．005，差异有统计学意义。结论NvCWS对HPV16型病毒基因的TC-1细胞荷瘤鼠肿瘤有明显的抑制作用；且对临床女性下生殖道HPV感染有一定的清除作用。     

Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study

AIMS: To monitor the association between the course of high risk human papillomavirus (HR-HPV) infection and the development of cervical neoplasia over time, from a baseline of normal cervical cytology. METHODS: This paper presents the follow up data from a previous cross sectional analysis. Women from a screening population who had normal cytology and who were HR-HPV positive were recalled after two to three years for cytology and HPV genotyping. The development of cervical neoplasia at follow up was related to the course of HPV infection (clearance, persistence, or sequential infection) and the presence of single or multiple HPV infections at baseline. A comparator control group of women who were HPV and cytologically negative at baseline were selected from the same population. RESULTS: Twelve cases of dyskaryosis were found in women who were HPV positive at baseline; four were high grade. Only three cases of low grade dyskaryosis were found in the control group. Women with type specific persistent infections were significantly more likely to develop cervical neoplasia than women who cleared the infection (p = 0.0001) or were sequentially infected with different types (p = 0.001). Women with multiple HPV infections at baseline were no more likely to develop cervical dyskaryosis than those with a single infection. CONCLUSIONS: Type specific persistent HR-HPV infection as monitored by genotyping can identify women at increased risk of cervical neoplasia more accurately than a single or repeated presence/absence HPV test. The cost effectiveness of such an approach should be investigated by an appropriate, large scale cost-benefit analysis.