Human papillomavirus quantification in urine and cervical samples by using the Mx4000 and LightCycler general real-time PCR systems

During the last decade, growing efforts have focused on human papillomavirus (HPV) detection using liquid hybridization, conventional PCR, and real-time PCR-based methods to increase the overall proportion of patients participating in cervical cancer screening procedures. We proposed a new general HPV DNA real-time PCR on the Mx4000 (Stratagene) and LightCycler (Roche Diagnostics) systems usable for both cervical scrape specimens and urine samples. A linear range was obtained from 5 DNA copies to 8 log(10) DNA copies/ml, and intra- and interassay variations were between 1.8 and 4%. Cervical carcinoma and HPV DNA screening was performed in 333 individual women referred for gynecological examination at the university hospitals of Angers and Brest and enrolled in the PapU study. Among cervical specimens (n = 333), 45% were positive for HPV DNA, with a mean viral load at 5.00 log/ml (+/- 1.73). Among urine samples (n = 177), 37% were positive with a significant 50-fold-lower mean viral load (3.77 +/- 1.32 log/ml; P < 0.0001). Kappa agreement for HPV DNA between cervical and urine specimens was excellent (93%). Thus, we developed a highly sensitive and quantitative general HPV DNA real-time PCR method that allows mass screening of patients with HPV infection. The ongoing longitudinal and prospective multicenter PapU study should give us the opportunity to validate this method adapted to HPV DNA screening in urine samples in a larger population.     

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia.

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.     

Comparison of three different PCR methods for quantifying human papillomavirus type 16 DNA in cervical scrape specimens

We compared real-time LightCycler and TaqMan assays and the GP5+/6+ PCR/enzyme immunoassay (EIA) to assess the human papillomavirus type 16 (HPV16) load in cervical scrape specimens. Both real-time PCR assays determined the HPV16 load in scrape specimens similarly. The level of agreement between these assays and the GP5+/6+ PCR/EIA was low (P = 0.004), suggesting that the latter method is not suited for quantifying HPV16 DNA.     

Human papillomavirus load measured by Linear Array correlates with quantitative PCR in cervical cytology specimens

Carcinogenic human papillomavirus (HPV) infections are necessary causes of most anogenital cancers. Viral load has been proposed as a marker for progression to cancer precursors but has been confirmed only for HPV16. Challenges in studying viral load are related to the lack of validated assays for a large number of genotypes. We compared viral load measured by Linear Array (LA) HPV genotyping with the gold standard, quantitative PCR (Q-PCR). LA genotyping and Q-PCR were performed in 143 cytology specimens from women referred to colposcopy. LA signal strength was measured by densitometry. Correlation coefficients and receiver operating characteristic (ROC) analyses were used to evaluate analytical and clinical performance. We observed a moderate to strong correlation between the two quantitative viral load measurements, ranging from an R value of 0.61 for HPV31 to an R value of 0.86 for HPV52. We also observed agreement between visual LA signal strength evaluation and Q-PCR. Both quantifications agreed on the disease stages with highest viral load, which varied by type (cervical intraepithelial neoplasia grade 2 [CIN2] for HPV52, CIN3 for HPV16 and HPV33, and cancer for HPV18 and HPV31). The area under the curve (AUC) for HPV16 Q-PCR at the CIN3 cutoff was 0.72 (P = 0.004), and the AUC for HPV18 LA at the CIN2 cutoff was 0.78 (P = 0.04). Quantification of LA signals correlates with the current gold standard for viral load, Q-PCR. Analyses of viral load need to address multiple infections and type attribution to evaluate whether viral load has clinical value beyond the established HPV16 finding. Our findings support conducting comprehensive studies of viral load and cervical cancer precursors using quantitative LA genotyping data.     

Detection of specific human papillomavirus types in paraffin-embedded sections of cervical carcinomas

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.     

Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction.

The authors describe the detection of human papillomavirus (HPV) 16 DNA in paraffin-embedded, formalin-fixed tissues of cervical squamous intraepithelial lesions (SILs) by in situ hybridization after amplification by the polymerase chain reaction (PCR). Using conventional in situ hybridization and a biotin-labeled probe, variable numbers of superficial cells and none of the basal cells in the SILs showed detectable HPV 16 DNA. When the in situ assay was done after amplification, increased numbers of superficial cells had detectable HPV DNA, and the hybridization signal was much more intense. HPV DNA was also detected in basal and parabasal cells at the site of the lesion whereas not detectable in directly adjacent, normal squamous epithelium. Amplified HPV DNA was demonstrated in formalin-fixed SiHa cells using a biotin-labeled probe, demonstrating the ability to detect one copy of HPV 16 DNA. This technique should allow for direct visualization in cells of other DNA sequences of low copy number from achival specimens otherwise undetectable by conventional in situ hybridization analysis.     

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.     

Clinical Validation of the HPV-Risk Assay,a Novel Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus DNA by Targeting the E7 Region

The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk human papillomavirus (HPV) types (i.e., HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and −68), and provides additional genotype information for HPV16 and HPV18. This study evaluated the clinical performance and reproducibility of the HPV-Risk assay with cervical scraping specimens and its utility with self-collected (cervico)vaginal specimens. The clinical performance of the HPV-Risk assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) with cervical scraping specimens was evaluated by a noninferiority analysis, relative to high-risk HPV GP5+/6+ PCR, following international guidelines for HPV test requirements for cervical cancer screening. The HPV-Risk assay showed clinical sensitivity for CIN2+ of 97.1% (95% confidence interval [CI], 89.1 to 99.3%; 67/69 samples) and a clinical specificity for CIN2+ of 94.3% (95% CI, 92.5 to 95.7%; 777/824 samples). The clinical sensitivity and specificity were noninferior to those of GP5+/6+ PCR (noninferiority score test, P = 0.006 and 0.0003, respectively). Intralaboratory reproducibility over time (99.5% [95% CI, 98.6 to 99.8%]; 544/547 samples, kappa = 0.99) and interlaboratory agreement (99.2% [95% CI, 98.6 to 99.8%]; 527/531 samples, kappa = 0.98) for the HPV-Risk assay with cervical scraping specimens were high. The agreement of the HPV-Risk assay results for self-collected (cervico)vaginal specimens and clinician-obtained cervical scraping specimens was also high, i.e., 95.9% (95% CI, 85.1 to 99.0%; 47/49 samples, kappa = 0.90) for self-collected lavage samples and 91.6% (95% CI, 84.6 to 95.6%; 98/107 samples, kappa = 0.82) for self-collected brush samples. In conclusion, the HPV-Risk assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered clinically validated for cervical screening purposes. The compatibility of the HPV-Risk assay with self-collected specimens supports its utility for HPV self-sampling.     

Correlation between laminin-5 immunohistochemistry and human papillomavirus status in squamous cervical carcinoma

BACKGROUND: Human papillomavirus (HPV) plays a critical role in the carcinogenesis of squamous cervical carcinoma. Integration of viral DNA into the host genome is a major contributing factor to malignant transformation. Viral load may influence integration. AIMS: To compare HPV status (type, viral load, integration status) between normal samples, carcinoma in situ and invasive carcinoma in order to elucidate the role of HPV in progression to invasive lesions. METHODS: The study population comprised 10 biopsy samples from each diagnostic group. Laminin-5 immunohistochemistry was performed to distinguish invasive carcinoma from non-invasive high-grade lesions. Real-time PCR was used to detect specific HPV types, viral load and integrated HPV, with quantification of viral E2 and E6 genes. RESULTS: Invasive carcinomas contained a higher number of laminin-5 immunoreactive cells as compared to non-invasive lesions. Almost all samples contained HPV, with a higher viral load and copy number of HPV16 integrated in E2 in cases of laminin-5 immunoreactivity and cases of invasive carcinoma. High HPV16 viral load was associated with more integrated copies in E2. CONCLUSIONS: HPV is important in progression from carcinoma in situ to invasive carcinoma. Viral load and HPV integration influence the development of cervical cancer towards invasiveness. Overall HPV status may be more predictive of patient outcome and may influence patient management.     

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.     

Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system

Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.     

Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer

We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10(2) to 10(7) HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.     

Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique

In contrast to cervical cancer, integration of human papillomavirus (HPV) DNA into the host genome has been considered a rare event in cancer precursor lesions (cervical intraepithelial neoplasia [CIN]). With our new real-time PCR method, we demonstrated that integrated HPV type 16 (HPV16) is already present in CIN lesions. The physical state of HPV16 and the viral load were simultaneously detected. A unique region of the E2 open reading frame (ORF) that is most often deleted during HPV16 integration is targeted by one set of PCR primers and a probe, and another set targets the E6 ORF. In episomal form, both targets should be equivalent, while in integrated form, the copy numbers of E2 would be less than those of E6. The method was tested with DNAs from 31 cervical lesions (non-CIN to CINIII) from 24 women prospectively followed up for 10 years. This report presents viral load and integration results from the largest series of CIN lesions described to date. Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Samples from another patient, with only integrated HPV16, rapidly progressed from CINI to CINIII in 2 years. In all other patients, episomal and integrated forms of HPV16 DNA were found to coexist. Rapid progression of the CIN lesions was closely associated with a heavy load of integrated HPV16. Thus, the method described here is a very sensitive tool with which to assess the physical state of HPV, which is useful in predicting disease progression.     

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.     

Quantitative analysis of human papillomavirus type 16 in cervical neoplasm: a study in Chinese population.

BACKGROUND: Human papillomavirus (HPV) infection was recognized as a major causal factor for the development and progression of squamous intraepithelial lesions (SIL). It is possible to use HPV test for the detection of cervical lesions as an adjunct to cervical cytology. OBJECTIVES: To evaluate the relation between HPV 16 viral load and the severity of cervical lesions in a Chinese population. METHODS: Study population was recruited from the colposcopy and general outpatient clinic. The presence of HPV 16 E6 and E7 in cytological specimens was detected using HPV 16 specific polymerase chain reaction (PCR). The viral load in the specimens that were positive for HPV 16 specific PCR, was quantified by using real-time PCR assay. RESULTS: The study recruited 394 women, in which 148 were high-grade SIL (HG-L), 121 were low-grade SIL (LG-L) and 125 were Normal. Sufficient DNA integrity was proven in 347 samples. Among 121 positive cases for HPV 16, 70 were HG-L, 34 were LG-L and 17 were Normal. Using quantitative real-time PCR, the percentages of samples with greater DNA copies were found to increase with the severity of diseases. There was also a significant difference in DNA copies among the three groups (HG-L versus Normal, p<0.001; HG-L versus LG-L, p<0.001). Area under receiver operating characteristic (ROC) curve of the HG-L versus LG-L and Normal was 0.836 indicating that quantitative PCR had a good diagnostic value in differentiating HG-L from the LG-L and Normal groups. CONCLUSIONS: Our data suggested HPV 16 viral load was significantly related to the severity of cervical lesions. Evaluation of viral burden could be a potential clinical tool in management of cervical lesions.     

A system for the detection and typing of human papillomavirus of the lower genital tract: in situ hybridization screening and polymerase chain reaction confirmation

We developed a simplified and non-isotopic in situ hybridization procedure for the detection of Human Papillomavirus (HPV) in routine Papanicolaou cervical smears. The assay involves one oligonucleotide (malignant probe) which recognizes high risk HPV 16 and 18, and another which detects HPV 6 and 11 (benign probe). We adapted the system to fulfill the requirements of gynecologists and cytologists, assimilating their protocols and simplifying the in situ hybridization assay. When we compared the detection levels reached by the in situ hybridization versus a ladder PCR assay in 156 clinical samples, original designed for this work, the kappa coefficient between both methods is 0.945, indicating a strong agreement between the ISH and the PCR assays.     

Novel method for detection, typing, and quantification of human papillomaviruses in clinical samples

We report the development of a novel detection and typing methodology for human papillomaviruses (HPV) based on real-time PCR with the self-probing fluorescent primers known as Scorpions. This technique is quick, simple, specific, sensitive, and capable of estimating viral load per cell. We report the results of over 100 typing reactions performed on cell lines, biopsies, and cervical cytobrush samples which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. We further report preliminary data suggesting a relationship between viral load per cell and grade of cervical disease.     

Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation

HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.     

两种检测头颈部鳞状细胞癌HPV16/18感染状态方法的对比

 目的 比较荧光定量PCR法（RT-PCR）和原位杂交法检测头颈部鳞状细胞癌中HPV（HPV）16/18亚型感染的差异。方法 采用RT-PCR法和原位杂交法对78例头颈部鳞状细胞癌患者的肿瘤组织进行HPV感染状态的检测,评价两种方法的一致性。结果 RT-PCR法检测到62.8%的头颈部鳞癌组织中含有HPV16/18 DNA。原位杂交法检测到47.4%的肿瘤组织中有HPV16DNA。用RT-PCR方法,唇、口腔、口咽及下咽的HPV16/18DNA阳性率分别为33.3%、66.67%、70%和57.14%;用原位杂交方法,唇、口腔、口咽、下咽的HPV16 DNA阳性率分别为33.3%、43.8%、60.0%和57.1%。总体上,两种方法检测HPV16/18DNA具有较高一致性（Kappa=0.595,P<0.001）。 结论 RT-PCR和原位杂交法检测HPV16/18DNA结果的一致性较高。