Anal HPV 16 and 18 viral load: A comparison between HIV‐negative and ‐positive MSM and association with persistence

Does anal HPV viral load explain the difference in anal HPV persistence between HIV‐negative and ‐positive men who have sex with men (MSM)? MSM ≥18 years were recruited in Amsterdam, the Netherlands, in 2010‐2011. Anal self‐swabs were collected every 6 months and genotyped (SPF₁₀‐PCR‐DEIA‐LIPA₂₅‐system). HPV16 and HPV18 load was determined with a type specific quantitative (q)PCR, and compared between HIV‐negative and ‐positive men using ranksum test. Persistence was defined as ≥3 positive samples for the same HPV‐type. Determinants of persistent HPV16/18 infection and its association with HPV16/18 load were assessed with logistic regression. Of 777 recruited MSM, 54 and 22 HIV negative men were HPV16 and HPV18 positive at baseline, and 64 and 39 HIV‐positive MSM. The geometric mean titer (GMT) of HPV16 was 19.6 (95%CI 10.1‐38.0) and of HPV18 8.6 (95%CI 2.7‐27.5) DNA copies/human cell. HPV16 and HPV18 load did not differ significantly between HIV‐negative and ‐positive MSM (P = 0.7; P = 0.8, respectively). In multivariable analyses HPV16 load was an independent determinant of HPV16 persistence (OR 1.8, 95%CI 1.3‐2.4). No difference in anal HPV viral load was found between HIV‐positive and HIV‐negative MSM. HPV 16/18 viral load is an independent determinant of type‐specific persistence.     

Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma

Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10³ cells in normal samples up to 56,354 copies per 10³ cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10³ cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.     

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.     

Significance of HPV 16 and 18 viral load quantitation in women referred for colposcopy

The clinical utility of HPV 16 and 18 viral loads remains debated. The aim of this study was to assess the clinical significance of HPV 16 and 18 viral load and to determine a cut‐off for optimal prediction of grade 2 or higher cervical intraepithelial neoplasia among patients referred to colposcopy. A total of 186 cervico‐vaginal specimens harboring HPV 16 and/or 18 obtained at the time of colposcopy from patients without previous cervical neoplasia were tested for HPV 16 and 18 detection and quantitation using quantitative duplex real‐time PCR method. Grade 2 or higher cervical intraepithelial neoplasia was diagnosed in 87 (46.8%) cases. Only HPV 16 median viral load increased significantly with the lesion grade: 9.1 × 10⁴ in normal cervix or grade 1 cervical intraepithelial lesion versus 4.0 × 10⁶ copies per million cells in grade 2 or higher cervical intraepithelial lesion (P < 0.001). The highest predictive value for grade 2 or higher cervical intraepithelial lesion was observed with a HPV 16 viral load cut‐off of 3.0 × 10⁶ copies per million cells (91% specificity, 58.2% sensitivity). Using this cut‐off, the highest predictive value of HPV 16 viral load was observed among those referred for previous low‐grade abnormal cervical cytology (96.4% specificity, 88% sensitivity). HPV 18 quantitation showed very poor predictive value. Specific attention should be given when performing colposcopic examination of women with an HPV 16 viral load higher than 3.0 × 10⁶ copies per million cells, especially among those referred after a low‐grade abnormal cytology. J. Med. Virol. 84:306–313, 2012. © 2011 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with INNO-LiPA HPV genotyping extra assay

Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.     

Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (kappa = 0.661) and HPV18 (kappa = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.     

Prevalence and viral load of oncogenic human papillomavirus types associated with cervical carcinoma in a population of North Italy

A cross‐sectional study was carried out in a population of North Italy to determine the prevalence of eight oncogenic human papillomavirus (HPV) types most commonly found in cervical carcinoma and to study the relationship between HPV DNA loads and severity of disease. A total of 597 cervical samples obtained from patients with pathological findings (n = 472) and from women with normal cytology (n = 125) were analyzed by means of normalized Real‐time PCR assays to quantify HPV‐16, ‐18, ‐31, ‐45, and ‐33 group (including ‐33, ‐52, ‐58, ‐67); the normalization of oncogenic HPV viral load was carried out by quantitation of a single copy gene. The two most common oncogenic HPV types found were 16 and 31 (24.3% and 22.9% of pathological samples, respectively); multiple infections were demonstrated in 22% of pathological samples. Overall, the HPV total viral load was found to increase with increasing severity of associated lesions, although a stronger association was observed only for HPV‐31 and HPV‐16 (γ = 0.49 and 0.41, respectively) as compared to HPV‐18 and ‐33 group (γ = 0.19 and 0.02, respectively). However, we found that high levels of HPV‐31 or 33 group DNA could be prognostic of minor oncogenic risk for high‐grade squamous intraepithelial lesions (H‐SIL) (age adjusted odds ratio [AORs] = 1.57 and 1.26, respectively) than HPV‐16 and HPV‐18 (AORs = 30 and 8, respectively). The AORs also increased with HPV total viral load and reached a maximum of AORs = 15.7. Thus, HPV load is a type‐dependent risk marker for the development of H‐SIL. J. Med. Virol. 81:278–287, 2009. © 2008 Wiley‐Liss, Inc.     

Two years outcome of women infected with high risk HPV having normal colposcopy following low‐grade or equivocal cytological abnormalities: Are HPV16 and 18 viral load clinically useful predictive markers?

Management of patients infected with high-risk HPV (hrHPV) despite normal colposcopy following abnormal cytology remains a clinical challenge. The aim of this study was to evaluate if, in that specific population, initial HPV 16 and HPV 18 viral loads are predictive of infection clearance over a 24-month follow-up. A total of 67 women infected with hrHPV having normal colposcopy following equivocal or low-grade cytological abnormalities were recruited and attended regular follow-ups based on repeat colposcopies and HPV testing. HPV16 and HPV18 infection were diagnosed in 36 (53.7%) and 7 (10.4%) cases, respectively. Viral load was quantified using the quantitative duplex real-time PCR method. Although this was not observed for HPV 18, initial HPV 16 viral load was highly associated to HPV 16 infection outcome (receiver operating characteristic curve analysis, area under curve: 0.90). Thus, women who had cleared their HPV 16 infection had significantly lower median initial HPV 16 viral load than those with persistent HPV 16 infection: 1.5 × 10(3) copies per million cells (CPMC) versus 3.8 × 10(6) CPMC, respectively (P = 0.006). The best prediction of HPV 16 clearance was obtained with an initial HPV 16 viral load of <7.5 × 10(4) CPMC: 86.7% specificity and 85.7% sensitivity. Finally, six patients were diagnosed with grade 2 or 3 cervical or vaginal intraepithelial neoplasia. Although all had a persistent hrHPV infection, neither HPV 16 nor 18 viral loads were found to be predictive of the risk of cervical or vaginal intraepithelial neoplasia. HPV16 viral load quantitation could represent a clinically useful marker in that very specific population.     

广州东部妇女宫颈人乳头瘤病毒16型感染及基因分析

目的 研究广州东部妇女中人乳头瘤病毒16型(HPV-16)宫颈感染分布,分析其早基因E6/E7的多态性,分析L1和E6基因定量与病程的关系.方法 通过导流杂交基因芯片技术检测宫颈脱落细胞的HPV-16感染;通过特异性扩增获取病毒早基因E6/E7序列,克隆测序并进行多态性分析;荧光定量PCR技术对E6基因和L1基因进行定量分析.结果 806例宫颈脱落细胞样本中HPV-16感染阳性36例(4.5%),其中18例(50.0%)宫颈细胞发生高度以上病变;7例(4例低度或以下病变,3例高度以上病变或浸润癌)阳性标本得到E6/g7序列有15个位点分别出现变异;高度病变组(A组,11例)与低度或以下病变组(B组,14例)的L1基因和E6基因定量数据对数值均有显著差异(P<0.05),但L1/E6比值差异无统计学意义(P=0.19).结论 本地区在17～62岁妇女中HPV-16感染阳性发生率约4.5%,50.0%发生高度以上宫颈病变,本研究显示病毒基因拷贝数与宫颈病变程度可能有关,L1/E6比值未能提示病毒整合的发生.     

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.     

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.     

Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women.     

Viral load and physical status of human papillomavirus (HPV) 18 in cervical samples from female sex workers infected with HPV 18 in Burkina Faso

Viral DNA load and physical status might be predictive of either high‐grade cervical lesions or disease progression among women infected by human papillomavirus (HPV) 16, but these virological markers have rarely been studied in HPV 18 infections. The relationships between HPV 18 DNA load, viral genome physical status and cervical squamous intraepithelial lesions were analyzed among female sex workers infected with HPV18 in Burkina Faso. HPV 18 E2 and E6 genes were quantitated by real‐time PCR. Among 21 women infected with HPV 18, 67% of whom were HIV‐1‐seropositive, 11 (52.4%) had a normal cytology, 8 (38.1%) had low‐grade squamous intraepithelial lesions, and 2 (9.5%) had high‐grade squamous intraepithelial lesions. Total viral load and integrated viral load were higher in women with squamous intraepithelial lesions than in women with normal cytology (P = 0.01 for both parameters). Total viral load and integrated viral load were higher in HIV‐1‐seropositive women than in those who were not infected with HIV (P = 0.01, and P, 0.01, respectively). Total viral load or integrated viral load >1,000 copies/ng of DNA were more frequent in women with squamous intraepithelial lesions than in women with normal cytology (7/10 vs. 1/11; P = 0.007) and in HIV‐1‐seropositive women (8/14 vs. 0/7 in HIV‐uninfected women; P = 0.02). Both HPV 18 DNA and integrated DNA loads might represent markers of cervical lesions. Prospective evaluations are needed to establish the value of these parameters to predict high‐grade lesion or lesion progression. J. Med. Virol. 81:1786–1791, 2009. © 2009 Wiley‐Liss, Inc.     

Prevalence,viral load,and physical status of HPV 16 and 18 in cervical adenosquamous carcinoma

Adenosquamous carcinoma of the uterine cervix is a rare mixture of malignant squamous and glandular epithelial elements and accounts for approximately 10% of cervical carcinomas. The aims of the present study were to evaluate the prevalence, physical status, and viral load of HPV 16 and 18 in adenosquamous carcinoma. Formalin-fixed paraffin-embedded tissue samples from 20 cases of histologically diagnosed adenosquamous carcinoma were examined. The squamous and glandular components were separately microdissected and analyzed for their HPV DNA subtype, viral load, and physical status using real-time polymerase chain reaction (PCR). The percentages of HPV 16- and 18-positive cases among all the HPV-positive cases were 36.8% (7/19) and 57.9% (11/19) in the squamous epithelial elements and 33.3% (6/18) and 61.1% (11/18) in the glandular elements, respectively. PCR analysis with E2 primers revealed that seven of eleven (63.6%) HPV 18-positive cases had the pure integrated form in both elements. The mean HPV 16 DNA copy numbers/cell was 7.22 in the squamous elements and 1.33 in the glandular elements (p = 0.04) while the corresponding mean HPV 18 DNA copy numbers/cell was 1.50 and 0.89, respectively. The prevalence of HPV 18 in adenosquamous carcinoma was high and many HPV 18-positive cases were the pure integrated form resulting in very low copy numbers/cell. It is possible that more aggressive transformation with early integration of HPV 18 results in cases with greater chromosomal instabilities, higher growth rates, and rapid progression.     

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.