Incident Detection of High-Risk Human Papillomavirus Infections in a Cohort of High-Risk Women Aged 25-65 Years.

BACKGROUND: The risk of incident high-risk human papillomavirus (HR-HPV) infection associated with recent sexual behaviors is undefined in mid-adult women (defined as women aged 25-65 years). METHODS: Triannually, 420 female online daters aged 25-65 years submitted vaginal specimens for HPV testing and completed health and sexual behavior questionnaires. The cumulative incidence of and risk factors for incident HR-HPV detection were estimated by Kaplan-Meier and Cox proportional hazards methods. RESULTS: The 12-month cumulative incidence of HR-HPV detection was 25.4% (95% confidence interval [CI], 21.3%-30.1%). Current hormonal contraceptive use was positively associated with incident HR-HPV detection. Lifetime number of male sex partners was also positively associated but only among women not recently sexually active with male partners. In analysis that adjusted for hormonal contraceptive use and marital status, women reporting multiple male partners or male partners who were new, casual, or had ≥1 concurrent partnership had a hazard of incident HR-HPV detection that was 2.81 times (95% CI, 1.38-5.69 times) that for women who reported no male sex partners in the past 6 months. Thus, among women with multiple male partners or male partners who were new, casual, or had ≥1 concurrent partnership, approximately 64% of incident HR-HPV infections were attributable to one of those partners. CONCLUSIONS: Among high-risk mid-adult women with recent new male partners, multiple male partners, or male partners who were casual or had ≥1 concurrent partnership, about two thirds of incident HR-HPV detections are likely new acquisitions, whereas about one third of cases are likely redetections of prior infections.

Variant-specific persistence of infections with human papillomavirus Types 31, 33, 45, 56 and 58 and risk of cervical intraepithelial neoplasia.

In our previous study of the etiologic role of oncogenic human papillomavirus (HPV) types other than HPV16 and 18, we observed a significantly higher risk of cervical intraepithelial neoplasia Grades 2-3 (CIN2/3) associated with certain lineages of HPV types 31/33/45/56/58 [called high-risk (HR) variants] compared with non-HR variants. This study was to examine whether these intra-type variants differ in persistence of the infection and persistence-associated risk of CIN2/3. Study subjects were women who had any of HPV types 31/33/45/56/58 newly detected during a 2-year follow-up with 6-month intervals. For each type, the first positive sample was used for variant characterization. The association of reverting-to-negativity with group of the variants and CIN2/3 with length of positivity was assessed using discrete Cox regression and logistic regression, respectively. Of the 598 newly detected, type-specific HPV infections, 312 became undetectable during follow-up. Infections with HR, compared with non-HR, variants were marginally more likely to become negative [adjusted hazard ratio = 1.3; 95% confidence interval (CI), 0.9-1.8]. The adjusted odds ratio associating with the development of CIN2/3 was 3.0 (95% CI, 1.2-7.4) for persistent infections with HR variants for 6 months and 10.0 (95% CI, 3.8-38.0) for persistent infections with HR variants for 12-18 months as compared with the first positive detection of HR variants. Among women with non-HR variants, there were no appreciable differences in risk of CIN2/3 by length of positivity. Findings suggest that the lineage-associated risk of CIN2/3 was not mediated through a prolonged persistent infection, but oncogenic heterogeneity of the variants.

Re-detection vs. new acquisition of high-risk human papillomavirus in mid-adult women.

To understand high-risk (hr) human papillomavirus (HPV) epidemiology in mid-adulthood, we assessed whether associations between incident detection of hrHPV DNA and recent sexual behavior differed according to whether or not there was serologic evidence of prior infection. From 2011 to 2012, we enrolled 409 women aged 30-50 years into a 6-month longitudinal study. We collected health and sexual behavior histories, enrollment sera for HPV antibody testing, and monthly self-collected vaginal swabs for HPV DNA genotyping. Generalized estimating equations logistic regression identified risk factors for type-specific incident hrHPV DNA, stratified by type-specific hrHPV serostatus at enrollment. Population attributable risks of hrHPV due to prior and recent exposure were estimated. When type-specific hrHPV serology was negative, recent sexual risk behavior was positively associated with incident hrHPV DNA (odds ratio in women reporting ≥3 recent sexual risk behaviors [e.g., new or multiple partners] vs. no recent sexual activity = 9.8, 95% CI: 2.4-40.6). No associations with recent sexual behavior were observed with positive type-specific hrHPV serology. Thirty percent of incident hrHPV DNA detection was attributable to prior infection (with positive serology) and 40% was attributable to recent sexual risk behavior (with negative serology). The proportion of incident hrHPV DNA detection attributable to recent sexual risk behavior decreased with increasing age. Among women with serologic evidence of prior infection, re-detection of the same hrHPV type is likely due to reactivation or intermittent detection of persistent infection. Without serologic evidence of prior infection, new detection is likely due to new acquisition or to intermittent detection of persisting infection.

Association of Human Papillomavirus 31 DNA Load with Risk of Cervical Intraepithelial Neoplasia Grades 2 and 3.

The association between human papillomavirus 31 (HPV31) DNA loads and the risk of cervical intraepithelial neoplasia grades 2 and 3 (CIN2-3) was evaluated among women enrolled in the atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) triage study (ALTS), who were monitored semiannually over 2 years and who had HPV31 infections detected at ≥1 visit. HPV31 DNA loads in the first HPV31-positive samples and in a random set of the last positive samples from women with ≥2 HPV31-positive visits were measured by a real-time PCR assay. CIN2-3 was histologically confirmed at the same time as the first detection of HPV31 for 88 (16.6%) of 530 women. After adjustment for HPV31 lineages, coinfection with other oncogenic types, and the timing of the first positive detection, the odds ratio (OR) per 1-log-unit increase in viral loads for the risk of a concurrent diagnosis of CIN2-3 was 1.5 (95% confidence interval [CI], 1.2 to 1.9). Of 373 women without CIN2-3 at the first positive visit who had ≥1 later visit, 44 had subsequent diagnoses of CIN2-3. The initial viral loads were associated with CIN2-3 diagnosed within 6 months after the first positive visit (adjusted OR, 1.5 [95% CI, 1.0 to 2.4]) but were unrelated to CIN2-3 diagnosed later. For a random set of 49 women who were tested for viral loads at the first and last positive visits, changes in viral loads were upward and downward among women with and without follow-up CIN2-3 diagnoses, respectively, although the difference was not statistically significant. Results suggest that HPV31 DNA load levels at the first positive visit signal a short-term but not long-term risk of CIN2-3.

Epidemiology of Human Papillomavirus Detected in the Oral Cavity and Fingernails of Mid-Adult Women.

BACKGROUND: Oral and fingernail human papillomavirus (HPV) detection may be associated with HPV-related carcinoma risk at these nongenital sites and foster transmission to the genitals. We describe the epidemiology of oral and fingernail HPV among mid-adult women. METHODS: Between 2011 and 2012, 409 women aged 30 to 50 years were followed up for 6 months. Women completed health and behavior surveys and provided self-collected oral, fingernail, and vaginal specimens at enrollment and exit for type-specific HPV DNA testing. Concordance of type-specific HPV detection across anatomical sites was described with κ statistics. Using generalized estimating equations or exact logistic regression, we measured the univariate associations of various risk factors with type-specific oral and fingernail HPV detection. RESULTS: Prevalence of detecting HPV in the oral cavity (2.4%) and fingernails (3.8%) was low compared with the vagina (33.1%). Concordance across anatomical sites was poor (κ < 0.20 for all comparisons). However, concurrent vaginal infection with the same HPV type (odds ratio [OR], 101.0; 95% confidence interval [CI], 31.4-748.6) and vaginal HPV viral load (OR per 1 log10 viral load increase, 2.2; 95% CI, 1.5-5.5) were each associated with fingernail HPV detection. Abnormal Papanicolaou history (OR, 11.1; 95% CI, 2.8-infinity), lifetime number of male vaginal sex partners at least 10 (OR vs. 0-3 partners, 5.0; 95% CI, 1.2-infinity), and lifetime number of open-mouth kissing partners at least 16 (OR vs. 0-15 partners, infinity; 95% CI, 2.6-infinity, by exact logistic regression) were each associated with oral HPV detection. CONCLUSIONS: Although our findings support HPV DNA deposition or autoinoculation between anatomical sites in mid-adult women, the rarity of HPV in the oral cavity and fingernails suggests that oral/fingernail HPV does not account for a significant fraction of HPV in genital sites.

Detection of Human Papillomavirus Infections at the Single-Cell Level.

OBJECTIVE: To explore the possibility of single-cell analysis of human papillomavirus (HPV) infection. METHODS: Two hundred and twenty cells were isolated by laser capture microdissection from formalin-fixed and paraffin-embedded cervical tissue blocks from 8 women who had HPV DNA detected in their cervical swab samples. The number of type-specific HPV copies in individual cells was measured by quantitative polymerase chain reaction with and without a prior reverse transcription. The cells were assayed and counted for more than once if the corresponding swab sample was positive for ≥2 HPV types. RESULTS: Infection with HPV16, HPV39, HPV51, HPV52, HPV58, HPV59 and HPV73 was detected in 12 (5.5%) of 220, 3 (9.4%) of 32, 3 (5.8%) of 52, 11 (22.9%) of 48, 9 (18.8%) of 48, 3 (9.4%) of 32 and none of 20 cells, respectively. The numbers of HPV genome copies varied widely from cell to cell. The coexistence of multiple HPV types was detected in 6 (31.6%) of 19 positive cells from 1 of the 6 women who had 2 or 3 HPV types detected in their swab samples. CONCLUSION: Given the heterogeneity of HPV status in individual cells, further clarification of HPV infection at the single-cell level may refine our understanding of HPV-related carcinogenesis.

Viral load and short-term natural history of type-specific oncogenic human papillomavirus infections in a high-risk cohort of midadult women.

Oncogenic human papillomavirus (HPV) viral load may inform the origin of newly detected infections and characterize oncogenic HPV natural history in midadult women. From 2007 to 2011, we enrolled 521 25-65-year-old-female online daters and followed them triannually with mailed health and sexual behavior questionnaires and kits for self-sampling for PCR-based HPV DNA testing. Samples from oncogenic HPV positive women were selected for type-specific DNA load testing by real-time PCR with adjustment for cellularity. Linear or logistic regression models were used to evaluate relationships between viral levels, health and sexual behavior, and longitudinal oncogenic HPV detection. Type-specific viral levels were borderline significantly higher in oncogenic HPV infections that were prevalent versus newly detected (p = 0.092), but levels in newly detected infections were higher than in infections redetected after intercurrent negativity (p < 0.001). Recent sex partners were not significantly associated with viral levels. Compared with prevalent infections detected intermittently, the likelihood of persistent (OR = 4.31, 95% CI: 2.20-8.45) or single-time (OR = 1.32, 95% CI: 1.03-1.71) detection increased per 1-unit increase in baseline log10 viral load. Viral load differences between redetected and newly detected infections suggest a portion of new detections were due to new acquisition, although report of recent new sex partners (a potential marker of new infection) was not predictive of viral load; oncogenic HPV infections in midadult women with new partners likely represent a mix of new acquisition and reactivation or intermittent detection of previous infection. Intermittent detection was characterized by low viral levels, suggesting that intermittent detection of persisting oncogenic HPV infection may be of limited clinical significance.

Human papillomavirus genital infections among men, China, 2007-2009.

To determine prevalence of genital human papillomavirus (HPV) infection among men in rural China, we analyzed genital swab specimens. Among 2,236 male residents of rural Henan Province, HPV infection prevalence was 17.5%. The most common oncogenic and nononcogenic types were HPV-16 and HPV-3, respectively. Infection was associated with younger age and multiple sex partners.

Persistence of newly detected human papillomavirus type 31 infection, stratified by variant lineage.

Variants of human papillomavirus (HPV) type 31 have been shown to be related both to risk of cervical lesions and racial composition of a population. It is largely undetermined whether variants differ in their likelihood of persistence. Study subjects were women who participated in the ASCUS-LSIL Triage Study and who had a newly detected HPV31 infection during a two-year follow-up with six-month intervals. HPV31 isolates were characterized by sequencing and assigned to one of three variant lineages. Loss of the newly detected HPV31 infection was detected in 76 (47.5%) of the 160 women (32/67 with A variants, 16/27 with B variants and 28/66 with C variants). The adjusted hazard ratio associating loss of the infection was 1.2 (95% CI, 0.7-2.1) for women with A variants and 2.1 (95% CI, 1.2-3.5) for women with B variants when compared with those with C variants. Infections with A and C variants were detected in 50 and 41 Caucasian women and in 15 and 23 African-American women, respectively. The likelihood of clearance of the infection was significantly lower in African-American women with C variants than in African-American women with A variants (p = 0.05). There was no difference in the likelihood of clearance between A and C variants among Caucasian women. Our data indicated that infections with B variants were more likely to resolve than those with C variants. The difference in clearance of A vs. C variants in African-Americans, but not in Caucasians, suggests a possibility of the race-related influence in retaining the variant-specific infection.

Association of human papillomavirus type 31 variants with risk of cervical intraepithelial neoplasia grades 2-3.

Although the lineages of human papillomavirus type 31 (HPV31) variants are recognized, their clinical relevance is unknown. The purpose of our study was to examine risk of cervical intraepithelial neoplasia Grades 2-3 (CIN2/3) by HPV31 variants. Study subjects were women who participated in the atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion Triage Study and who had HPV31 infections detected at one or more visits. They were followed semi-annually over 2 years for detection of HPV DNA and cervical lesion. HPV31 isolates were characterized by DNA sequencing and assigned into 1 of 3 variant lineages. CIN2/3 was histologically confirmed in 127 (27.0%) of the 470 HPV31-positive women, 83 diagnosed at the first HPV31-positive visit and 44 thereafter. The odds ratio for the association of 2-year cumulative risk of CIN2/3 was 1.7 (95% CI: 1.0-2.9) for infections with A variants and 2.2 (95% CI: 1.2-3.9) for infections with B variants as compared to those with C variants. Among women without CIN2/3 at the first HPV31-positive visit, the risk of subsequent CIN2/3 was 2.2-fold greater for those with A variants (95% CI: 1.0-4.8) and 2.0-fold greater for those with B variants (95% CI: 0.9-4.9) as compared to those with C variants. Similar associations were observed when CIN3 was used as the endpoint. The findings from our study help to tag HPV31 variants that differ in risk of CIN2/3 and to explain in part why some HPV31 infections regress spontaneously and others lead to disease progression.

Prevalence and risk factors for oncogenic human papillomavirus infections in high-risk mid-adult women.

BACKGROUND: The epidemiology of high-risk (hr) human papillomavirus (HPV) infections in mid-adult women with new sex partners is undefined. METHODS: We analyzed baseline data from 518 25- to 65-year-old women online daters. Women were mailed questionnaires and kits for self-collecting vaginal specimens for polymerase chain reaction-based hrHPV testing. Risk factors for infection were identified using Poisson regression models to obtain prevalence ratios (PRs). RESULTS: The prevalence of hrHPV infection was 35.9%. In multivariate analysis restricted to sexually active women, the likelihood of hrHPV infection was associated with abnormal Papanicolaou test history (PR = 1.42, 95% confidence interval [CI]: 1.10-1.84), lifetime number of sex partners >14 (compared with 1-4; PR = 2.13, 95% CI: 1.13-4.02 for 15-24 partners; and PR = 1.91, 95% CI: 1.00-3.64 for ≥25 partners), male partners with ≥1 concurrent partnership (PR = 1.34, 95% CI: 1.05-1.71), and male partners whom the subject met online (PR = 1.39, 95% CI: 1.08-1.79). Age was inversely associated with infection only in women who were sexually inactive (PR = 0.67 per 5-year age difference, adjusted for Papanicolaou history and lifetime number of partners). Compared with sexually inactive women, the likelihood of infection increased with increasing risk level (from low-risk to hr partners; P < 0.0001 by trend test). In multivariate analysis, infection with multiple versus single hrHPV types was inversely associated with ever having been pregnant (PR = 0.64, 95% CI: 0.46-0.90) and recent consistent condom use (PR = 0.56, 95% CI: 0.32-0.97), and positively associated with genital wart history (PR = 1.43, 95% CI: 1.03-1.99). CONCLUSIONS: Measures of both cumulative and recent sexual history were associated with prevalent hrHPV infection in this hr cohort of mid-adult women.

Viral load in the natural history of human papillomavirus type 16 infection: a nested case-control study.

BACKGROUND: Viral load may influence the course of human papillomavirus type 16 (HPV-16) infection. METHODS: This case-control study was nested within the 2-year Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study, in which women were followed semiannually for HPV and cervical intraepithelial neoplasia (CIN). Case patients (n = 62) were women diagnosed with CIN3 following HPV-16-positive detection at a follow-up visit. HPV-16-positive controls (n = 152) without CIN2 or CIN3 were matched to cases based on the follow-up visit in which viral load was measured. Real-time polymerase chain reaction was used for HPV-16 DNA quantification. RESULTS: The risk of CIN3 increased with increasing HPV-16 DNA load at the follow-up visit (odds ratio, 1.63; 95% confidence interval, 1.33-1.99 per 1 log(10) unit increase); the association was not affected by whether HPV-16 was present at enrollment. When HPV-16 was present at both enrollment and follow-up, viral load remained high among cases (P = .77) but decreased substantially among controls (P = .004). Among women with HPV-16 found initially during follow-up, viral load in the first HPV-16-positive sample was associated with short-term persistence; load was higher in those with infection, compared with those without infection, 1 visit after the initial positivity (P = .001). CONCLUSIONS: Viral load of newly detected infections and changes in viral load predict persistence and progression of HPV-16 infections.

Intrahepatic sarcomatoid cholangiocarcinoma: A case report of the youngest patient on record and a review of the condition's characteristics.

As a rare disease, intrahepatic sarcomatoid cholangiocarcinoma (s-CCC) represents less than 1% of malignancies of the hepatobiliary system and its main clinical symptoms include abdominal pain and fever. Results of pathological examinations, despite being the "gold standard", can easily be confused with hepatocellular carcinoma (HCC). This report is about a 32-year-old male patient who was hospitalized due to occupancy of segment V of the liver for three days and had a history of chronic hepatitis B (CHB) over a 20-year span. Magnetic resonance imaging (MRI) showed a 43 mm × 52 mm-sized liver mass in the V segment, with patchy peripheral enhancement during the arterial phase and rapid wash-out during the portal and late phases. A laparoscopic hepatectomy of segment V, along with cholecystectomy, was performed. Histopathological and immunohistochemical examinations indicated a malignant neoplasm that was positive for vimentin and cytokeratin, with these features providing a positive diagnosis for intrahepatic sarcomatoid cholangiocarcinoma. After surgery, an adjuvant therapy of albumin-paclitaxel combined with gemcitabine regimen was given. No recurrence was found six months after the surgery, with follow-up still ongoing. This report aims to improve the awareness, diagnosis, and treatment of s-CCC.

Downregulation of Cdc2/CDK1 kinase activity induces the synthesis of noninfectious human papillomavirus type 31b virions in organotypic tissues exposed to benzo[a]pyrene.

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16(INK4) and p27(KIP1), which normally mediate pRb hypophosphorylation. In contrast, p21(WAF1) and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.

Human papillomavirus type 16 variants in paired enrollment and follow-up cervical samples: implications for a proper understanding of type-specific persistent infections.

Prospective studies of the persistence of human papillomavirus (HPV) variants are rare and typically small. We sequenced HPV-16 variants in longitudinal pairs of specimens from 86 women enrolled in the ASCUS-LSIL Triage Study. A change of variants was identified in 4 women (4.7% [95% confidence interval, 1.3%-11.5%]). Among women with intervening HPV results (n = 60), a variant switch occurred in 2 of 11 who had evidence of intervening negativity for HPV-16, compared with 1 of 49 who consistently tested positive (P = .11). These results suggest the possibility that rare misclassification of transient infections as persistent infections occurs in natural history studies of type-specific HPV infections.

Human Papillomavirus (HPV) type 16 and type 18 DNA Loads at Baseline and Persistence of Type-Specific Infection during a 2-year follow-up.

BACKGROUND: Studies of viral load-associated persistence of human papillomavirus (HPV) infection are rare, with inconsistent results reported. METHODS: The study subjects were 741 and 289 women who were positive for HPV type 16 (HPV-16) and HPV type 18 (HPV-18), respectively, at the time of enrollment into in the ASCUS-LSIL (Atypical Squamous Cells of Undetermined Significance-Low-Grade Squamous Intraepithelial Lesion) Triage Study and who returned 1 or more times for HPV testing during a biannual 2-year follow-up. The numbers of HPV-16 and HPV-18 copies per nanogram of cellular DNA at baseline were measured by use of real-time polymerase chain reaction. RESULTS: Women with, compared with women without, persistent infection at month 6 of follow-up had a higher viral load at enrollment (P< .001, for HPV-16; P=.01, for HPV-18). The association of each 1-log(10) increase in viral load with persistence of HPV-16 or HPV-18 during the first 6 months of the study was statistically significant among women with multiple HPV types at enrollment (for HPV-16: odds ratio [OR], 1.53 [95% confidence interval {CI}, 1.29-1.82]; for HPV-18: OR, 1.35 [95% CI, 1.09-1.68]) but not among women with monotype infections (in tests assessing the interaction between viral load and coinfection, P=.002 for HPV-16 and P=.34 for HPV-18). Among women who continued to have positive results at month 6, 12, or 18, persistence of infection for another 6 months was unassociated with the viral load at baseline. CONCLUSION: Prevalent infection with a higher viral load of HPV-16 or HPV-18 was associated with short- but not long-term persistence.

A Pooled Analysis to Compare the Clinical Characteristics of Human Papillomavirus-positive and -Negative Cervical Precancers.

Given that high-risk human papillomavirus (HPV) is the necessary cause of virtually all cervical cancer, the clinical meaning of HPV-negative cervical precancer is unknown. We, therefore, conducted a literature search in Ovid MEDLINE, PubMed Central, and Google Scholar to identify English-language studies in which (i) HPV-negative and -positive, histologically confirmed cervical intraepithelial neoplasia grade 2 or more severe diagnoses (CIN2+) were detected and (ii) summarized statistics or deidentified individual data were available to summarize proportions of biomarkers indicating risk of cancer. Nineteen studies including 3,089 (91.0%) HPV-positive and 307 (9.0%) HPV-negative CIN2+ were analyzed. HPV-positive CIN2+ (vs. HPV-negative CIN2+) was more likely to test positive for biomarkers linked to cancer risk: a study diagnosis of CIN3+ (vs. CIN2; 18 studies; 0.56 vs. 0.24; P < 0.001) preceding high-grade squamous intraepithelial lesion cytology (15 studies; 0.54 vs. 0.10; P < 0.001); and high-grade colposcopic impression (13 studies; 0.30 vs. 0.18; P = 0.03). HPV-negative CIN2+ was more likely to test positive for low-risk HPV genotypes than HPV-positive CIN2+ (P < 0.001). HPV-negative CIN2+ appears to have lower cancer risk than HPV-positive CIN2+. Clinical studies of human high-risk HPV testing for screening to prevent cervical cancer may refer samples of HPV test-negative women for disease ascertainment to correct verification bias in the estimates of clinical performance. However, verification bias adjustment of the clinical performance of HPV testing may overcorrect/underestimate its clinical performance to detect truly precancerous abnormalities.

Sequence variation of human papillomavirus type 16 and measurement of viral integration by quantitative PCR.

Given that the integration of human papillomavirus type 16 (HPV16) into the host genome occurs preferentially with the disruption of the E2 gene, a ratio of E2 to E7 gene copies is often used as a marker for integration. It is largely undetermined, however, whether ratio estimates are affected by HPV intratypic variations. We assembled four plasmid constructs, each containing a DNA fragment from an HPV16 European, Asian-American, African-1, or African-2 variant. These constructs and nine cervical swab samples were assayed by real-time PCR with two primer-probe sets for each gene: a specific set, fully complementary to the HPV16 prototype, and a degenerate set, incorporating degenerate bases at positions where nucleotides differed among the variants. The ratio of E2 to E7 gene copies for the European variant construct was close to 1, no matter which sets of primers and probes were used. While the ratios for the African-1 and Asian-American variant constructs remained close to 1 with the degenerate sets of primers and probes, the ratios were 0.36 and 2.57, respectively, with the specific sets of primers and probes. In addition, a nucleotide alteration at the position immediately following the 3' end of the E2 forward primer binding site was found to be responsible for an underestimation of E2 gene copies for the African-2 variant construct. Similar patterns were found in nine cervical samples. In conclusion, mismatches between the primers and probes and their targets due to HPV16 intratypic variations would introduce errors in testing for integration; this situation can be sufficiently ameliorated by incorporating degenerate bases into the primers and probes.

Quantitative human papillomavirus 16 and 18 levels in incident infections and cervical lesion development.

Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV-16 and HPV-18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990 to 2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri-annually, and women were referred for colposcopically directed biopsy when indicated. Quantitative real-time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV-16 and HPV-18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV-16 mRNA levels at the time of incident HPV-16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2-3 (HR per 1 log(10) increase in mRNA = 6.36, 95% CI = 2.00-20.23). Increasing HPV-16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV-16 E7 DNA levels nor HPV-18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR] = 10.05, 95% CI = 1.09-92.56) and increased HPV DNA (VLR = 16.80, 95% CI = 1.46-193.01). In newly detected HPV-16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre-cancer.

Changes in DNA Level of Oncogenic Human Papillomaviruses Other Than Types 16 and 18 in Relation to Risk of Cervical Intraepithelial Neoplasia Grades 2 and 3.

BACKGROUND: Epidemiologic data addressing clinical relevance of viral load fluctuation of oncogenic types other than human papillomavirus (HPV) types 16 and 18 are limited. METHODS: A type-stratified set of infections by non-HPV16/18 oncogenic types that were detected at ≥2 visits was randomly selected from women who were enrolled in a clinical trial and followed every 6 months for 2 years for detection of HPV and cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3). Type-specific viral load was measured on both first and last HPV-positive cervical swab samples. RESULTS: CIN2/3 was initially confirmed at the last HPV-positive visit for 67 of 439 infections. The increase in risk of CIN2/3 was associated with high, relative to low, viral load at both first and last positive visits [ORadjusted = 3.67; 95% confidence interval (CI), 1.19-11.32] and marginally associated with a change of viral load from low to high levels (ORadjusted = 3.15; 95% CI, 0.96-10.35) for infection by species group alpha-9 non-HPV16 oncogenic types but not species group alpha-5-7 non-HPV18 oncogenic types. Among women with an initial diagnosis of CIN2/3 at the first positive visit, CIN2/3 was more frequently redetected at the last positive visit for infections with, compared with without, high DNA load of species group alpha-9 non-HPV16 oncogenic types at both visits (P exact = 0.04). CONCLUSIONS: In agreement with data on baseline viral load, the viral load change-associated risk of CIN2/3 differs by HPV species groups. IMPACT: These findings underscore the importance of distinguishing species groups in future studies of clinical relevance of HPV DNA load.