Human Papillomavirus DNA In Cervical Smears Research Articles

Concentration levels of IL-10 and TNFα cytokines in patients with human papilloma virus (HPV) DNA+ and DNA− cervical lesions

The present study was performed to assess the immune response in women with human papilloma virus (HPV) DNA+ and DNA− cervical lesions. Eighty women with cervical lesions (age range = 25–70 years) and 20 healthy individuals (control group) were enrolled in the study. Lesions were cytologically classified into four groups: ASC-US (20), CINI (30), CINII-III (16), and cervical carcinoma (14) prior to HPV DNA detection. Estimation of interleukin (IL)-10 and tumor necrosis factor (TNF)-α levels in cervical secretions and serum of the studied patients was performed utilizing ELISA. PCR screening kits were used to detect HPV DNA in cervical smears obtained from the studied cases with the different lesions. IL-10 levels in cervical secretions of HPV DNA+ were significantly greater than those from DNA− patients (i.e., 88.73 vs 24.00 pg/ml) and from controls (i.e., 88.73 vs 8.27 pg/ml) and the levels were higher in DNA− patients than in controls (i.e., 24.00 vs 8.27 pg/ml). In comparison, serum IL-10 levels in these patients did not significantly differ from control values (i.e., 13.69 vs 12.16 vs 9.99 pg/ml, respectively). TNFα levels in cervical secretions of the HPV DNA+ and DNA− cases did not significantly differ from values for the controls (i.e., 12.18 vs 9.90 vs 7.90 pg/ml, respectively). Serum TNFα of these patients also did not differ significantly from controls (i.e., 11.59 vs 11.90 vs 10.83 pg/ml, respectively). The detected levels of IL-10 in cervical secretions of patients with HPV DNA+ lesions was significantly higher than in their sera, while secretion TNFα levels were nominally greater than sera values. Lastly, higher levels of IL-10 were observed in secretions of 10–14 (71.4%) patients who had progressive cervical lesions (HSIL and cervical cancer stages) who were HPV DNA+ than observed in 20 of 66 (30.0%) of DNA− patients with similar progressive lesions. In general, the higher levels of IL-10 than of TNFα suggested a potential down-modulation of tumor-specific immune responses to HPV-infected lesions. This phenomenon appears to provide a tumor ‘progressive’ microenvironment in these particular patients.

Model-Based Estimation of Viral Transmissibility and Infection-Induced Resistance From the Age-Dependent Prevalence of Infection for 14 High-Risk Types of Human Papillomavirus

Viral transmissibility and natural resistance to infection are key determinants in assessing the population impact of human papillomavirus (HPV) vaccination, yet information on these parameters is scarce. Using data from 2 large-scale surveys on sexual behavior in the Netherlands (carried out in 2005-2006), the authors employed a Bayesian framework to fit a transmission model to the cross-sectional age-dependent prevalence of HPV DNA in cervical smears (data collected in 1992-2002), assuming that the prevaccine situation reflected an endemic equilibrium, and calculated type-specific estimates of transmissibility and infection-induced resistance. The posterior median transmission probability per heterosexual partnership covered a range of 0.43-0.94 among the 14 high-risk types of HPV. The transmission probability of HPV-16 was estimated at 0.80 (95% posterior interval: 0.60, 0.99) and that of HPV-18 at 0.93 (95% posterior interval: 0.72, 1). The model predicted that the decrease in HPV prevalence with age could not solely be explained by sexual activity and screening but also by resistance to reinfection, which is lost at a rate of 0.014-0.047 (1%-5%) per year. These results support the notion that HPV infection is highly transmissible, and they suggest a gradual loss of type-specific immunity over time. Because high transmission potential is associated with a low impact of herd immunity, extensive vaccination coverage will be required to substantially reduce cervical cancer incidence.

Detection of human papillomavirus in cervical cell specimens by hybrid capture and PCR with different primers.

The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n=42) and abnormal (n=83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (chi2, P<0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique--78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific--the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.

Longitudinal study of patients after surgical treatment for cervical lesions: detection of HPV DNA and prevalence of HPV-specific antibodies

The principal aims of this study were to test whether persistence of human papillomavirus (HPV) DNA is predictive of recurrent disease in women after surgical treatment for cervical lesions, to distinguish between persistent and newly acquired HPV infection, and to observe the effect of surgical treatment on levels of HPV-specific antibodies. A group of 198 patients surgically treated for low-grade and high-grade squamous intraepithelial lesions and 35 age-matched controls were monitored for 18 months at 6-month intervals. The presence of HPV DNA in cervical smears was detected by means of consensus polymerase chain reaction, and serum levels of HPV-specific antibodies to HPV types 16, 18, 31, 33, and 45 were measured. In ten patients positive for HPV type 16 in consecutive samples, the HPV 16 variants were identified using a polymerase chain reaction specific for the long control region. Data regarding demographics, risk factors for cervical cancer, and risks related to HPV exposure were collected through a patient questionnaire. Subjects persistently positive for HPV DNA were more likely to present with cytological and/or colposcopical abnormalities. A higher reactivity to HPV-specific antibodies was observed in these women at the 18-month follow-up visit. All ten patients with HPV 16 infection detected in consecutive samples showed persistence of either the same prototype or the same variant during the follow-up period. Risky sexual behavior and smoking were more common in patients than in controls. Persistent HPV infection as demonstrated by both HPV DNA detection and antibody detection appears to be a risk factor for the recurrence of pathological findings in women after surgery. An individually based approach to surgical treatment is an important factor in the outcome of disease at follow-up.

Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison.

BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for...

Increased IL-6 and IL-8 Levels in Cervicovaginal Secretions of Patients with Cervical Cancer

Objective.Conflicting data exist on IL-6 production by human papillomavirus (HPV) immortalized cell lines and several cervical carcinoma cell lines. However, no information has been reported on the levels of cytokines in cervicovaginal washings in relation to cervical neoplasia. The aim of this study was to investigate whether local production of IL-6 could be found and whether the level of this cytokine was related to the severity of cervical neoplasia. IL-8 was measured to obtain additional information on an inflammatory cytokine with possible epithelial origin.Methods.Cervicovaginal washings and sera were obtained from 35 patients with invasive cervical cancer, 62 patients with cervical intraepithelial neoplasia (CIN), and 25 control subjects. IL-6 and IL-8 levels were determined by ELISA. HPV DNA in cervical smears was detected by a HPV-16-specific PCR method and additionally by CPI/IIG PCR. Histological analysis of the inflammatory infiltrate was performed on hematoxylin–eosin-stained tissue sections.Results.In the patients with cervical cancer, those with CIN, and the controls, the median IL-6 concentration in cervicovaginal washings was 171 pg/ml (interquartile range: 54–780), 22 pg/ml (<2–73), and < 2 pg/ml (<2–<2), respectively. For IL-8, the levels were 2756 pg/ml (1651–7107), 489 pg/ml (248–1158), and 631 pg/ml (346–897), respectively. In most subjects the local levels were much higher than in serum. Local IL-6 and IL-8 levels were significantly higher in patients with cervical carcinoma compared with CIN patients and controls. Likewise, local IL-6 levels were increased in patients with CIN compared with controls. No relation was found between cytokine levels and CIN grade or between cytokine levels and the inflammatory infiltrate scored by histological analysis.Conclusions.There is local production of IL-6 and IL-8 in cervicovaginal secretions, and the production of IL-6 was related to the severity of cervical neoplasia.

Early regression of cervical lesions in HIV-seropositive women receiving highly active antiretroviral therapy.

Advanced HIV disease is associated with a high prevalence of cervical squamous intra-epithelial lesions (SIL) and of infection with oncogenic human papillomavirus (HPV) genotypes. Triple-combination antiretroviral therapy results in decreased plasma HIV viral load, increased CD4 cell counts and partial restoration of immune functions in patients with severe HIV disease. This study investigated the outcome of SIL in HIV-seropositive women undergoing triple combination antiretroviral treatment. Forty-nine women who started triple-combination antiretroviral therapy, including a protease inhibitor, were examined prior to and after a median 5-month treatment. We collected cytological, colposcopic and histologic data and assessed the presence of HPV DNA in cervical smears by PCR and Southern blot hybridization (SBH). The prevalence of SIL decreased from 69 to 53% during follow-up (P < 0.0001). Among 13 women who initially presented with high-grade SIL, conversion to lower grade was observed in two women and a full regression to normality was observed in one. Cytology also returned to normality in nine out of 21 women who initially presented with low-grade SIL. The high prevalence of HPV infection as detected by SBH and PCR was similar at the first and second examinations and the same high-risk viral genotypes were identified at both examinations in all infected patients but one. There was a higher increase in absolute CD4 cells in the subgroup of patients whose lesions regressed (99 versus 50 x 10(6)/l, P=0.03). Our observations demonstrate that active antiretroviral therapy may result in a reduced prevalence of cervical squamous intra-epithelial lesions despite the absence of clearance of HPV infection.

A prospective study on the risk of cervical intra-epithelial neoplasia among healthy subjects with serum antibodies to HPV compared with HPV DNA in cervical smears.

To estimate the risk of developing cervical intra-epithelial neoplasia (CIN) among women exposed to human papillomavirus (HPV) type 16, we performed a prospective study in a population-based cohort of more than 15,000 women followed for 34.9 months. Seventy-four women developed CIN during follow-up and were matched for age, time of sampling and area of residence with 148 women who remained CIN-free during follow-up. The blood samples taken at enrollment were tested for serum antibodies to HPV types 16, 18 and 33 capsids. Cervical smears or biopsies were analyzed for the presence of HPV DNA by nested PCR using HPV general primers and by HPV 16- and 18-type-specific PCR HPV serology and HPV-PCR were in good agreement, particularly when the blood sample and the Pap smear were taken less than 6 months apart. HPV DNA was found in 88% of cases and 4% of controls, whereas HPV 16 DNA was present in 44% of cases and in 1 of 142 controls. HPV-16-seropositive women had a 3-fold increased risk of developing CIN. The risk was highest among women younger than 35 years of age, of whom an estimated 3.4% of HPV-16-seropositive and 0.5% of seronegative women developed CIN. Since the risk associated with HPV-16 seropositivity (a measure of past or present infection) was 35-fold lower than that of HPV DNA (present infection), most infections appear to be eliminated before CIN develops. In conclusion, HPV 16 infection does confer an excess risk of CIN development, and HPV DNA detection has a high predictive value for the presence of high-grade CIN.

Non-isotopic detection of HPV DNA in cervical smears using dot-blot hybridization

Infection of the cervix with specific types of the human papillomavirus (HPV) results in condylomatous, dysplastic and/or neoplastic epithelial changes. To enable routine screening of premalignant cervical lesions for HPV DNA, we have developed a sensitive method of detecting HPV -6, -11, -16, -18 and -33 in cervical scrapes using a non-isotopic, dot-blot hybridization assay. Cloned, genomic HPV DNA is labelled with biotin-11-dUTP by nick translation, and used to screen for HPV types under high stringency conditions. DNA is extracted from cervical cells and spotted on nitrocellulose membranes. Cervical cells, from 20 patients attending colposcopy were screened for HPV types. All patients had a dysplasia or condyloma. HPV DNA was isolated from 80%. The method visualized an amount of target DNA as low as 1 pg without background. Compared with Southern blot hybridization utilizing radiolabelled probe, this assay is equally sensitive and specific. The technique is easy to do, and may significantly contribute to the management of cervical dysplasia.