Subtype-specific prognostic impact of different immune signatures in node-negative breast cancer.

BACKGROUND: The role of different subtypes of immune cells is still a matter of debate. METHODS: We compared the prognostic relevance for metastasis-free survival (MFS) of a B-cell signature (BS), a T-cell signature (TS), and an immune checkpoint signature (CPS) in node-negative breast cancer (BC) using mRNA expression. Microarray-based gene-expression data were analyzed in six previously published cohorts of node-negative breast cancer patients not treated with adjuvant therapy (n = 824). The prognostic relevance of the individual immune markers was assessed using univariate analysis. The amount of independent prognostic information provided by each immune signature was then compared using a likelihood ratio statistic in the whole cohort as well as in different molecular subtypes. RESULTS: Univariate Cox regression in the whole cohort revealed prognostic significance of CD4 (HR 0.66, CI 0.50-0.87, p = 0.004), CXCL13 (HR 0.86, CI 0.81-0.92, p < 0.001), CD20 (HR 0.76, CI 0.64-0.89, p = 0.001), IgκC (HR 0.81, CI 0.75-0.88, p < 0.001), and CTLA-4 (HR 0.67, CI 0.46-0.97, p = 0.032). Multivariate analyses of the immune signatures showed that both TS (p < 0.001) and BS (p < 0.001) showed a significant prognostic information in the whole cohort. After accounting for clinical-pathological variables, TS (p < 0.001), BS (p < 0.05), and CPS (p < 0.05) had an independent effect for MFS. In subgroup analyses, the prognostic effect of immune cells was most pronounced in HER2+ BC: BS as well as TS showed a strong association with MFS when included first in the model (p < 0.001). CONCLUSION: Immune signatures provide subtype-specific additional prognostic information over clinical-pathological variables in node-negative breast cancer.

Prognostic significance of interferon regulating factor 4 (IRF4) in node-negative breast cancer.

PURPOSE: The transcription factor IRF4 regulates immunoglobulin class switch recombination as well as plasma cell differentiation. We examined the prognostic significance of IRF4 expression in node-negative breast cancer (BC). METHODS: IRF4 expression was evaluated by immunostaining in a cohort of 197 node-negative BC patients not treated in adjuvant setting, referred to as Mainz cohort. The prognostic significance of immunohistochemically determined IRF4 expression for metastasis-free survival (MFS) was examined by Kaplan-Meier survival analysis as well as univariate and multivariate Cox analysis adjusted for age, pT stage, histological grade, ER, and HER2 status. For verification of immunohistochemical results, IRF4 mRNA expression was evaluated using microarray-based gene expression profiling in four previously published cohorts (Mainz, Rotterdam, Transbig, Yu) consisting of 824 node-negative breast cancer patients in total, who were not treated with adjuvant therapy. The prognostic significance of IRF4 mRNA expression on metastasis-free survival (MFS) was examined by univariate and multivariate Cox analysis in the Mainz cohort and by a meta-analysis of all node-negative BC patients and different molecular subtypes. IRF4 mRNA levels were compared to immunohistochemically determined IRF4 expression in 140 patients of the Mainz cohort using Spearman correlation. RESULTS: Immunohistochemically determined high IRF4 expression was associated with higher MFS in univariate Cox regression (HR 0.178, 95% CI 0.070-0.453, p < 0.001). IRF4 maintained its significance independently of established clinical factors for MFS (HR 0.088, 95% CI 0.033-0.232, p < 0.001). Immunohistochemically, determined IRF4 correlated moderately with IRF4 mRNA expression (ρ = 0.589). Higher expression of IRF4 was associated with better MFS in a meta-analysis of the total cohort (HR 0.438, 95% CI 0.307-0.623, p < 0.001). Prognostic significance was more pronounced in the HER2+ molecular subtype (HR 0.215, 95% CI 0.090-0.515, p = 0.001) as compared to the luminal A (HR 0.549, 95% CI 0.248-1.215, p = 0.139), luminal B (HR 0.444, 95% CI 0.215-0.916, p = 0.028), and basal-like subtypes (HR 0.487, 95% CI 0.269-0.883, p = 0.018). Further, IRF4 expression showed independent prognostic significance in a multivariate analysis of the Mainz cohort (HR 0.236, 95% CI 0.105-0.527, p < 0.001). CONCLUSIONS: IRF4 had independent prognostic significance in node-negative BC. Higher expression of IRF4 was associated with improved outcome. The prognostic impact differed between diverse molecular subtypes and was most pronounced in HER2+ breast cancer.

Targeted resequencing of candidate genes using selector probes.

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

Prediction of disturbed eating attitudes in adolescent girls: a 3-year longitudinal study of eating patterns, self-esteem and coping.

OBJECTIVE: The purpose was to examine the extent to which yearly assessments of eating patterns and attitudes, self-esteem and coping strategies over a 3-year period among adolescent girls predicted the degree of disturbed eating attitudes at the year 3-assessment. Our main hypothesis was that such attitudes year 3 would be predicted by eating attitudes, restrained, emotional, and external eating behaviour, as well as by low self-esteem and coping by acting out or avoidance. METHOD: Three-hundred and seventy- eight Swedish adolescent girls were assessed once a year for three years. RESULTS: The results suggest that eating patterns and attitudes were the strongest predictors of disturbed eating attitudes year 3. Self-esteem and coping had a limited predictive value for eating attitudes year 3, and the effect of self-esteem appeared to be mediated by coping. DISCUSSION: The results suggest that early eating patterns (e.g., more disturbed eating attitudes and restrained eating behaviors) and attitudes are potentially important predictors for the development of more serious eating disturbances among adolescent girls.

Expression profiling of microdissected cell populations selected from basal cells in normal epidermis and basal cell carcinoma.

BACKGROUND: Basal cell carcinomas (BCCs) are prevalent tumours with uniform histology that develop without any known precursor lesion. Alterations in the sonic hedgehog-patched1 signalling pathway are accepted as necessary events for tumorigenesis, and mutations in the patched1 gene are frequently present in tumours. OBJECTIVES: To analyse transcript profiles in BCC. METHODS: We used laser-assisted microdissection to isolate and collect cell populations defined under the microscope. Peripheral cells from nests of BCC were selected to represent tumour cells, and normal keratinocytes from epidermis basal layer were used as control. Extracted RNA was amplified and hybridized on to a cDNA microarray. Results Our results show that BCC cells express a transcript signature that is significantly different from that of normal keratinocytes, and over 350 genes with various functions were identified as differentially expressed. The compiled data suggest an upregulation of the Wnt signalling pathway as a major event in BCC cells. Furthermore, tumour cells appear to have an increased sensitivity to oxygen radicals and dysregulated genes involved in antigen presentation. RESULTS: were validated at both the transcriptional level using real-time polymerase chain reaction and at the protein level using immunohistochemistry. CONCLUSIONS: We show that microdissection in combination with robust strategies for RNA extraction, amplification and cDNA microarray analysis allow for reliable transcript profiling and that antibody-based proteomics provides an advantageous strategy for the analysis of corresponding differentially expressed proteins. We found that expression patterns were significantly altered in BCC cells compared with basal keratinocytes and that the Wnt signalling pathway was upregulated in tumour cells.

Psychometric properties of the Eating Disorders in Obesity questionnaire: validating against the Eating Disorder Examination interview.

Inconclusive results of how weight-loss treatment (WLT) results are affected by participants' eating disorders and/or binge eating are partly due to the variation caused by the multitude of assessment instruments used. The objective of the present study was to evaluate the psychometric properties of a short DSM-IV-based assessment instrument designed to be used specifically in WLT settings, the Eating Disorders in Obesity (EDO) questionnaire. Participants were 97 patients seeking WLT at four surgical and one non-surgical clinics. Participants were assessed by the EDO and the Eating Disorder Examination (EDE) interview . The validity and reliability of the EDO was measured as concordance with the EDE, and test-retest agreement of the EDO, respectively. Validity as well as reliability was found to be good for both eating disorders diagnoses and binge eating as a distinct symptom. Results suggest that the EDO is a short, easily administered instrument with good psychometric properties which makes it a suitable, economical method of assessing eating disorders and binge eating in clinical WLT settings.

Initial interactions of subgenus D adenoviruses with A549 cellular receptors: sialic acid versus alpha(v) integrins.

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

Adenovirus type 37 uses sialic acid as a cellular receptor.

Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) alpha2, have recently been identified. In the absence of CAR, MHC-I alpha2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expressing CHO cells and MHC-I alpha2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via alpha(2-->3)-linked sialic acid saccharides rather than alpha(2-->6)-linked ones, since (i) alpha(2-->3)-specific but not alpha(2-->6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with alpha(2-->3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of alpha(2-->3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I alpha2.

Sexual behaviour and papillomavirus exposure in cervical intraepithelial neoplasia: a population-based case-control study.

Sexual history is an established risk determinant for cervical neoplasia. It is not clear if human papillomavirus (HPV) exposure entirely explains the sexual behaviour-related risk or if other sexually transmitted agents may act as cofactors for HPV in carcinogenesis. The aim of this study was to elucidate whether HPV exposure or HPV persistence explains the sexual history-related risk of high-grade cervical intraepithelial neoplasia (CIN) using a population-based case-control study of most of the 254 women referred to colposcopy in the Vasterbotten county in Sweden because of an abnormal cervical smear during October 1993 to December 1995 and 320 age-matched women from the general population. The women were interviewed for sexual history and tested for presence of serum antibodies to HPV-16, -18 and -33 as well as for presence of HPV DNA in cervical brush samples. HPV-16, -18 and -33 seropositivity was specific for the corresponding type of HPV DNA, dependent on the lifetime sexual history and associated with a two- to threefold increased risk of CIN 3. There was no sexual history-related risk of CIN among HPV-seropositive women and adjustment for HPV DNA presence explained the sexual history-related risk of CIN. In conclusion, HPV exposure appeared to explain the sexual history-related risk of high-grade CIN.

The first human isolate of Puumala virus in Scandinavia as cultured from phytohemagglutinin stimulated leucocytes.

A virus isolate was recovered from blood leucocytes of a patient with nephropathia epidemica (NE). Leucocytes were isolated from EDTA-blood by dextran sedimentation and cultured on monolayers of Vero E6 cells in the presence of phytohemagglutinin (PHA) in roller tubes during the first 72 hours of incubation followed by rolling culture for three weeks in total. Thereafter the first subculture was done in a plastic flask and afterward at at least 6 week intervals. Antigen was first detected after 6 months and 2 weeks of culture. When tested by monoclonal antibodies and patient sera the isolate had the characteristics of a PUU virus. PCR amplification using PUU-specific primers and subsequent partial sequencing of the S and M segments revealed that the Umeå/305/human/95 virus differs from the Finnish PUU Sotkamo rodent prototype virus and is similar but not identical to rodent strains of PUU virus acquired from the same region as the patient isolate. It is we concluded that the first human isolate of the etiologic agent of NE in Scandinavia was recovered from blood leucocytes stimulated with PHA by long-term culture in Vero E6 cells. The isolate belongs to the PUU serotype of hantaviruses as shown by its serologic profile and partial sequencing data.

Nonradioisotopic detection and typing of human papillomaviruses by use of polymerase chain reaction and single-strand conformation polymorphism.

The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques.

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.

The influence of sexual and social factors on the risk of Chlamydia trachomatis infections: a population-based serologic study.

BACKGROUND: Genital chlamydia infections often are asymptomatic, which promotes their spread in the population. In women, the possible consequences of infection are pelvic inflammatory disease and infertility. Most studies on the prevalence of Chlamydia trachomatis have been based on clinical series, and prevalences tend to vary with the clinical setting. Few seroepidemiologic studies have emerged from industrialized countries. GOAL OF THIS STUDY: To assess the prevalence of Chlamydia trachomatis using culture and serology, and its relationship with possible risk factors. STUDY DESIGN: This was a population-based study involving completion of a self-administered questionnaire, analysis of cervical samples for Chlamydia trachomatis, and serologic tests for Chlamydia trachomatis antibodies. RESULTS: The prevalence of Chlamydia trachomatis infection was 2.7%, and the seroprevalence was 24.7% among the sexually active women. Seropositivity was correlated with sexual behavior variables, and the incidence of serologic cross-reactivity with respiratory infections (strain TWAR) was low. Multivariate logistic regression analysis showed the number of sexual partners, age at first coitus, history of therapeutic abortion, and previous pelvic inflammatory disease to be independently correlated with seropositivity. CONCLUSION: Early sexual experience and multiple lifetime sexual partners are independent risk factors for Chlamydia trachomatis infection.

The silent suffering women--a population based study on the association between reported symptoms and past and present infections of the lower genital tract.

OBJECTIVES: To assess the prevalence of lower genital tract symptoms and the association between reported symptoms and past and present signs of sexually transmitted diseases (STD) in young women. DESIGN: All women belonging to the 19-, 21-, 23- and 25-year age cohorts and living in the catchment area of the community health centre, were invited by mail to take part in a population-based study. The participants answered a structured questionnaire and a gynaecologic examination was performed. Samples for wet smear, cervical Pap smear, HPV DNA determination and Chlamydia trachomatis culture were taken at the gynaecologic examination. The presence of genital warts was noted. A blood sample was analysed for antibodies against C trachomatis and HSV-2. SETTING: The community health care centre was located in Umeå, a city in Northern Sweden. RESULTS: Of the 886 women who were eligible, 611 (70%) participated in the investigation. One out of four women reported symptoms from the lower genital tract. The most commonly reported symptoms were itching, followed by discharge, and soreness. The most commonly reported STD was C trachomatis (15%). The most prevalent present STD was HPV infection (20%) whereas C trachomatis infection could be isolated from 2.7% of the women. Antibodies against C trachomatis and HSV-2 were present among 22% and 6% of the women, respectively. There was a significant correlation between the women's complaint of vaginal discharge and previous C trachomatis infection, lack of lactobacilli and presence of leucocytosis in wet smear. CONCLUSIONS: We have in a population-based study of young healthy women found that one out of four women had some kind of lower genital tract complaint. Itching was the most commonly reported symptom and was associated with pseudohyphae and acetowhite patches. Reported vaginal discharge and soreness were associated with the history of a past C trachomatis infection and signs of a disturbed vaginal flora.

Human papillomavirus infection is transient in young women: a population-based cohort study.

The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from a cohort of 276 young women was determined by a general two-step polymerase chain reaction. HPV infection fluctuated among young women during a 2-year interval. The total prevalence of HPV infection decreased from 21% to 8.3%. The most prevalent HPV types at enrollment were HPV-16 (3.3%) and HPV-6 (2.9%). At follow-up, the most common type was HPV-16 (2.9%), while no HPV-6 was detected. In 2 women only, the same HPV type persisted. Regression of HPV infection was found in 80% of the women. A new HPV type-specific infection was detected in 7.2% of the women and was independently associated with a new sex partner or an abnormal smear since enrollment.

Lifetime number of partners as the only independent risk factor for human papillomavirus infection: a population-based study.

BACKGROUND AND OBJECTIVES: Previous studies of relationships between genital human papillomavirus infection and tentative risk factors have yielded conflicting results, possibly because of inaccuracy of the viral detection methods used and differences in selection criteria. GOAL OF THIS STUDY: To determine human papillomavirus prevalence and identify risk factors in a group of young Swedish women. STUDY DESIGN: This was a population-based study involving completion of a structured questionnaire, analysis of cervical scrapings for human papillomavirus and Chlamydia trachomatis, and serologic tests for C. trachomatis and herpes simplex virus antibodies. RESULTS: The prevalence of human papillomavirus infection was 22% among the sexually active women and 4% among the virgins. A number of factors were associated with human papillomavirus prevalence in univariate analysis, but logistic regression analysis showed that lifetime number of male sexual partners was the only independent risk factor for human papillomavirus infection (adjusted odds ratio, 7.45; 95% CI, 2.79-19.92 for six or more partners vs. one partner). CONCLUSION: Human papillomavirus infection is a prevalent sexually transmitted disease among young Swedish women, and the lifetime number of male sexual partners is a major risk factor.

Prediction of behavior-management problems in 3-year-old children.

The aim was to determine power of nondental background factors to predict behavior-management problems at the first dental visits of 3-yr-old children. A total of 273 children from three kinds of residential area--city, town, and rural area--in Sweden took part. The parents were interviewed before the child's dental visit. The behavior of the child was rated by registering the degree of acceptance according to the method of HOLST & CROSSNER. The following steps were rated: entering the dental treatment room, mirror in mouth, probe on fingernail and tooth, air-blower on hand and in mouth, sitting in the dental chair, and examination. The behavior was then analyzed in relation to the answers of the interviews, and a logistic regression model was used to calculate the power of the variables, separately or combined, to predict behavior-management problems. Seventy-six percent of the children cooperated well at the dental examination; 13% reacted reluctantly, and 11% reacted negatively. Two interview variables had statistically significant predictive power: the parent's expectation of a negative reaction from the child in the dental situation and the child's anxiety when meeting unfamiliar people. Sixty-nine percent of child patients accepted the examination while sitting alone in the chair. Sitting alone used as a predictor for cooperation showed sensitivity 0.80, specificity 0.71, predictive value for positive test 0.44, and predictive value for negative test 0.94.

Comparison of a one-step and a two-step polymerase chain reaction with degenerate general primers in a population-based study of human papillomavirus infection in young Swedish women.

The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from young women was determined by polymerase chain reaction (PCR) by using general primer pairs localized within the L1 region. With a one-step general PCR, 5.9% (35 of 590) of young women in a population-based study were found to contain HPV DNA. The proportion of HPV-positive women increased with age, from 1.4% (1 of 69) among women aged 19 years to 9.2% (13 of 142) among women aged 25 years. Among the cervical scrapes from women with normal cytology, 5.6% (30 of 539) harbored HPV DNA. A total of 5 of 19 (26.3%) of the women with pathological signs were positive for HPV DNA. By a two-step PCR, using nested general primers, 20.3% (118 of 581) of all women were shown to contain HPV DNA. The proportion of HPV-positive women also increased with age, from 17.4% (12 of 69) among women aged 19 years to 31.9% (43 of 135) among women aged 25 years, when the two-step PCR was used. Some 19.2% (102 of 530) of cervical scrapes from women with normal cytology contained HPV DNA. Among the women with pathological signs, 16 of 19 (84.2%) were positive for HPV DNA. The HPV DNA-positive specimens were demonstrated to contain HPV type 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 55, or 56. The most prevalent HPV types were 6 (2.0%) and 16 (2.7%). More than one type was found in 16 specimens. Sixty HPV-positive samples could not be typed.