Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

RATIONALE: Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. METHODS: An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). RESULTS: Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. CONCLUSIONS: The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated.

An assessment of the accuracy and usability of a novel optical wound measurement system.

AIMS: Measurement of wound size can predict healing and provide information to guide treatment. This study assesses a novel optical wound imaging system that creates a three-dimensional image of the ulcer. METHODS: Using a new camera-based digital system and traditional elliptical wound measurements, 36 foot ulcers from 31 patients (aged 44-94 years, median 70 years) were examined during a 12-week period at two centres. Median diabetes duration was 18 years (range 6-56 years). Seventeen percent had Type 1 diabetes, 93% had peripheral neuropathy and 57% had peripheral artery disease. Twenty-five were reviewed consecutively, resulting in 76 ulcer examinations. Median ulcer size was 94 mm(2), with size ranging from 3.1 to 2195 mm(2). RESULTS: Pearson, Spearman and Kendall rank coefficients showed a strong correlation (in all cases P < 0.001) between digital measurements of wounds against traditional hand-measured estimates. Intra-observer variation of wound length using digital elliptical measurement (DEM) gave a coefficient of variation of < 3.0%. Interobserver variation of wound length using DEM was < 6.5%. Variation from a standard known-size wound area was < 8.0% across 30 trials. CONCLUSIONS: This study shows a strong correlation between digital and traditional measurement techniques. The system can be easily deployed in routine clinical practice, providing an objective visual record, allowing remote in-depth analysis.

Absence of human papillomavirus in esophageal carcinomas from southwestern Kenya.

Esophageal squamous cell cancer is highly prevalent in south-western Kenya. The role of human papillomavirus (HPV) in esophageal cancers from this region was evaluated. Biopsies of 29 esophageal squamous cell cancers were assayed for HPV DNA sequences by reverse line blot polymerase chain reaction, using 27 HPV type-specific probes. Viral sequences were found in none of the specimens. These results suggest the HPV is unlikely to be an etiologic factor for esophageal squamous cell cancers in this region.

Membrane transporters as machines: degenerate singularities as a requirement for function.

It is suggested that Membrane Transporter functionality is based on low energy paths between proteins of different conformations. A simple extension of the Born-Oppenheimer approximation is used to reduce the protein structure problem to one of the kinematics of engineering mechanisms. Such low energy paths between conformations with the same handedness imply the existence of degenerate singularities in the engineering mechanism. The requirement for degeneracy leads to a number of conjectures. These include the structure and function of chaperones for constructing such proteins and the thermodynamic properties of membrane transporters.

Poliovirus vaccination during pregnancy, maternal seroconversion to simian virus 40, and risk of childhood cancer.

Before 1963, poliovirus vaccine produced in the United States was contaminated with simian virus 40 (SV40), which causes cancer in animals. To examine whether early-life SV40 infection can cause human cancer, the authors studied 54,796 children enrolled in the US-based Collaborative Perinatal Project (CPP) in 1959-1966, 52 of whom developed cancer by their eighth birthday. Those children whose mothers had received pre-1963 poliovirus vaccine during pregnancy (22.5% of the children) had an increased incidence of neural tumors (hazard ratio = 2.6, 95% confidence interval: 1.0, 6.7; 18 cases) and hematologic malignancies (hazard ratio = 2.8, 95% confidence interval: 1.2, 6.4; 22 cases). For 50 CPP children with cancer and 200 CPP control children, the authors tested paired maternal serum samples from pregnancy for SV40 antibodies using a virus-like particle enzyme immunoassay and a plaque neutralization assay. Overall, mothers exhibited infrequent, low-level SV40 antibody reactivity, and only six case mothers seroconverted by either assay. Using the two SV40 assays, maternal SV40 seroconversion during pregnancy was not consistently related to children's case/control status or mothers' receipt of pre-1963 vaccine. The authors conclude that an increased cancer risk in CPP children whose mothers received pre-1963 poliovirus vaccine was unlikely to have been due to SV40 infection transmitted from mothers to their children.

Diagnosis of human papillomavirus infection by dry vaginal swabs in military women.

OBJECTIVE: Human papillomavirus (HPV) assays are likely to be used with increasing frequency in clinical management of women with abnormal Papanicolaou smears and in cervical cancer screening. Our objective was to simplify the method of collection of female genital tract specimens. The utility of vaginal dry swabs for HPV diagnosis was evaluated. METHODS: Specimens for cytology and for HPV identification were collected by a clinician from 189 female soldiers attending a military clinic. Three methods of specimen collection for HPV identification were compared: a vaginal dry swab (v-DRY), and vaginal and cervical swabs placed into specimen transport medium (v-STM and c-STM). Swabs were shipped to a STD laboratory for processing. Specific HPV types were identified by a consensus primer based PCR based method. Results from 165 women were evaluable. RESULTS: HPV prevalence by the three methods was similar and ranged from 44.8% to 50.9%. 53 (32.1%) women were HPV positive and 60 (36.4%) women were HPV negative by all three collection methods. With respect to the risk categories of specific HPV types, there was greater agreement between the results from the two vaginal (v-DRY and v-STM) samples (kappa values of 0.69-0.81) than between the cervical (c-STM) and either of the vaginal samples (kappa values of 0.37-0.55). The HPV yield from c-STM was somewhat greater than that from the vaginal specimens but the correlation between cytological abnormalities and HPV was high for all three methods. CONCLUSION: A dry vaginal swab may be an acceptable method of specimen collection for HPV diagnosis.

Intra-laboratory reproducibility of human papillomavirus identification in cervical specimens by a polymerase chain reaction-based assay.

BACKGROUND: polymerase chain reaction (PCR)-based assays for human papillomavirus (HPV) sequences are in wide use in clinical and epidemiological studies. The reproducibility of these assays is not extensively studied. OBJECTIVES: to estimate the intra-laboratory reproducibility of generic and type-specific HPV diagnoses by the MY09/MY11/HMB01 consensus L1 primer-based PCR assay. STUDY DESIGN: systematically collected specimens (n=207) were masked and retested. RESULTS: when specimens negative in both initial and repeat assays were excluded from analysis, the diagnostic reproducibility was 98. 6% for beta-globin, 90.7% for generic HPV (any HPV type), and 76.9% for type-specific HPVs. The reproducibility of type-specific diagnosis increased with increase in signal strength in the hybridization reaction of the initial assay. When a specimen contained five or more HPV types in the initial assay, it was rare to identify all of the HPV types in the repeat assay. CONCLUSIONS: the degree of reproducibility of the PCR diagnosis should be taken into account in the interpretation of HPV data in clinical and epidemiological studies.

Concordance of human papillomavirus in the cervix and urine among inner city adolescents.

OBJECTIVES: To evaluate concordance for human papillomaviruses (HPVs) between cervix and urine in sexually active adolescents. METHODS: Cervical swabs and urine were collected from 80 adolescents in Baltimore, MD. Specimens were tested for 34 HPVs by PCR and for cancer-associated HPVs by Hybrid Capture (HC II) Probe B. Cervical vs. urine prevalence was evaluated by logistic regression with general estimating equations. Risk factors for prevalence and viral burden were evaluated by Fisher's exact and Kruskal-Wallis tests, respectively. RESULTS: HPV prevalence by PCR, for any HPV, was very high in the cervix (90.0%) and somewhat lower in urine (75.0%) (odds ratio, 1.07; 95% confidence interval 1.07 to 1.34). Only one adolescent was HPV-positive in urine alone. Among HPV-PCR positives at either or both sites, concordance was 82% for presence of any HPV and 40% for specific HPV types. Multiple infections were common at both sites. On an average, HC II viral burden (relative light unit ratio) was 9-fold higher in cervix than in urine (median, 47.3 vs. 4.9; P = 0.005) but correlated poorly between the two sites of the same individual (r = 0.14). Compared with normal adolescents, those with squamous intraepithelial lesions had a much higher prevalence of HPV by HC II in cervix (100% vs. 28.6, P<0.0001) as well as in urine (86.7% vs. 35.4%, P = 0.002) and a significantly higher viral burden in the cervix (median, 141.8 vs. 7.3, P = 0.0045) but not in urine (median, 22.7 vs. 4.38; P = 0.13). CONCLUSION: There was a very high prevalence of HPV in cervix and urine of sexually active adolescents. Testing urine for HPV may be useful in epidemiologic investigations and in monitoring of infected women.

Evidence for a causal association between human papillomavirus and a subset of head and neck cancers.

BACKGROUND: High-risk human papillomaviruses (HPVs) are etiologic agents for anogenital tract cancers and have been detected in head and neck squamous cell carcinomas (HNSCCs). We investigated, retrospectively, an etiologic role for HPVs in a large series of patients with HNSCC. METHODS: Tumor tissues from 253 patients with newly diagnosed or recurrent HNSCC were tested for the presence of HPV genome by use of polymerase chain reaction (PCR)-based assays, Southern blot hybridization, and in situ hybridization. The viral E6 coding region was sequenced to confirm the presence of tumor-specific viral isolates. Exons 5-9 of the TP53 gene were sequenced from 166 specimens. The hazard of death from HNSCC in patients with and without HPV-positive tumors was determined by proportional hazards regression analysis. RESULTS: HPV was detected in 62 (25%) of 253 cases (95% confidence interval [CI] = 19%-30%). High-risk, tumorigenic type HPV16 was identified in 90% of the HPV-positive tumors. HPV16 was localized specifically by in situ hybridization within the nuclei of cancer cells in preinvasive, invasive, and lymph node disease. Southern blot hybridization patterns were consistent with viral integration. Poor tumor grade (odds ratio [OR] = 2.4; 95% CI = 1.2- 4.9) and oropharyngeal site (OR = 6.2; 95% CI = 3.1-12.1) independently increased the probability of HPV presence. As compared with HPV-negative oropharyngeal cancers, HPV-positive oropharyngeal cancers were less likely to occur among moderate to heavy drinkers (OR = 0.17; 95% CI = 0.05-0.61) and smokers (OR = 0.16; 95% CI = 0.02-1.4), had a characteristic basaloid morphology (OR = 18.7; 95% CI = 2.1-167), were less likely to have TP53 mutations (OR = 0.06; 95% CI = 0.01-0. 36), and had improved disease-specific survival (hazard ratio [HR] = 0.26; 95% CI = 0.07-0.98). After adjustment for the presence of lymph node disease (HR = 2.3; 95% CI = 1.4- 3.8), heavy alcohol consumption (HR = 2.6; 95% CI = 1.4-4.7), and age greater than 60 years old (HR = 1.4; 95% CI = 0.8-2.3), all patients with HPV-positive tumors had a 59% reduction in risk of death from cancer when compared with HPV-negative HNSCC patients (HR = 0.41; 95% CI = 0.20-0.88). CONCLUSIONS: These data extend recent molecular and epidemiologic studies and strongly suggest that HPV-positive oropharyngeal cancers comprise a distinct molecular, clinical, and pathologic disease entity that is likely causally associated with HPV infection and that has a markedly improved prognosis.

Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7.

Human papillomavirus (HPV) infection has been causally associated with cervical cancer. We tested the effectiveness of an HLA-A*0201-restricted, HPV-16 E7 lipopeptide vaccine in eliciting cellular immune responses in vivo in women with refractory cervical cancer. In a nonrandomized Phase I clinical trial, 12 women expressing the HLA-A2 allele with refractory cervical or vaginal cancer were vaccinated with four E786-93 lipopeptide inoculations at 3-week intervals. HLA-A2 subtyping was also performed, and HPV typing was assessed on tumor specimens. Induction of epitope-specific CD8+ T-lymphocyte (CTL) responses was analyzed using peripheral blood leukapheresis specimens obtained before and after vaccination. CTL specificity was measured by IFN-gamma release assay using HLA-A*0201 matched target cells. Clinical responses were assessed by physical examination and radiographic images. All HLA-A*0201 patients were able to mount a cellular immune response to a control peptide. E786-93-specific CTLs were elicited in 4 of 10 evaluable HLA-A*0201 subjects before vaccination, 5 of 7 evaluable HLA-A*0201 patients after two vaccinations, and 2 of 3 evaluable HLA-A*0201 cultures after all four inoculations. Two of three evaluable patients' CTLs converted from unreactive to reactive after administration of all four inoculations. There were no clinical responses or treatment toxicities. The ability to generate specific cellular immune responses is retained in patients with advanced cervical cancer. Vaccination with a lipidated HPV peptide epitope appears capable of safely augmenting CTL reactivity. Although enhancements of cellular immune responses are needed to achieve therapeutic utility in advanced cervical cancer, this approach might prove useful in treating preinvasive disease.

HPV prevalence among Mexican women with neoplastic and normal cervixes.

Genital human papillomavirus (HPV) infection is causally linked to cervical cancer, yet little is known regarding HPV prevalence in cancerous and normal women in Mexico, a country with a high cervical cancer incidence. We studied 185 Mexican women among the patients attending gynecological outpatient clinics in four hospitals in Mexico City. Each woman had a Pap smear, a colposcopy, and, when necessary, a biopsy. HPVs were identified by a consensus-primer-based polymerase chain reaction (PCR) assay. HPV was detected in 87% of 69 cancers, 83% of 24 high-grade squamous intraepithelial lesions (HGSILs), 33% of 21 low-grade squamous intraepithelial lesions (LGSILs), and 17% of 71 normals. Twenty-one of the 32 HPV types tested were detected at least once. The ratio of high-risk:low-risk types was 87:6 in HGSILs and cancers, compared to 11:8 for LGSILs and normals. In invasive cancers, HPV types found at the highest frequency were, in descending order: HPV-16, -18, and -45, followed by -39, -59, and -58 with the same frequency. HPV-16 and related types were present in 52% of the cancer cases, as well as in 79% of HGSILs, and HPV-18 and related types were present in 36% of the cancers but in only 12.5% of the HGSILs. HPV-16 was predominant in squamous carcinomas, and HPV-18 and related types were predominant in adenosquamous carcinoma. Both biopsies and scrapes were tested for HPVs in 63 women, all of them with cervical neoplasia. Identical HPV results were obtained in 89% of the samples, but additional types were often identified in scrapes. HPV prevalence and type distribution in cervical cancer in Mexico was similar to the reported worldwide, as well as in other Latin American countries.

Human papillomavirus, anal squamous intraepithelial lesions, and human immunodeficiency virus in a cohort of gay men.

Cross-sectional associations between human papillomavirus (HPV), anal squamous intraepithelial lesions (SIL), and human immunodeficiency virus (HIV) were studied in a cohort of gay men. HPV DNA was detected by generic and type-specific polymerase chain reaction (PCR) probes and hybrid capture assay (HC). HPV virus load was estimated by HC relative light unit (RLU) ratio. HPV prevalence, number of HPV types detected, and HC RLU ratios were each greater in HIV-positive than HIV-negative participants. Further, among HIV-positive men, HC RLU ratio was inversely associated with CD4 cell count. SIL was more frequent in HIV-positive participants, particularly those with a CD4 cell count <200/microL and was positively associated with HPV. Men with a high HC RLU ratio were nearly 3 times more likely to have SIL than were those both PCR- and HC-negative. These data support that HIV augments HPV-associated anal disease in this population.

Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter.

The enterohepatic circulation of bile acids is maintained by Na(+)-dependent transport mechanisms. To better understand these processes, a full-length human ileal Na(+)-bile acid cotransporter cDNA was identified using rapid amplification of cDNA ends and genomic cloning techniques. Using Northern blot analysis to determine its tissue expression, we readily detected the ileal Na(+)-bile acid cotransporter mRNA in terminal ileum and kidney. Direct cloning and mapping of the transcriptional start sites confirmed that the kidney cDNA was identical to the ileal Na(+)-bile acid cotransporter. In transiently transfected COS cells, ileal Na(+)-bile acid cotransporter-mediated taurocholate uptake was strictly Na+ dependent and chloride independent. Analysis of the substrate specificity in transfected COS or CHO cells showed that both conjugated and unconjugated bile acids are efficiently transported. When the inhibition constants for other potential substrates such as estrone-3-sulfate were determined, the ileal Na(+)-bile acid cotransporter exhibited a narrower substrate specificity than the related liver Na(+)-bile acid cotransporter. Whereas the multispecific liver Na(+)-bile acid cotransporter may participate in hepatic clearance of organic anion metabolites and xenobiotics, the ileal and renal Na(+)-bile acid cotransporter retains a narrow specificity for reclamation of bile acids.

Investigation of human urine for genomic sequences of the primate polyomaviruses simian virus 40, BK virus, and JC virus.

Recent reports of the detection of simian virus 40 (SV40) nucleotide sequences in ependymomas, choroid plexus tumors, osteosarcomas, and mesotheliomas have raised the possibility that SV40, which naturally infects Asian macaques, is circulating among humans. This possibility was examined by performing polymerase chain reaction assays on urine samples of 166 homosexual men, 88 of them human immunodeficiency virus (HIV)-seropositive, for genomic sequences of SV40 as well as of human polyomaviruses BK virus (BKV) and JC virus (JCV). Tests with masked urine specimens spiked with SV40-transformed cells were included to monitor the SV40 assay. SV40, BKV, and JCV sequences were identified, respectively, in 0, 14%, and 34% of the urine specimens. JCV viruria was far more common (37%) than BKV viruria (5%) in HIV-seronegative persons. HIV infection and more severe immunosuppression were associated with a higher frequency of BKV viruria. In summary, SV40 viruria was not detected among homosexual men who shed human polyomaviruses at a high frequency.

Prevalence of penile human papillomavirus DNA in husbands of women with and without cervical neoplasia: a study in Spain and Colombia.

To investigate the role of men in cervical cancer, 816 husbands of women enrolled in four case-control studies of cervical neoplasia in populations at high (Colombia) and low (Spain) risk for cervical cancer were interviewed. Exfoliated cells from the penis were obtained and analyzed by polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Penile HPV DNA prevalences were higher in husbands of women with cervical neoplasia than in husbands of controls. Husbands of controls in Colombia had a 5-fold higher penile HPV DNA prevalence than the corresponding husbands in Spain. Strong dose-response relationships were found between penile HPV DNA prevalence and all sexual behavior-related variables in Spain but not in Colombia. Sexual promiscuity is the most important risk factor for penile HPV infections. Differences in HPV DNA prevalence in the male populations of Spain and Colombia are consistent with their 8-fold difference in cervical cancer incidences.

Simian virus 40 and pleural mesothelioma in humans.

It has been reported that DNA of SV40, a virus of Asian macaques that is tumorigenic for rodents and can transform human cells in vitro, is present in pleural mesotheliomas and in several other cancers. To verify these observations, we tested paraffin sections from mesothelioma tissues of 50 patients for SV40 DNA using PCR with two separate sets of primers. The analytic sensitivity for detection of SV40 DNA was 1-10 genome copies. We also tested the specimens for beta-globin by PCR to assess the suitability of the specimen DNAs for amplification. beta-Globin amplification was detected in 48 of the 50 specimens, but SV40 DNA was not detected in any tumors, with either of two SV40 primer sets. Furthermore, sera from 34 additional patients with mesothelioma, 33 patients with osteosarcoma (another cancer reported to be SV40-related) and 35 controls were tested for SV40 antibodies by a plaque neutralization assay. The serological data, like the DNA results, did not support an association of SV40 with mesothelioma or with osteosarcoma; antibodies to SV40 were detected in three mesothelioma patients, in one osteosarcoma patient, and in one control. These findings call into question the association of SV40 with mesothelioma.

Cervical abnormalities, human papillomavirus, and human immunodeficiency virus infections in women in Malawi.

Cervical lavage samples and Pap smears were obtained from 284 women in Malawi to evaluate the association between human papillomavirus (HPV) and human immunodeficiency virus (HIV) infections. Squamous intraepithelial lesions were present in 15% (17/16) of HIV-seropositive and 7% (11/152) of HIV-seronegative women (P=.05) and in 23% (19/83) of HPV polymerase chain reaction (PCR)-positive and 4% (6/156) of HPV PCR-negative women (P<.001). HPV DNA was detected in 23% of HIV-uninfected women but in 60% of HIV-infected women with <300 CD4 cells/mm(3) (P<.002). High-risk HPV types 16 and 18 constituted half of the identified types. HPV DNA in previously HPV-positive women was detected more often than in HIV-seropositive abnormal cervical cytology than uninfected ones and were more likely to have persistent HPV infections. Early detection of HPV and regular monitoring of HIV-related cervical lesions may be important in HIV-infected women.

Accuracy and interlaboratory reliability of human papillomavirus DNA testing by hybrid capture.

Epidemiologists and clinicians wishing to introduce human papillomavirus (HPV) testing into cervical cancer prevention programs need standardized, reliable, and accurate HPV DNA tests that can detect the full spectrum of pathogenic HPV types. The Hybrid Capture System assay from Digene (hybrid capture assay) is a nonradioactive kit designed to detect 14 HPV types in two groups: a mix of 9 high-risk types associated with anogenital cancer (HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56) and another group of 5 low-risk types associated with condyloma acuminatum (HPV types 6, 11, 42, 43, and 44). The assay yields quantitative data meant to reflect viral concentration. In a study of 199 cervical specimens from women with concurrent Pap smears, we assessed the reliability of the new assay by comparing the hybrid capture assay results from three laboratories. We assessed the accuracy of the hybrid capture assay in comparison with a reference standard of HPV DNA content (multiple testing by several methods in two reference laboratories). We also compared the hybrid capture assay results with the concurrent cytologic diagnoses on the basis of an independent review of each smear by five pathologists. Pairwise interlaboratory agreement rates on HPV positivity for either high-risk or low-risk types ranged from 87 to 94%, and kappa values ranged from 0.61 to 0.83. Among specimens positive for high-risk types (the most important clinical outcome), the interlaboratory correlations of the quantitative data ranged from 0.60 to 0.90. Test results from all three laboratories were strongly associated with those of the HPV DNA reference standard and with the concurrent cytopathologic diagnoses. The most common errors were sporadic, apparently false-positive results.

A measurement system for the recognition of arm gestures using accelerometers.

This paper describes a strategy to measure arm movements using accelerometers for the computer recognition of arm gestures. Gesture recognition is being investigated as an alternative method of computer input for people with severe speech and motor impairment; the emphasis is on the needs of people with athetoid cerebral palsy who have difficulties with existing computer input devices. An initial model-based approach to estimate the kinematic motion of the arm from acceleration measurements is given, followed by the chosen measurement scheme. The current system considers the forearm as a rigid body and uses two data streams derived from four linear accelerometers. By treating these signals as outputs from postulated mechanical models, the data are reduced to two series of step inputs that are appropriate for pattern classification and recognition. Although this does not in any way model the arm, the concepts used are based on the type of driving signals expected in the control of arm gestures. Initial experimental results show that the information content of gestures is preserved by this data parameterization.

Comparison of ViraPap, Southern hybridization, and polymerase chain reaction methods for human papillomavirus identification in an epidemiological investigation of cervical cancer.

In order to provide a reliable diagnosis for the presence and type of human papillomavirus (HPV) DNA in a case-control study of cervical cancer in Colombia and Spain, 926 cervical scrapes from female subjects were examined by ViraPap (VP) and Southern hybridization (SH), and 510 of these (263 cases and 247 controls) were also tested by polymerase chain reaction (PCR) using the HPV L1 consensus primers. HPV DNA prevalence was much higher in cases than in controls by each of the three tests. There was complete agreement between the results of the three tests for 64.9% of the 510 specimens; 53.5% were negative and 11.4% were positive (regardless of type) by all tests. An additional 29.0% of the specimens were positive by PCR: 19.4% by PCR alone, 6.7% by PCR and VP, and 2.9% by PCR and SH. SH and/or VP gave positive results for 6.0% of the specimens for which the PCR finding was negative: 2.7% by SH alone, 2.5% by VP alone, and 0.8% by both VP and SH. When specimens which were positive by VP alone or only by SH at low-stringency conditions were excluded, PCR confirmed all but four specimens which were positive by other tests. The concordance between type-specific diagnosis by SH and PCR was 86% when HPVs were typed in both tests. HPV-16 accounted for over 80% of the typed HPVs in each test. The presence of blood in case specimens did not appear to inhibit HPV positivity by VP or by PCR at the dilution tested. Low amounts of cellular DNA of specimens resulted in some underestimation of HPV positivity by VP and SH but not by PCR. Compared with that of PCR, the sensitivities for case specimens were 38% by SH and 50% by VP; the sensitivity for control specimens, although it could not be measured precisely because there were few positive specimens, appeared to be lower than for case specimens. It was concluded that PCR-based tests are best suited for epidemiological investigation of HPVs.