Diagnostic sensitivity of polymerase chain reaction and Southern blot hybridization for the detection of human papillomavirus DNA in biopsy specimens from cervical lesions.

Human papillomaviruses (HPVs) are associated with benign and malignant neoplasms of the cervix. One of the criteria for their etiologic role requires an assessment of whether virtually all or only a small fraction of lesions contain viral genomes. DNA preparations from colposcopically directed punch biopsies of cervical lesions were analyzed by Southern blot hybridization and the polymerase chain reaction (PCR) for the presence of HPV DNA. The biopsy specimens represented different pathologic entities (koilocytosis, condyloma, cervical intraepithelial neoplasia, and invasive carcinoma). In Southern blot hybridization with radioactive probes for HPV 11, 16, 18, 31, and 33, HPV DNA was detected in 74% of the biopsy specimens (42 of 57 cases), with the predominant types being HPV 16 and HPV 18. In contrast, after PCR amplification with primers yielding fragments of characteristic size for HPV 11, 16, and 18, the analysis of the same 57 biopsy specimens revealed that all samples were positive for at least one HPV type. To exclude false-positive PCR results, controls without HPV DNA were interspersed at regular intervals, and results were evaluated only if these controls remained HPV negative. To exclude false-negative results due to failure of the reaction, a target sequence within the c-Ha-ras-1 gene was used as an internal control. All HPV typing results obtained by Southern blot hybridization were in agreement with HPV typing by PCR. The higher number of positive samples in the latter analysis stems from the increased sensitivity of PCR, which was which was effective in identifying as few as 10-100 HPV DNA molecules; in contrast, the sensitivity of Southern blot hybridization was 1 pg, or approximately 10(5) molecules of HPV DNA. The authors conclude that, with sufficiently sensitive diagnostic methods, HPV DNA can be detected in most, if not all, neoplastic cervical lesions.     

Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA

A total of 268 biopsies from the genital region was screened for the presence of human papillomavirus DNA. The specimens included carcinoma of the vulva, vagina, cervix, corpus uteri, ovaries and penis, and Bowen's carcinomas, Bowenoid papuloses, Bowen's disease, cervical intraepithelial neoplasias (CIN I to III), Buschke-Löwenstein tumors, a cervical polyp, decidua, endometrium and histologically normal biopsies. Of 45 carcinomas, 18 contained either HPV 16 and/or 18 and 3 HPV 6-related sequences.In a few individual biopsies double or even triple infections were noted. Unusual was the presence of HPV 2-related DNA in one biopsy from Bowen's disease, whereas 2 condylomata acuminata contained HPV 3-related DNA and one contained HPV DNA related to a group of epidermal HPV's found in epidermodysplasia verruciformis lesions.     

Evaluation of two commercially available nucleic acid hybridization assays for the detection and typing of human papillomavirus in clinical specimens.

Southern blot (Oncor, Gaithersburg, MD) and dot blot (Life Technologies, Gaithersburg, MD) nucleic acid hybridization assays were compared for their ability to detect and type human papillomavirus (HPV) DNA in 50 cervical swab specimens and 11 biopsy specimens. Overall agreement between the two methods was 78.7%. With the use of Southern blot analysis, HPV 6, 11, 16, or 18 was detected in 22 specimens, however, 4 were untypable because of abnormal or smeared band patterns. Dot blot analysis detected HPV 6/11, 16/18, or 31/33/35 in those same 22 specimens and in 9 additional specimens. Eight of the 13 specimens in which HPV was not detected or untypable by Southern blot contained type 31/33/35 by dot blot. Based on convenience of specimen collection and transport, ease of performance and the ability to detect HPV types 31, 33, and 35, the authors are currently using the dot blot assay for the detection and typing of HPV in clinical specimens.     

In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia

The authors examined 18 cervical intraepithelial neoplasms (CIN) for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and DNA-DNA in situ hybridizations for HPV DNA sequences and compared the epithelial distribution of HPV 16 DNA sequences with HPV 6/11 sequences in selected condylomas. Fifteen of the 18 CIN lesions contained HPV 16 DNA as determined by Southern blot hybridization. With the use of biotinylated HPV 16 DNA probes, 10 of the 18 were positive by in situ hybridization, 9 of which were also positive by Southern blot hybridization. In situ hybridization to HPV 16 probes was found primarily in areas of CIN which contained either maturation or koilocytotic atypia, although in two cases hybridizing sequences were detected in superficial cells from epithelium with no discernible maturation. Staining in both condylomas and CIN lesions varied in distribution and intensity. However, in some CIN lesions staining from cell to cell varied considerably. This greater variability in staining appeared to correlate with greater morphologic variations which characterize CIN, and which may influence greater variation in HPV DNA replication. Thus, some differences in patterns of hybridization for HPV DNA between CIN and condylomas may be explained by morphologic differences in the two classes of lesions. Differences in viral gene expression between condylomas and CIN and their relationship to morphologic findings remain to be clarified.     

Typing of human papillomaviruses in cervical intraepithelial neoplasia grade 3 biopsies from Cape Town

DNA from 98 cervical intraepithelial neoplasia grade 3 (CIN 3) biopsies was screened, using the Southern blotting hybridization technique, for human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, and 35. HPV 16 was detected in 16 biopsies (16%), HPV 33 in eight biopsies (8%), HPV 31 in two biopsies (2%), and HPV 18 in one biopsy (1%). One of the biopsies contained both HPV 31 and 18. Six biopsies (6%) contained an HPV type very similar but not identical to HPV 16, and 35 biopsies were positive for HPV, but the Pst 1 restriction fragments were distinct from any of the HPV types used as probes. HPV was not detected in 32% (32/98) of the biopsies screened.     

Typing and molecular characterization of human papillomaviruses in genital warts from South African women.

Condyloma acuminata from 12 women were examined for the presence of human papillomavirus DNA. Six of the women had HPV 6a, two had HPV 11a, one had a new HPV 6 subtype, and one a new HPV 11 subtype. The new HPV 6 subtype could be distinguished from other HPV 6 subtypes on the basis of Pst I fragments as well as Hind II fragments. The new HPV 11 subtype had a different Hpa II restriction endonuclease pattern. Restriction maps of both new subtypes were constructed. Two of the biopsies did not contain detectable HPV DNA when probed with HPV 6, HPV 11, and HPV 16. Biopsies were taken from normal tissue 1 cm away from the condyloma in 11 of the patients. Only one of these normal tissue biopsies was positive for HPV by Southern blot hybridization.     

Infection of the oral mucosa with defined types of human papillomaviruses

Biopsies from 9 different oral papillomatous proliferations were analysed for human papilloma viral (HPV) sequences of types 1 to 19 and 21 to 26 by Southern blot analysis with 32p-labelled cellular DNA. HPV sequences were detected in 7 out of 9 biopsies obtained from individual patients. Of three cases with the clinical diagnosis focal hyperplasia Heck, two contained HPV-6 related sequences and one HPV 13. In addition, one tongue base papilloma contained HPV 11. A papilloma of the palate revealed HPV 11 sequences. HPV 6 could be demonstrated twice in the ramaining 4 oral papillomatous proliferations. Two biopsies remained negative. The data shows that HPV DNA can be regularly demonstrated in oral papillomas.     

Evaluation of methods for detecting human papillomavirus deoxyribonucleotide sequences in clinical specimens.

Specimens from 26 condylomatous lesions, 24 invasive cancer cells, and 33 cervices, without evidence of the diseases, were tested for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by Southern blot hybridization, in situ filter hybridization, or in situ tissue hybridization methods. A total of 89% (23 of 26) of the condylomatous lesions contained HPV DNAs, as determined by one or more of the methods. The positive rates for the detection of HPV DNA in condylomas by the different methods were 82% for Southern blot hybridization, 62% for in situ filter hybridization, and 72% for in situ tissue hybridization. Among the specimens from patients with cancer, HPV DNA was found in 83% (19 of 23) by one or more of the methods. Positive rates of 89 and 70%, respectively, were obtained for cancer lesions tested by the filter in situ and Southern blot hybridization methods; however, only 30% of those lesions were positive by the in situ tissue hybridization method. Thirteen percent of the control cervices were positive for HPV DNA by one or more of the assays. With respect to all disease categories, the methods had comparable sensitivities and specificities, except for the in situ tissue hybridization method, which revealed a specificity of 72% for condylomatous lesions and 30% for invasive cancer cells.     

A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction.

Ninety-six colposcopically directed biopsies from squamous epithelial carcinoma of the uterine cervix and 22 age-matched normal control biopsy specimens were examined by both Southern blot hybridization and polymerase chain reaction (PCR) for the presence of different human papillomavirus (HPV) DNA types. Cancer of the uterine cervix, which is the most common malignant disease in Indian women, showed a high frequency (98%) of HPV as compared to those reported from other parts of the world. HPV type 16 was found to be the dominant (64%) type while the frequency of HPV type 18 was very low (3%). On individual typing of HPV, no biopsy was found to contain any other known HPV types under stringent conditions of hybridization except a single case of HPV type 11. Only one case of double infection with HPV types 16 and 18 was recorded. Under low stringency conditions of hybridization with a mixed probe of HPV types 16 and 18, 29 additional biopsies were found to be positive. Southern blot hybridization alone detected HPV DNA in 92% of the cases but none in the controls. By PCR, six (6.25%) more cases and four (18.18%) healthy women were found to be positive for HPVs. Analysis of the physical state of HPV 16 indicated integration in about 70% of carcinoma cases while 30% of them were in episomal form. The findings suggest that infection with HPV is an important etiologic factor for the development of cervical cancer, that a number of such tumours may arise without HPV infection, and that integration of the viral DNA into host genome is not always essential for malignant progression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin's disease

We used slot blot hybridization, Southern blot hybridization, and in situ hybridization to investigate the presence of Epstein-Barr virus (EBV) genomes in biopsy tissues from patients with Hodgkin's disease. Slot blot hybridization performed on DNA of tissue specimens from 16 patients revealed that biopsy tissue from 3 (19 percent) contained EBV DNA. Southern blot hybridization with a DNA probe containing the 500-base-pair tandem repeated sequences located at the termini of the EBV genome confirmed the findings of the slot blot hybridization in the three positive tissue specimens and indicated the monoclonality of the EBV-infected cells in such tissues. In situ hybridization performed on the three positive specimens and on two from a previous study localized EBV nucleic acid to the Reed-Sternberg cells and variants in all specimens, with intense hybridization to Reed-Sternberg cells in two, less intense but consistent hybridization to Reed-Sternberg cells in two, and focal hybridization to Reed-Sternberg cells in one. We conclude that EBV genomes are present within Reed-Sternberg cells and variants in some patients with Hodgkin's disease and that the infected cells are monoclonal.     

Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.     

Human papillomavirus DNA is present in a subset of unselected breast cancers

OBJECTIVE: The major molecular events in the genesis of most breast cancers are unknown. However, human papillomaviruses (HPV) have been reported to be found in a significant portion of breast cancers of women with concomitant cervical intraepithelial neoplasia III. To investigate a potential HPV-breast cancer link, we carried out a small survey to identify HPV in unselected, general breast cancer tissues. STUDY DESIGN/METHODS: Deoxyribonucleic acid (DNA) was isolated from 17 breast cancer tissues (and one cervical swab) taken from our local, randomly selected patient population. Two different previously characterized broad-spectrum primer sets (targeting the E6/E7 or L1 regions) were used to amplify HPV DNA, and another primer set was used to amplify the ColE1/pBR322 origin of replication by polymerase chain reaction amplification. The polymerase chain reaction product DNA was analyzed by dot blot hybridization with HPV-16, -18, -31, or pRB322 DNA probes. Total cellular DNA was also analyzed by one- and two-dimensional Southern blot analysis. Finally, the E6/E7 polymerase chain reaction products were cloned, sequenced, and compared to previously cloned HPV types. RESULTS: Polymerase chain reaction/dot blot analysis by both the HPV E6-E7 and L1 primer sets identified the same 6 out of 17 (35%) breast cancers as being HPV positive. ColE1/pBR322 origin targeted polymerase chain reaction/dot blot analysis failed to identify plasmid contamination. One- and two-dimensional Southern blot analysis showed that the breast cancers specimens contained significant levels of HPV DNA and that the viral DNA was largely episomal. The sequences of the HPV clones demonstrated that HPV-16, -18, and possibly type 11 were present within the breast cancer specimens. Furthermore, the HPV sequences cloned from the cervical swab and breast cancer of the same patient were found to be identical. CONCLUSIONS: These data suggest that HPV may be associated with a significant subset of breast cancers, and further suggest that additional studies are warranted.     

Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA.

A methodologic study was performed to compare the polymerase chain reaction (PCR) and Southern blot hybridization, two commonly used testing strategies for the detection of human papillomavirus (HPV) infection. Three laboratories tested masked aliquots of exfoliated cervical cell specimens obtained from 120 women by cervicovaginal lavage. The study population included 32 women with condylomatous atypia or cervical intraepithelial neoplasia and 88 control women with no known history of cervical neoplasia. Two laboratories used PCR with different sets of consensus primers for HPV detection. The third laboratory used low-stringency Southern blot hybridization to identify all HPV types, followed by high-stringency Southern and/or dot blot hybridization to confirm specific HPV types. One of the PCR primer sets detected HPV types with a differential efficiency that was not predicted by analysis of DNA sequences or direct testing of HPV-containing plasmids. In contrast, the second PCR primer set was shown to be a much broader consensus system, detecting the same HPV types as Southern blotting, though requiring much less clinical specimen. Over 80% of women with cervical intraepithelial neoplasia or condylomatous atypia were found to be HPV infected both by Southern blotting and by the second PCR primer set. Among the control women, 11% were HPV positive by Southern blotting, while 31% were positive with the second set of primers. Most of the HPV infections found only by PCR were not due to HPV type 6, 11, 16, 18, 31, 33, or 45. These known HPV types were uncommon among normal women in the study population, even as determined by the PCR method.     

Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping

Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay.     

Human papillomavirus type 13 DNA in focal epithelial hyperplasia among Mexicans

Summary Human papillomavirus (HPV) type 13 DNA was detected in focal epithelial hyperplasia lesions of the oral mucosa in seven half-caste mexicans. The lesions contained intracellular papillomavirus-like particles with a diameter of about 50 nm. DNA extracted from biopsies contained unintegrated HPV type 13 DNA genomes as revealed by Southern blot hybridization. The HPV 13 DNA that was isolated in the present study had the same restriction enzyme cleavage map as HPV 13 DNA, previously described by others. It was moreover confirmed that HPV type 13 genome is related to the genomes of HPV types 6 and 11.With 3 Figures     

Human papillomavirus types 16 and 18 sequences in early cervical neoplasia

A total of 100 colposcopic biopsies from patients with abnormal Papanicolau's tests were surveyed for the presence of human papillomavirus (HPV) types 16 and 18 sequences by spot-blot hybridization. HPV 16 and 18 DNA sequences were detected in 58% of the biopsies. None of the cervical intraepithelial neoplasia grade I (CIN I) contained HPV 16 while 50% of the CIN III lesions (carcinoma in situ, CIS) contained HPV 16. HPV 18-related sequences were equally represented in CIN I, II, and III. Southern-blot hybridization of total undigested cellular DNA revealed the presence of HPV DNA sequences only in an episomal form. While the restriction enzyme patterns in HPV 16-positive samples were mostly identical to the originally cloned sequence, the restriction enzyme pattern for HPV 18-positive samples were different from that of HPV 18 but identical to each other. Furthermore, this DNA hybridized more strongly to HPV 18 under nonstringent conditions, suggesting a new type.     

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

Optimization of human papillomavirus genotype detection in cervical scrapes by a modified filter in situ hybridization test.

Optimal conditions for the screening of cervical scrapes for human papillomavirus (HPV) were investigated by using filter in situ hybridization. Since integrated and episomal HPV can be found, cell lines containing viral DNA in an integrated form (HPV in CaSki) or in an episomal state (BK virus-induced hamster tumor cells) were used for optimization experiments. An increase in sensitivity was achieved by alkaline denaturation and neutralization before the specimens were spotted onto the membrane. This increase was 5-fold for the episomal virus and 16-fold for the integrated virus in the model system, as compared with other methods. To evaluate this method on clinical material, 1,963 cervical scrapes were screened for the presence of HPV 6/11 and HPV 16. Nineteen scrapes were positive for HPV 6/11 or HPV 16; and in 1,810 scrapes, no HPV 6/11 or HPV 16 could be detected by the modified filter in situ hybridization technique. Scrapes from which the interpretation of the modified filter in situ hybridization results were equivocal (n = 71, 3.6%) or in which positivity was detected for both HPV 6/11 and HPV 16 (n = 63, 3.2%) were further analyzed by the DNA dot spot technique. Eight scrapes with an equivocal result and only one scrape showing a double positivity by the modified filter in situ hybridization technique could be confirmed in the dot spot assay. In the total group 12 scrapes were positive for HPV 6/11 DNA, 15 were positive for HPV 16 DNA, and 1 was positive for both HPV 6/11 and HPV 16 DNA. Southern blot analysis on modified filter in situ hybridization-positive and -negative scrapes revealed a 100% correlation.     

Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: Correlation between southern blot hybridization,polymerase chain reaction,and serology

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic pep-tide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences. © 1994 Wiiey-Liss, Inc.     

Detection of human papillomavirus DNA in genital lesions by using a modified commercially available in situ hybridization assay.

A modified, commercially available DNA-DNA in situ hybridization test that uses biotinylated probes for the identification of human papillomavirus (HPV) DNA types 6/11, 16/18, and 31/33/35 was evaluated. HPV DNA was detected in 314 of 787 (40%) histologically abnormal genital biopsy specimens by using the ViraType in situ assay (Life Technologies, Gaithersburg, Md.), in which the hybridization time was increased from 2 to 16 h. Ninety percent of positive condyloma acuminata specimens contained HPV type 6/11 DNA. The prevalences of HPV DNA for cervical intraepithelial neoplasia I, II, and III lesions by this in situ hybridization test were 42, 54, and 55%, respectively. The combined prevalence of HPV type 16/18 and 31/33/35 DNAs increased with the severity of the lesion, while the prevalence of type 6/11 DNA decreased. HPV type 6/11 DNA was found only in 1 of 16 (6%) positive cervical intraepithelial neoplasia III specimens. HPV type 16/18 and 31/33/35 DNA was detected in 11 of 16 (69%) and 4 of 16 (25%) in situ hybridization-positive cervical intraepithelial neoplasia III specimens, respectively. Thus, the observation that certain "higher-risk" HPV genotypes are associated with upper-grade cervical precancer lesions was confirmed by this commercial hybridization system. In general, the assay was found to be well suited for use in the clinical laboratory. The ViraType in situ procedure modified for a longer hybridization time may be helpful in identifying lesions containing higher-risk HPV strains.