Detection of human papillomavirus in reprocessed routine Papanicolaou smears by DNA hybridization

DNA dot blot hybridization was used for the detection of human papillomavirus (HPV) types 6, 11, 16, and 18 in reprocessed routinely collected Papanicolaou smears. DNA was extracted from the smears with alkaline lysis and applied onto a nitrocellulose filter. The specificity and sensitivity of the dot blot hybridization on reprocessed Papanicolaou smears were confirmed by Southern blot analysis of selected samples, using CaSki, SiHa cell lines as positive controls and HF 32 as negative controls. From 42 normal smears and 44 abnormal smears with koilocytosis present, 9 (21%) and 43 (98%), respectively, were positive for HPV 6/11/16 or 18. These results show that reprocessed Papanicolaou smears in combination with DNA hybridization have potential application in retrospective studies on the prevalence and distribution of HPV infection.     

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.     

HPV detection by polymerase chain reaction (PCR) in verrucous lesions of the upper aerodigestive tract.

Nineteen formalin-fixed, paraffin-embedded verrucous lesions of the upper aerodigestive tract (UADT) were evaluated for the presence of human papillomavirus (HPV) 6b/11, 16, and 18 DNA sequences using the polymerase chain reaction (PCR), in-situ hybridization, and dot blot analysis. HPV DNA was confirmed in two dysplastic papillomas only; both cases contained HPV 6b/11. E6-E7 portions of HPV DNA was not reproducibly detected in any of the 11 verrucous carcinomas, 4 verrucous hyperplasias, or 2 mature papillomas. In-situ hybridization and dot blot analysis confirmed HPV 6b/11 in the two dysplastic papillomas and failed to identify HPV in the other verrucous lesions.     

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.     

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.     

Performance of commercial reverse line blot assays for human papillomavirus genotyping

The performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (Roche Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA) (Innogenetics), and the reverse hybridization assay (RH) (Qiagen), was evaluated using quantitated whole genomic human papillomavirus (HPV) plasmids (types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, and 68b) as well as epidemiologic samples. In a plasmid titration series, LiPA and RH did not detect 50 international units (IU) of HPV type 18 (HPV18) in the presence of 5 × 10(4) IU or more of HPV16. HPV DNA (1 to 6 types) in the plasmid challenges at 50 IU or genome equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA, and 95.4% by RH, with positive reproducibility of 99.8% (kappa = 0.992), 88.2% (kappa = 0.928), and 88.1% (kappa = 0.926), respectively. Two instances of mistyping occurred with LiPA. Of the 120 epidemiologic samples, 76 were positive for high-risk types by LA, 90 by LiPA, and 69 by RH, with a positive reproducibility of 87.3% (kappa = 0.925), 83.9% (kappa = 0.899), and 90.2% (kappa = 0.942), respectively. Although the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that LA has an advantage for internationally comparable genotyping studies.     

Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women

Cervical cancer is a leading cause of cancer‐related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV‐positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%). J. Med. Virol. 81:678–684, 2009 © 2009 Wiley‐Liss, Inc.     

Detection and typing of human papillomavirus using the Vira Type "in situ" kit: comparison with a conventional dot blot technique

A new commercial kit (Vira Type "in situ", Life Technologies, Inc., Molecular Diagnostics Division, Guithersburg, Maryland, USA) for the detection of human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33 and 35 in routinely processed human anogenital tissue was compared with a conventional dot blot assay for HPV 6, 11, 16 and 18. Both systems use double-stranded genomic DNA probes for the detection of type specific HPV DNA. The probes used on the dot blots were labelled with 32P and visualised autoradiographically. The Vira Type probes were labelled with biotin and visualised using a streptavidin-alkaline phosphatase conjugate with NBT-BCIP substrate. Biopsy specimens from the cervix, vagina, and vulva of 46 women were processed by both methods and compared. The histological diagnoses ranged from benign changes, to dysplasia, and invasive carcinoma. Overall, 50% of biopsy specimens were positive for HPV DNA by dot blot hybridisation; only 39% were positive by Vira Type in situ hybridisation. Three of the specimens positive by the Vira Type "in situ" kit showed no cross hybridisation and were the same HPV type as the dot blot. A further 13 showed hybridisation, but the showed cross hybridisation, but the to the dot blot results. One biopsy specimen was positive for different HPV types by the two tests and one was positive by Vira Type and negative by dot blot. Six biopsy specimens were negative by Vira Type but positive by dot blot. It is concluded that the Vira Type "in situ" kit has a similar specificity but lower sensitivity than the dot blot hybridisation method for the detection of HPV DNA.     

Genital human papilloma virus infection in Oslo studied by dot blot DNA hybridization and the polymerase chain reaction.

Samples from patients with genital condyloma acuminata or with cervical condylomas and/or dysplasia and from women without cytological/clinical evidence of cervical affection were examined by dot blot DNA hybridization or the polymerase chain reaction (PCR). The PCR was much more sensitive than dot blot, more than doubling the human papilloma virus (HPV) findings. HPV DNA, mainly HPV 6/11, was detected in 18 of 19 biopsies of condyloma acuminata, whereas HPV 16 was most frequently detected in the 21 cervices (76%) with condyloma and/or dysplasia. HPV 16 was detected in eight of 103 cervical smears with no signs of infection. The prevalence of HPV 16 in cervical samples was somewhat higher than expected. This suggests that, in Oslo, HPV 16 is a common HPV type in women with cytologically normal cervices. HPV 18 was relatively rare and was detected only in combination with other HPVs.     

Human papillomavirus (HPV) genotyping using paired exfoliated cervicovaginal cells and paraffin-embedded tissues to highlight difficulties in attributing HPV types to specific lesions

Defining type-specific human papillomavirus (HPV) infections within cervical tissues is important for understanding the pathogenesis of cervical neoplasia and assessing the effectiveness of prophylactic vaccines with limited type-specific spectra. We compared HPV DNA-testing results from 146 matched exfoliated-cell and formalin-fixed-tissue specimens collected by cervicovaginal lavage (CVL) within 90 days of each other from women with histologically confirmed cervical intraepithelial lesions (CIN). The CVL specimens were HPV typed using a MY09/11 L1 consensus primer PCR method followed by dot blot hybridization. The tissue specimens were HPV typed using an SPF(10) line probe assay HPV detection system. Of the 146 specimen pairs with evidence of CIN in the tissue, 91.8% were positive for one or more HPV types in both the tissue and cellular specimens. Tissue sections were more likely to be HPV negative (P < 0.01). Typing directly from tissue sections resolved multiple infections detected in exfoliated cells to a single HPV type in only 46.9% of cases. Combined use of both specimen types to attribute lesions to HPV type 16 (HPV-16) and/or -18 led to 43.1% attributed to HPV-16 and/or -18 by both specimen types and 19.9% attributed to HPV-16 and/or -18 by one, but not both, specimen types. Unambiguous attribution of cervical lesions to a single, specific HPV type remains a difficult proposition. Use of multiple specimen types or the development of highly sensitive and robust in situ hybridization HPV-testing methods to evaluate the certainty of attribution of lesions to HPV types might provide insights in future efforts, including HPV vaccine trials.     

Human papillomavirus DNA in anal carcinomas. Comparison of in situ and dot blot hybridization.

Because the sensitivities of individual hybridization techniques differ considerably, their role in accounting for the published frequencies of human papillomavirus (HPV) DNA in anal squamous cell carcinomas, ranging from 0 to 61%, must be investigated. With the use of biotinylated probes to HPV 6, 11, 16, 18, and 33, three hybridization techniques were performed on the same paraffin-embedded tissue blocks selected from 13 cases of anal squamous cell carcinoma. HPV DNA was detected in 0%, 62%, and 85% of cases with the use of in situ hybridization with horseradish peroxidase, in situ hybridization with alkaline phosphatase, and dot blot hybridization, respectively. By dot blot hybridization, 69% had HPV 16/6 and 15% had HPV 6/11. An HPV DNA frequency range of 0-85% in the same group of tumors with the use of three hybridization techniques indicates the influential role of the method on HPV DNA prevalences. HPV DNA was identified regardless of patient gender or type of squamous cell carcinoma. The presence of HPV 16 in 82% of the positive cases in supportive evidence of the carcinogenic role of the HPV in anal squamous cell carcinoma.     

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.     

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.     

Comparative analysis of human papillomavirus detection by polymerase chain reaction and Virapap/Viratype kits

Cervical samples from 270 women referred by area physicians were analyzed for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by the polymerase chain reaction (PCR) followed by Southern hybridization. Samples from 154 patients were concurrently analyzed by a commercial filter hybridization technique (Virapap and Viratype Kits, Life Technologies, Bethesda Research Labs, Gaithersburg, MD). The sensitivity of the Southern blot procedure combined with PCR was significantly higher than that of the Virapap and Viratype methods. HPV was detected in 67% of women who had positive results for dysplasia by PCR and in 47% by the Virapap method. HPV types 16/18 were found more commonly than types 6/11 in every diagnostic category. More than one HPV type was detected in 12% of HPV-positive patients. The prevalence of HPV in cytologically negative or indefinite patients as measured by PCR was 22% and 40%; in contrast, by the Virapap method, these values were 7% and 10%. These results demonstrate that PCR combined with Southern hybridization provides a higher level of sensitivity than methods that use hybridization without amplification of HPV DNA and also show that the prevalence of HPV is highest in cytologically positive smears.     

Prevalence of human papillomavirus types 6, 11, 16, 18, 31, and 33 in a cohort of Greek women

To study HPV prevalence and HPV types 6, 11, 16, 18, 31, and 33 distribution in cervical smears in a cohort of Greek women. One thousand six hundred thirty-six samples were cytologically evaluated and molecularly analyzed, by PCR based assay. Abnormal cytology was identified in 997 women and 75.4% of them were HPV DNA positive, while 639 had normal cytology and 24.6% were HPV DNA positive. HPV was detected in 62.9% of 256 ASCUS smears, 89.3% of 516 LSIL, 86.7% of 60 HSIL and 47.3% of 165 with cervical carcinoma. Overall, HPV 11 was the most common type (13.4%), followed by 18 (10.3%), 6 (7.2%), 16 (6.4%), 31 (3.4%) and 33 (3.4%). Multiple infections with two (11.3%) or more types, primarily 11 and 18 (4.8%), were also identified. Low-risk types 11 and 6 were common in ASCUS (36.6% and 26.4%, respectively), and high-risk types 16 and 18 in HSIL (42.3% and 30.8%, respectively) and in cancer (51.3% and 41%, respectively). Multiple infections were detected in 2.2% of normal and 31.7% of HSIL. HPV prevalence was 75.4% in abnormal and 24.6% in normal cervical smears. HPV 16 and 18 were the most common types in cancer. Single infection with type 11 and multiple infections with 11 and 18 were more frequent.     

Prevalence of HPV cervical infection in a family planning clinic determined by polymerase chain reaction and dot blot hybridisation

The overall prevalence of human papillomavirus (HPV) cervical infection in 131 women attending a family planning clinic was 7% (HPV 6/11, 16, 18, 31) by dot blot hybridisation, 53% (HPV 11, 16, 31) by polymerase chain reaction (PCR), and 56% by the two methods combined. HPV 16 and 18 were the commonest types (4% each) by dot blot, HPV 16 (39%) by PCR. Fifteen percent of subjects had mildly abnormal cervical cytology (grades 1A, 2A, or 3). There was no significant correlation between cytological abnormality and HPV positivity, or between cytological or HPV status and other postulated risk factors for cervical neoplasia. It is concluded that PCR is considerably more sensitive than dot blot DNA hybridisation in detecting HPV cervical infection in such a "low risk" setting, where HPV copy number may be low. Firm conclusions cannot be drawn from our results regarding a causal role for HPV or other factors in the development of cervical neoplasia.     

Detection and localization of human papillomavirus in penile condylomas and squamous cell carcinomas using in situ hybridization with biotinylated DNA viral probes

Human papillomavirus (HPV) has been previously demonstrated in male genital neoplasms using Southern blot hybridization (SBH) and in situ hybridization with radiolabeled probes (ISH-R). In this study we used in situ hybridization with biotinylated DNA viral probes (ISH-B), a technique that can be applied to routinely collected and processed tissue. Thirty cases of exophytic penile condyloma acuminatum and nine cases of invasive squamous cell carcinoma of the penis were examined for the presence of HPV using ISH-B for HPV types 6, 11, 16, 18, 31, and 33. HPV DNA was found in 25 of 30 (83%) penile condylomas; HPV type 6 in 13 (43%); and HPV type 11 in 12 (40%). Slight cross-reactivity between HPV types 6 and 11 was noted. None of the condyloma cases was positive for HPV types 16, 18, 31, or 33. One of the nine patients with squamous cell carcinoma of the penis was positive for HPV 16. In situ hybridization with biotinylated DNA viral probes is a highly sensitive method for detecting and localizing HPV in penile condylomas. This method, however, may not be as sensitive as SBH for detecting HPV in invasive penile squamous cell carcinomas.     

Detection and typing of human papillomavirus infection of the uterine cervix by dot blot hybridisation: comparison of scrapes and biopsies

Paired scrapes and biopsies from 100 women attending a routine colposcopy clinic were examined by dot blot DNA hybridisation for infection with human papillomavirus (HPV) types 6/11, 16, 18, and 31; 51% of the scrapes and 50% of the biopsies were positive for HPV infection. Scrapes detected more HPV 18 (10% vs. 2%, P = less than 0.05) and HPV 31 (7% vs. 3%, not significant) than did the biopsies, but biopsies detected more HPV 16 (42% vs. 33%, not significant). Comparison of the results for each patient revealed that the correlation between scrapes and biopsies was not very close: only 34 patients were HPV-positive by both sampling methods, whereas 67 were positive if the results were combined. This report discusses the implications of these findings.     

Detection of human papillomavirus (HPV) type 16 and 52b in cervical cancer tissues by Southern blot hybridization and polymerase chain reaction (PCR)

DNA samples from cancer tissues diagnosed histologically as squamous cell carcinoma of the uterine cervix were examined for the presence of human papillomavirus (HPV) genome DNA by Southern blot hybridization. Of 63 specimens, 32 were found to hybridize with HPV DNA probes; 23 specimens (35%) with HPV type 16 (HPV16), one (2%) with HPV type 18 (HPV18), four (7%) with HPV type 52b (HPV52b), and four others (7%) weakly with HPV52b. Specimens negative for HPV DNA with Southern blot hybridization were subjected to polymerase chain reaction (PCR) to determine the presence of HPV DNA under more sensitive conditions. After PCR using one set of primers specific for HPV16 and HPV52b, 7 out of 31 specimens were found to have HPV16 DNA. None was positive for HPV52b DNA. Our results indicate that HPV52b, as well as HPV16 and HPV18, is associated with squamous cell carcinoma of uterine cervix, and more sensitive determination of HPV infection can be made by amplification of the viral genome by PCR.     

Detection of human papillomavirus deoxyribonucleic acid in the female genital tract

A total of 336 biopsies, scrapes and exfoliated cells from the cervix and from the lower genital tract were screened for human papilloma (HP) viral sequences of types 6, 11, 16 and 18 by Southern blot, dot blot and filter in situ (FISH) hybridizations with cloned ³²P-radiolabeled HPV DNA probes. The specimens included cervical intraepithelial neoplasias (CIN I–III), carcinoma in situ and invasive carcinoma of the cervix and vagina, adenocarcinomas, vulvar and vaginal condylomata acuminata and healthy epithelial samples. The oncogenic HPV 16 was found in 46% of the cervical carcinomas. Most of the type 16 occurences (75%) represented the third stage of inooperable cases. Similarly, HPV 18 was also most frequently present in this stage as well as in carcinoma in situ and in CIN III (25%, 18%). At the same time, in condylomata acuminata, types 6 and 11 were detectable in 88.7% of cares. In all, 13.5% of the normal samples harboured HPV DNA.