Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.     

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 and typing of human papillomavirus DNA in the uterine cervix of japanese women by nonradioactive dot blot and southern blot hybridization

HPV infection was examined with Cytobrush in exfoliated cervical cells sampled from 347 women. HPV DNA analysis was conducted in two steps. The presence of HPV DNA was demonstrated by dot blotting and typing of HPV DNA was made by Southern blotting using biotinylated probes. Of 167 cases with cervical intra-epithelial neoplasia (CIN) and cervical carcinoma, HPV DNA was detected and typed in 25 cases (15.0%). HPV 16, 18, and 33 were found mainly, and no HPV 6 or 11 was detected. The frequency of HPV DNA was 7.1% of CIN I, 28.6% of CIN II, 54.5% of CIN III, and 50.0% of invasive carcinoma. HPV occurred in patients newly diagnosed as CIN at 25.8% and in those of diagnosed as CIN in the past and followed up, 7.6%. Of 180 healthy women, the screening test of F08830 was positive for HPV DNA in four women (2.2%). The present system proved to be useful for facilitating large-scale clinical research.     

Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck^®. The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck^®, particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.     

Detection and typing of human papillomaviruses by in situ hybridization with biotinylated oligonucleotide mixtures

The value of biotinylated Oligonucleotide probes for screening and typing by in situ hybridization of the most frequent genital human papillomavirus infections (HPVs 6,11,16,18, 31, and 33) was assessed. Optimal hybridization conditions were defined on a panel of paraffin-embedded tissue sections previously characterized with HPV full genome probes. Mixtures of oligonucleotides rather than single oligonucleotides were used to improve sensitivity and specificity. All HPV-positive specimens were detected by the screening mixture with a sensitivity and specificity similar to that of full genome probes. Typing mixtures were highly specific for each HPV type. This study confirms the potential of Oligonucleotide probes for detecting and typing HPV infections. © 1995 Wiley-Liss, Inc.     

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.     

Large-scale screening for human parvovirus B19 DNA in clinical specimens by dot blot hybridization and polymerase chain reaction

Large-scale screening for human parvovirus B19 (619) DNA in serum samples was carried out by both dot blot hybridization and the polymerase chain reaction (PCR). Dot blot hybridization was undertaken with a digoxigenin-labeled DNA probe. Serum samples from four patients were pooled and tested by a dot blot hybridization assay. When a dot was positive, each of the four samples was tested separately to identify the positive sample. The PCR template was the DNA extracted from mixed serum samples from 10 patients. When B19 DNA was positive by PCR, each of the ten samples was tested separately. A total of 7, 969 serum samples were tested by dot blot hybridization and 15 samples (11 patients) were positive for B19 DNA; 7, 038 serum samples were tested by PCR and 71 samples (50 patients) were positive. Large-scale screening for B19 DNA by PCR suggested a broader spectrum of clinical manifestations associated with B19 infection. © Wiley-Liss, Inc.     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

肺癌组织中人乳头瘤病毒16和18型DNA相关序列的检测

采用聚合酶链反应技术（ＰＣＲ）及用ＰＣＲ法制备生物素标记探针的斑点杂交法,检测５０例肺癌、１８例肺良性病及４例胎肺组织中高危险型人乳头瘤病毒（ＨＰＶ）１６、１８型ＤＮＡ相关序列,以探讨ＨＰＶ与肺癌之间发生的关系。结果３２％的肺癌组织中检测出ＨＰＶ１６、１８ＤＮＡ,其中ＨＰＶ１６ＤＮＡ阳性１０例,ＨＰＶ１８ＤＮＡ阳性５例,１例同时含ＨＰＶ１６、１８ＤＮＡ。２７例鳞癌组织中ＨＰＶＤＮＡ阳性率为４８．２％。而肺良性病组织及胎肺组织均未发现ＨＰＶＤＮＡ。ＨＰＶＤＮＡ呈阳性的肺癌患者绝大多数为重度吸烟者。提示原发性肺癌的发生可能与ＨＰＶ感染有关。     

Prevalence of infection with high-risk human papillomavirus in women in Colombia

The prevalence of human papillomavirus (HPV) infections in 2109 females inhabiting five cities of Colombia was determined. Of the 49.2% with an HPV infection, 59.8% were infected with more than one viral type. Species 7 (of the the genus Alphapapillomavirus ) was associated with multiple infections. Analysis of the socio-demographic data revealed a statistically significant protective effect associated with the status of civil union (civil recognition of cohabitation without marriage), and indigenous ethnicity proved to be a risk factor for HPV infection. This is the first study comparing HPV infection among women from geographical regions of Colombia with different socio-cultural structures.     

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 of human papillomavirus deoxyribonucleic acid by filter in situ hybridization during pregnancy

Samples taken from 101 healthy pregnant women (49 over and 52 under the 20-week gestational period) and 108 healthy nonpregnant women were tested for human papillomavirus (HPV) types. Using 6, 11, 16, and 18 HPV DNA probes, 3-5 x 10(5) exfoliated cells scraped from the cervix were tested by filter in situ hybridization (FISH). Thirty-five of the pregnant women (34.6%) had evidence of the presence of HPV DNA: with 11.8% (12/101) HPV 6; 7.9% (8/101) HPV 11; 8.9% (9/101) HPV 16; and 5.9% (6/101) HPV 18 positivity. HPV DNA was detected in 20.4% (22/108) of the non-pregnant women. Compared with the healthy, nonpregnant group, the higher level of asymptomatic cervical HPV infection was mainly due to the accumulation of HPV 16 and 18 nucleic acids during the gestational period: with detection of HPV 16 in 8/49 cases (16.3%) and of HPV 18 DNA sequences in 4/49 (7.6%) cases. Screening 6-8 weeks after delivery indicated a decline of HPV positivity. Of the 4/12 HPV type 16 positive mothers, only one retained the presence of HPV 16 DNA, whereas neither of the 2/12 type 18 positive women reacted after birth with the type 18 radioactive probe.     

人乳腺癌HPV感染的超微结构和DNA分子原位杂交研究

目的：对乳腺癌的HPV感染进行形态学定位研究。方法：对46例乳腺癌切除标本及7例良性病变标本进行透射电子显微镜以及HPV DNA分子原位杂交检测。结果：透射电镜下，14例（30．40％）乳腺癌细胞核内有HPV样病毒颗粒，颗粒直径30－50nm，或弥漫散布，或聚集成团呈假结晶状排列。颗粒圆形或略不规则，含有HPV样颗粒的癌细胞核异型性较明显。7例良性病变细胞核内未见到HPV样颗粒。HPV DNA分子原位杂交显示，21例（45．65％）乳腺癌细胞核内HPV31／33 DNA阳性，1例软肉瘤样化生性癌细胞核同时有HPV31／33及HPV16／18阳性；1例纤维腺癌（14．29％）也显示HPV31／33 DNA阳性（X^2=2．7431，P＜0．05）。电镜下85％病毒样颗粒阳性病例中可检测到HPV DNA存在，两种检测结果符合率达73．91％。结论：乳腺肿瘤内存在未组装的、裸型病毒样颗粒。该研究对阐明乳腺癌的发病原因以及对乳腺癌的预防和治疗均有重要意义。     

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: A method suitable for semiautomation

Screening for high-risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow-up. Thirty-four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high-grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR-enzyme-linked immunosorbent assay (ELISA)-based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR-ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR-ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia. © 1996 Wiley-Liss, Inc.     

Comparison of two self-sampling methods for human papillomavirus (HPV) DNA testing among women with high prevalence rates

One major advantage of molecular assays for human papillomavirus (HPV) DNA detection is that these assays can be performed on self-collected samples unlike cytology or visual inspection with acetic acid (VIA). This cross-sectional study was carried out between March 2017 and April 2019 to compare the diagnostic performance in self-collected urine and vaginal samples for HPV DNA detection. Viral DNA was extracted from processed samples using a Qiagen viral DNA extraction Kit (QIAamp DNA Mini Kit). To detect four common high-risk HPV types (16, 18, 31, 45), multiplex real-time polymerase chain reaction (PCR) targeting the LCR/E6/E7 region of the HPV genome was performed in ABI 7500 cycler (Applied Biosystems). The negative samples were screened by conventional PCR targeting the L1 capsid region to exclude other HPV types. The overall agreement between the two self-collecting sampling methods was 64.04% with a κ value of 0.29 pointing towards a fair agreement (P < .01). The sensitivity of HPV DNA detection in urine samples was 57.95% (47.52%, 67.72), and specificity was 84.6% (66.47%, 93.85%) when compared with vaginal samples. The study concludes that self-collected vaginal HPV DNA testing is more sensitive than unpreserved-urine samples for HPV DNA detection in a hospital-based setting.     

Cervical adenoid basal tumors comprised of adenoid basal epithelioma associated with various types of invasive carcinoma: clinicopathologic features, human papillomavirus DNA detection, and P16 expression

Adenoid basal tumors are uncommon cervical lesions that some pathologists consider invasive carcinomas but others consider "epitheliomas" due to their low-grade histological appearance and rarely documented malignant behavior. We report the clinicopathologic features of 10 tumors comprised of both typical low-grade adenoid basal tumors (epitheliomas) intimately associated with invasive carcinomas having infiltrative growth, increased cytological atypia and mitotic activity, and various types of differentiation, including adenoid basal/squamous, pure squamous, adenoid cystic, and small cell neuroendocrine. Tumors were evaluated for the presence of human papillomavirus (HPV) DNA and immunohistochemical p16 expression. The patients in the study group ranged in age from 45 to 81 years (mean, 65 years). Most of the patients presented with abnormal cervicovaginal smears. The initial diagnosis was made on specimens obtained by cervical biopsy, laser electrocautery excision procedure (LEEP), or cone biopsy in 8 patients. Two 2 patients were incidentally diagnosed in hysterectomy specimens. All 10 patients had squamous intraepithelial lesions (9 high-grade, 1 low-grade). In all cases diagnosed in LEEP or cone biopsy specimens, the invasive carcinoma component was present in the excisional specimen and extended to the margins. Seven patients diagnosed on excisional or biopsy specimens who underwent hysterectomy had residual tumor in the cervix, ranging from microscopic foci to deeply invasive. No lymph node metastases were identified in 4 patients who were staged. Seven patients with follow-up were alive without evidence of disease after follow-up intervals of 8 to 84 months (mean, 45 months; median, 29 months). One patient with a component of small cell carcinoma died of other causes without evidence of disease at 18 months. HPV 16 DNA was detected in both the adenoid basal epithelioma and invasive carcinoma components in 9 tumors by in situ hybridization, and HPV 33 was detected by polymerase chain reaction in 1 tumor. All tumors expressed p16 diffusely. Adenoid basal tumors are high-risk HPV-related tumors that can be comprised of both a low-grade adenoid basal tumor, which can be designated as epithelioma, and invasive carcinomas of various types. The invasive component is usually evident in the excisional biopsy specimen, allowing for recognition of a tumor that needs further management.     

Evaluation of the detection of human papillomavirus genotypes in cervical specimens by hybrid capture as screening for precancerous lesions in HIV-positive women

Given the frequency and persistence of human papillomavirus (HPV) infection and associated cytological alterations in HIV-1-positive women, the incidence of uterine cervix neoplasm is likely to increase along with patient survival. More appropriate screening programs, which, in addition to Pap smears (PS), also include tests to detect and type HPV, are needed for the early identification of precancerous cervical lesions. This prospective study involved 168 HIV-positive (group A) and 100 HIV-negative women (group B). Cervicovaginal samples were collected for a PS and HPV DNA search. The detected virus was typed as high–intermediate oncogenic risk HPV (HR-HPV) and low-risk HPV (LR-HPV) using hybrid capture (HC) (Murex-Digene) and in-house PCR tests. The HC-detected prevalence of HPV was 111/168 (66%:HR 75.6%) in group A and 15/100 (15%:HR 42.9%) in group B (P < 0.0001). Polymerase chain reaction (PCR) was positive in 91% and 48%, respectively. No significant difference was observed between drug addicts and heterosexual HIV-1-positive women (P = 0.09). HPV was detected in 94% of the 57 HIV-positive women with cytological alterations. HR-HPV was found in 41/49 women with low-grade and 7/8 with high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively). In women with a negative PS, HPV was detected in 57/111 cases (HR 63%) of group A and in 13/98 of group B (6 cases of HR). Of the 54 group A women who underwent biopsy, histology revealed that 41 had LSIL (18 with negative PS, 19 with LSIL, and 4 with HSIL; HR-HPV in 73% and LR-HPV in 17%), nine had HSIL (5 LSIL and 4 HSIL on cytology; HR-HPV in 89% and LR-HPV in 11%), and four were negative (all cytology negative; 3 HR-HPV and 1 LR-HPV). HR-HPV was more frequent as immunodepression worsened. These results show that cytological evaluation alone underestimated histological alterations in 23/50 women (42.6%), whereas the combination of Pap smear and HPV detection reduced this underestimate to 5%. J. Med. Virol. 56:133–137, 1998. © 1998 Wiley-Liss, Inc.