Cytological evaluation using liquid‐based cytology in the male urogenital tract infected with human papillomavirus

This study was performed to determine the associations between human papillomavirus (HPV) infection and cytological changes in the male glans and urethral samples. Two rubbed samples of the glans and distal urethra were collected from 160 patients with urethritis, and the collected cells were placed into preservative solution for liquid‐based cytology. DNA was extracted from all samples, and β‐globin gene amplification, HPV‐DNA test, and HPV genotyping were performed. After papanicolaou staining of each sample, cytological findings were assessed based on nine non‐classic signs, and compared with the results of HPV‐DNA test. Among the β‐globin positive samples, HPV (any type) was detected in 33 and 25% of the glans and urethra samples, respectively. High‐risk HPV‐DNA was detected in 25 and 15% of the glans and urethra samples, respectively. In the glans samples, cytological signs of HPV infection were observed in 17% of low‐risk HPV‐positive samples and in 58% of high‐risk HPV‐positive samples. Cytological atypia suspected to indicate penile intraepithelial neoplasia were observed in 17 and 33%, respectively. On the other hand, abnormal cells were observed in 29% of low‐risk HPV‐positive and 83% of high‐risk HPV‐positive urethral samples. Cytological atypia suspected to be urethral intraepithelial neoplasia was observed in 29 and 65%, respectively. In situ hybridization demonstrated the presence of HPV‐DNA in the morphologically abnormal cells in 31 and 35% of high‐risk HPV‐positive glans and urethral samples, respectively. Cytological changes similar to cervical intraepithelial neoplasia in females could be detected in the HPV‐positive glans and urethral samples. Diagn. Cytopathol. 2014;42:491–497. © 2013 Wiley Periodicals, Inc.     

Detection of human papillomaviruses in exfoliated cervicovaginal cells by in situ DNA hybridization analysis.

A total of 851 specimens of exfoliated cervicovaginal cells and 27 specimens of male urethral smears obtained from 706 individuals with various clinical findings were examined for the presence of human papillomavirus (HPV) types 6, 11, 16, 18, 31, and 33 by in situ DNA hybridization analysis. The nonradioactive DNA in situ hybridization method used in this study showed no detectable cross-hybridization either among different types of HPV (except between types 6 and 11) or between HPV DNA and human cellular DNA. Furthermore, this system was found to be more sensitive than the Southern blotting method in detecting HPV. HPV was found in 233 of 276 (84.4%) and in 34 of 47 (72.3%) samples of cervicovaginal cells from patients with urogenital condylomata and cervical dysplasia, respectively. HPV was also detected in 6 of 39 (15.4%) women with normal cytological findings who were also symptom-free. Young women who were at low risk but were infected with HPV showed significantly reduced ratios of helper-inducer T lymphocytes to suppressor-cytotoxic T lymphocytes compared with those of uninfected normal controls (1.28 +/- 0.31 versus 2.47 +/- 0.64; P less than 0.001). This in situ DNA hybridization method can have broad application to the screening of HPV in early lesions and in normal-looking tissues and may be used to identify patients at risk of more serious or possibly malignant progression.     

Polymerase chain reaction detection of human papillomavirus: quantitation may improve clinical utility.

A case-control study compared detection by polymerase chain reaction (PCR) specific for human papillomavirus (HPV) type 16 with restriction enzyme analysis and Southern blot hybridization detection of HPV type 16. Cervicovaginal lavage samples from 64 women with histopathologic evidence of a cervical squamous intraepithelial lesion and 55 samples from cytologically healthy women were studied. Several methods of PCR product analysis, including radioactive and nonradioactive probing, were compared. The sensitivity of HPV detection by PCR when the amplified DNA fragment was visualized on a gel was equivalent to those of detection by restriction enzyme and Southern blot analyses. Hybridization of the PCR product with radioactively or nonradioactively labeled oligonucleotide probes increased the sensitivity of HPV detection by 100-fold. However, an increase in the sensitivity of the assay preferentially identified low levels of the virus in cytologically healthy women. Therefore, the value of HPV detection in identifying women with cervical neoplastic disease was greater, and the odds ratio for the presence of a cervical squamous intraepithelial lesion was higher when the less sensitive modalities were used. These results suggest that quantitation of HPV by PCR may maximize the clinical significance of a positive test result. Further studies will be needed to determine the optimal level of virus detection which has the highest positive predictive value of clinical disease.     

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.     

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.     

Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes

The presence of human papillomavirus (HPV) nucleotide sequences in paraffin sections of genital biopsies was examined by in situ hybridization using non-isotopic, digoxigenin-labeled probes representing HPV types 11, 16 and 18. Digoxigenin-labeling of the probes was performed using DNA labeling and a commercially provided detection kit. Hybridization was performed under stringent conditions. The hybrids were detected by using anti-digoxigenin alkaline phosphatase conjugate and visualized with enzyme catalyzed color reaction. In situ hybridization with digoxigenin-labeled probes was a useful technique for identification of HPV infection. The results were compared with the results obtained with radiolabeled DNA probes. The sensitivity of the digoxigenin-labeled probes was equal to the sensitivity of the radiolabeled probes. The background with digoxigenin-labeled probes was very low. Using nonradioactive probes the localization of hybrids at the cellular level was better than 35S-labeled probes.     

The sensitivity of in situ hybridization for detection of human papillomavirus

We studied a sensitivity of HPV DNA detection by in situ hybridization method using 3H labeled HPV DNA. The materials were CaSki cells and SiHa cells which were derived from as a negative control. The total cellular DNAs extracted from these cell lines were estimated copy numbers of HPV 16 DNA using Southern blot hybridization. In our result, CaSki cell has 400 copies/cell, SiHa cell were appeared to have 1-5 copies/cell. Simultaneously these cells were fixed by periodate-buffered lysine-paraformaldehyde-glutaraldehyde (PLPG) and were detected HPV 16 DNA using in situ hybridization. We detected HPV 16 DNA in CaSki cells and SiHa cells by in situ hybridization also. We concluded that the sensitivity of our in situ hybridization technique is 1-5 copies/cell.     

One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization.

This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.     

Detection of five different human papillomavirus types in cervical lesions by in situ hybridization. A study of 110 cases using sulfonated probes

Several findings suggest an etiologic relationship between genital tract squamous cell carcinoma and certain types of human papillomavirus (HPV). Detection of these HPV types in cervical lesions considered as preneoplastic states (ie, cervical intraepithelial neoplasia or CIN) is extremely important but difficult because the morphology of these states is highly heterogeneous and clinical course is rarely predictable. In situ hybridization (ISH) is the only technique allowing correlation between HPV type and tissue or cell morphology. In this report, 110 biopsy specimens from uterine cervix lesions were studied: 66 CIN, 10 invasive carcinoma, 28 metaplasia, and six condyloma acuminata. A new ISH technique based on direct modification of DNA probes by sulfonation was used. The hybridized DNA was revealed first by a specific monoclonal antibody against sulfonated DNA, and then by an alkaline phosphatase system. In order to determine the sensitivity level of this method, 14 biopsy specimens were also submitted to Southern blot hybridization. Five probes were used separately (HPV 6, 11, 16, 18, and 33) for each biopsy specimen. Results of ISH were correlated with morphologic criteria such as number of koilocytes and mitoses. Oncogenic HPV was found exclusively in CIN. The number of labeled cells varied with CIN grade. These data suggest that, whatever the grade, CIN represents a unique preneoplastic process, and that HPV replication depends on the squamous maturation of the pathologic epithelium.     

Human papillomavirus detection in urine samples from male patients by the polymerase chain reaction.

Human papillomavirus (HPV) detection was performed using the polymerase chain reaction technique on urine samples from 17 male patients with condylomata acuminata in the meatus urethrae. Urine samples from 14 male laboratory volunteers were analyzed as controls. The DNA was extracted and purified from urine sediments, centrifuged at 1,800 and 100,000 x g, and subjected to 40 cycles of amplification with HPV 6 and HPV 11 type-specific anticontamination primers and the heat-stable Taq DNA polymerase. HPV was detected in the urine of 15 (88%) patients. In all positive patients the urine sediments of both the 1,800 and 100,000 x g centrifugation steps contained HPV DNA. Eight patients were found to be positive for HPV 6 DNA, six were positive for HPV 11 DNA, and one was positive for both HPV 6 and HPV 11 DNA. None of the males in the control group was positive for either HPV 6 or HPV 11 DNA. The results demonstrate that HPV can be transported by the urine, probably in exfoliated HPV-infected cells. A similar mechanism may occur during ejaculation, allowing sexual transmission of HPV viruses harbored in the cells of the male genital tract.     

Ultrastructural visualization of human papillomavirus DNA in verrucous and precancerous squamous lesions.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.     

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

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

High frequency of human papillomavirus 6/11, 16, and 18 infections in precancerous lesions and squamous cell carcinoma of the conjunctiva in subtropical Tanzania

Dysplastic lesions and epithelial neoplasms of the conjunctiva account for approximately 2% of all malignant tumors in subtropical Tanzania. We examined the pathophysiologic role of human papillomavirus (HPV) in the development of conjunctival carcinoma in subtropical Tanzania, which has a high HPV prevalence. Tissue samples from 14 patients were obtained from the cancer registry archives at the medical center of the university in Dar es Salaam, Tanzania. A highly sensitive nonradioactive in situ hybridization technique (ImmunoMax) was applied to paraffin-embedded tissue samples to identify HPV DNA in conjunctival epithelial dysplasia and epithelial neoplasms. In each case, conventional morphologic evaluation revealed a transitional lesion extending from koilocytic dysplasia to severe dysplasia or invasive squamous cell carcinoma. Highly specific, morphologically easily distinguishable labeling of HPV-6/11, HPV-16, and HPV-18 was found in most cases. Coinfections were observed frequently. The signals showed varying intensities and different patterns of distribution. In general, higher signal intensity was found in dysplasia grades 1 and 2 and in well-differentiated areas of the invasive component of conjunctival carcinoma compared with less differentiated areas. This observation underlines the central role of HPV-16 and HPV-18 in the oncogenesis of conjunctival cancers in subtropical Tanzania.     

Typing of human papillomavirus by pyrosequencing

The possibility of using a new bioluminometric DNA sequencing technique, called pyrosequencing, for typing of human papillomaviruses (HPV) was investigated. A blinded pyrosequencing test was performed on an HPV test panel of 67 GP5+/GP6+ PCR-derived amplification products. The 67 clinical DNA samples were sequenced up to 25 bases and sequences were searched using BLAST. All of the samples were correctly genotyped by pyrosequencing and the results were unequivocally in accordance with the results obtained from conventional DNA sequencing. Pyrosequencing was found to be a fast and efficient tool for identifying individual HPV types. Furthermore, pyrosequencing has the capability of determining novel HPV types as well as HPV sequence variants harboring mutation(s). The method is robust and well suited for large-scale programs.     

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections.

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and 35S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.     

Unsuccessful effort to detect human papillomavirus DNA in urinary bladder cancers by the polymerase chain reaction and in situ hybridization

The association of human papillomavirus (HPV) with urinary bladder carcinogenesis is now a controversial issue. In order to certify the presence of HPV DNA in urinary bladder cancers, the polymerase chain reaction (PCR) using five primer sets for detecting various HPV types was used in this study as weli as in sltu hybridizetion (ISH) for HPV 16 and 18 detection. In the PCR study of 93 DNA samples extracted from formalin-fixed and paraffin-ernbedded urinary bladder cancem, no HPV DNA was detected in these tumor samples. The ISH study was also performed on the same tumor samples, but failed to demonstrate any HPV 16-or 18-positive signals in ail except one of the tumor samples. However, the FCR failed to demonstrate HPV 16 DNA even in the bladder cancer positive for HPV 16 DNA by the ISH. This ISH technique was able to demonstrate HPV 16 and 18 DNA in eight of 13 paraffinembedded cervical cancers, in which HPV 16 or 18 DNA had already been detected by the PCR. Our HPV study using PCR and ISH revealed that the HPV status of urinary bladder carcinomas was far different from that of cervical cancers.     

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dotlike positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA. Acta Pathol Jpn 41: 757-762, 1991.     

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.     

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.     

TCT查验子宫颈涂片中HPV感染的研究

目的评价液基薄层细胞检测技术(TCT)检测宫颈涂片中人乳头状瘤病毒(human papilloma virus,HPV)感染的敏感性与准确性。方法对1030例外阴尖锐湿疣患者的子宫颈涂片,采用TCT检测技术进行细胞病理学诊断,其诊断标准依据The Bethesda System(TBS)分类法。同期行阴道镜指引下的子宫颈活组织检查和聚合酶链反应(PCR)技术检测宫颈拭子中的HPVDNA。结果应用PCR检测1030例宫颈拭子HPVDNA,阳性者37例(35.92%)。宫颈活组织学检查结果为ASCUS57例(5.53%);上皮内病变CIN11例,占总数的1%;占HPV( )百分率为29.73;占57例为非典型麟状细胞阳性的19.3。结论TCT作为一种细胞病理学检测技术,用于诊断宫颈HPV感染或亚临床感染,有临床应用价值。