Human papillomavirus DNA determination of anal condylomata, dysplasias, and squamous carcinomas with in situ hybridization

Infection with types 6, 11, 16, and 18 of the human papillomavirus (HPV) is associated with condylomatous, dysplastic, or carcinomatous changes in the genital tract. Emerging evidence suggests that a similar series of lesions develops in the anal canal after exposure to the same HPV types. In situ hybridization was performed with the use of biotinylated DNA probes to HPV 6, 11, 16, and 18, so as to determine the frequency of HPV DNA in 45 perianal and/or anal condylomata, 6 anal intraepithelial neoplasias, and 13 anal squamous cell carcinomas. Of the 33 perianal and/or anal condylomata in which HPV DNA was detected, 13 contained HPV 6 and 11, 12 HPV 6, 7 HPV 11, and 1 HPV 6, 11, and 18. Two of four severe anal dysplasias contained HPV 16, whereas one case each of mild and moderate anal dysplasia contained HPV 6. No HPV DNA was detected in the anal squamous cell carcinomas. The study demonstrated the presence of HPV DNA in 73% of condylomata and 67% of anal dysplasias. The observations suggest that the cloacogenically derived anal epithelium is susceptible to infection by the same HPV types as infect the similarly derived epithelium of the lower female genital tract and that these HPV types result in some similar lesions, i.e., condylomata and dysplasias in both sites. A role in the genesis of anal cancer was not found in this study.     

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.     

Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction.

To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma, the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detect HPV DNA in formalin-fixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16.7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the L1 open reading frame of the HPV genomes, the frequency of positive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using type-specific primers and probes for HPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing of cases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer.     

Comparative analysis of human papillomavirus detection by dot blot hybridisation and non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. METHODS: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. RESULTS: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). CONCLUSIONS: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.     

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.     

Comparison of DNA dot blot hybridization and lancefield capillary precipitin methods for group B streptococcal capsular typing

Group B streptococci (GBS) (Streptococcus agalactiae) are a major cause of sepsis and meningitis in neonates and infants and of invasive disease in pregnant women, nonpregnant, presumably immunocompromised adults, and the elderly. Nine GBS serotypes based on capsular polysaccharide antigens have been described. The serotype distributions among invasive and colonizing isolates differ between pediatric and adult populations and have changed over time. Thus, periodic monitoring of GBS serotype distributions is necessary to ensure the proper formulation and application of an appropriate GBS vaccine for human use and to detect the emergence of novel serotypes. Since the mid-1990s, the proportion of GBS isolates that are nontypeable by standard serologic methods has increased, creating a need for more sensitive typing methods. We describe a typing method that uses DNA dot blot hybridization with probes generated by PCR from the GBS capsular genes for serotypes Ia, Ib, and II to VIII. PCR primers were designed to amplify type-specific GBS capsular gene sequences. Gene probes were constructed from the PCR products and used to classify isolates based on hybridization profiles. A total of 306 previously serotyped invasive and colonizing isolates were used to compare our dot blot capsular typing (DBCT) identification method with Lancefield serotyping (LS). A dot blot capsular type was assigned to 99% (303 of 306) of the isolates, whereas 273 of 306 isolates (89%) were assigned a Lancefield serotype. The overall agreement between the methods was 95% (256 of 270 isolates typeable by both methods). We conclude that the DBCT method is a specific and useful alternative to the commonly used LS method.     

Detection of enterotoxigenic Escherichia coli by dot blot hybridization with biotinylated DNA probes.

A dot blot hybridization test was developed for the detection of enterotoxigenic E. coli without the use of radioisotopes. Three biotin-labeled DNA (Bio-DNA) probes corresponding to structural genes specifying heat-labile and heat-stable enterotoxins of porcine and human origin were prepared by random priming; label incorporation was significantly higher than that obtained from the use of nick translation. Bio-DNA probes were highly specific when reacted with protein- and RNA-free DNA preparations in a dot blot hybridization assay. The Bio-DNA probe, in which 40% of available thymidines were replaced by a biotin-labeled deoxyuridine, readily detected 160 pg of target DNA mixed with 6 micrograms of carrier DNA. The minimum amount of total DNA required for reliable identification of a single-copy enterotoxin gene of porcine origin within a 5,000-kilobase chromosome was found to be approximately 5 micrograms. Complete agreement among the results of Bio-DNA probe hybridization, [32P]DNA hybridization, and biological assay was demonstrated for 15 (100%) of the clinical isolates. This procedure provides a more suitable approach for the diagnosis of enterotoxigenic E. coli infections in clinical settings than the hybridization assay based on 32P-labeled DNA probes.     

Human papillomavirus DNA in respiratory papillomatosis detected by in situ hybridization and the polymerase chain reaction.

The authors have demonstrated the presence of human papillomavirus (HPV) types 6 and 11 in 10 of 13 (77%) juvenile laryngeal papillomatosis by in situ DNA hybridization using as probes the radiolabeled DNAs of HPVs 6, 11, 16, and 18. Of six specimens from adult laryngeal papillomatosis assayed by the same technique, only 33% were positive. Immunohistochemistry to detect HPV capsid antigens performed on serial sections gave positive signals in 44% (8 of 18) of the specimens, all from juvenile lesions. These results were in agreement with in situ hybridization, except in two cases. When both series (juvenile and adult) were analysed by amplification of a 450-bp fragment corresponding to the L1 ORF of the HPV genomes directed by the polymerase chain reaction, the frequency of positive specimens rose to 100%. Our data agree with the concept that HPV is implicated in the etiology of laryngeal papillomatosis.     

In situ hybridization analysis of human papillomavirus in anal squamous cell carcinoma

To investigate the association between human papillomavirus (HPV) infection and anal carcinoma, we applied a sensitive in situ hybridization technique to detect HPV messenger RNA (HPV m-RNA) in formalin-fixed, paraffin-embedded sections from 18 patients. Using tritium-labeled probes, HPV m-RNA was detected in 12/18 (67%) patients. HPV 6 was detected in four patients, coexisting with HPV 18 in two cases, and HPV 16 was found in eight patients. In six patients, hybridization failed to demonstrate the presence of HPV. With respect to histology, HPV 6 was detected in 1/4 cases of well differentiated invasive squamous cell carcinoma. Ten of thirteen moderately or poorly differentiated invasive squamous cell carcinomas demonstrated HPV m-RNA (HPV 16, eight cases; HPV 6, one case; HPV 6 and 18, one case). HPV 31 was not detected in any specimens. These results suggest that HPV infection may play an important role in the pathogenesis of anal carcinoma.     

Chemiluminescence dot blot hybridization assay for detection of B19 parvovirus DNA in human sera.

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.     

Rapid detection of cytomegalovirus DNA in urine samples with a dot blot hybridization immunoenzymatic assay.

A dot blot hybridization immunoenzymatic assay for the rapid detection of cytomegalovirus DNA in urine samples was developed by using a digoxigenin-labeled probe which was immunoenzymatically visualized by antidigoxigenin Fab fragments labeled with alkaline phosphatase. A total of 516 urine samples from different groups of subjects were analyzed, and the hybridization assay was able to yield results within 24 h. The results obtained were compared with results for detection of cytomegalovirus antigens in infected cell cultures.     

Comparison of different in situ hybridization techniques for the detection of human papillomavirus DNA in cervical smears.

Different hybridization methods were used for detection of human papillomavirus (HPV) DNA in cervical smears. The results obtained by filter in situ hybridization (FISH) are consistent with most of the reports recently published. To overcome the unsatisfactory limitations of this method, especially the difficulties to distinguish clearly between positive and negative signals, we developed an in situ hybridization protocol using a cytospin and 35S-labelled as well as biotinylated DNA-probes. For direct comparison of different methods, the samples were obtained from two groups of patients. One group were women with reiterated Papanicolaou smears III, IV; the other were women with reiterated Pap III, IV and additional histological scoring. In all cases but one, the different methods used have shown the same results. In one case the hybridization on slides using 35S-labelled as well as biotinylated probes gave a negative result, whereas the FISH method using a 32P-labelled probe allowed to detect of HPV 16 and 18 DNA only when more than 1 x 10(6) cells were present per filter. Our data demonstrate that in situ hybridization on slides is a specific and sensitive technique, which enables a clear distinction between positive and negative results using a small number of cells and which, especially with biotinylated probes, is suitable for application in routine work.     

Human papillomavirus DNA in oesophageal carcinomas in South Africa

Using morphological criteria, the presence of human papillomavirus (HPV) in oesophageal carcinomas has been inferred in patients from Finland and South Africa. However, studies to demonstrate the viral antigen in tissue sections of these tumours have proved disappointing. This study investigates 48 archival oesophageal carcinoma biopsies from South Africa for the presence of HPV DNA using non-isotopic in situ hybridization (NISH) with HPV DNA probes to HPV 6, 11, 16, 18, 31, and 33. HPV DNA sequences were detected in 25/48 (52 per cent) oesophageal cancers. HPV 16 was present in 84 per cent of the HPV-positive cancers. A NISH type 2 signal pattern (punctate/dot) was present in all HPV-positive tumours. This signal pattern was previously shown to represent integrated HPV DNA within host chromosome. Integrated HPV DNA in oesophageal cancers has also been demonstrated in patients from China and Japan. In addition, the prevalence of HPV DNA in oesophageal cancers from high-risk countries like South Africa (52 per cent) and China (49 per cent) would appear to be consistent.     

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 human papillomavirus DNA in gynaecological swabs by filter in situ hybridization.

Gynaecological smears from the endo- and ectocervix of women with and without cytological and colposcopic abnormalities of the epithelium were investigated for human papillomavirus (HPV) types 6, 11, 16, and 18 by filter in situ hybridization (FISH). The data were compared with cytological, colposcopic, and histological findings. Of the 266 gynaecological smears, HPV DNA was detected in 84 (32%); of 101 cytologically and colposcopically HPV negative cases, HPV DNA was found in 10%. Of 56 women, cytologically and colposcopically positive for HPV infection, HPV DNA was detected in 68%. The sensitivity of the method was controlled by comparing the results of FISH with those of Southern-blot analysis of five cervical tumour biopsies. The data presented demonstrate the necessity of FISH for identification of the HPV type that might be of prognostic value in cervical pathology. Cytological and colposcopic positivity is a reliable sign in about 70% of the cases where HPV infection was proved by FISH.     

Demonstration of human papillomavirus DNA in a peripheral ameloblastoma by in situ hybridization.

The peripheral ameloblastoma (PA) is a rare, extraosseous, odontogenic lesion usually located on the mandibular or maxillary gingiva. It often appears as an asymptomatic, firm, pink-red nodule with occasional secondary involvement of the underlying bone. Although histologically identical to primary intraosseous ameloblastoma, the PA is notably less aggressive in biologic behavior and rarely recurs following conservative treatment. It has a proposed dual histogenesis, while its etiology remains obscure. Several benign oral mucosal lesions have been found to be associated with various types of human papillomavirus (HPV). The following study uses a commercially available HPV DNA in situ hybridization technique to confirm the presence of HPV in a previously reported case of PA. Results were positive for HPV types 16/18 but negative for HPV types 6/11. The implications of these results with regard to etiology are discussed.     

Problems in detection of cytomegalovirus in urine samples by dot blot hybridization.

A hybridization assay for the detection of cytomegalovirus (CMV) in urine specimens was established. Two different DNA fragments were used as hybridization probes: the HindIII L fragment (11.7 kilobases) and the EcoRI J fragment (10.6 kilobases) of the human CMV strain AD169. These probes were used in an isolated and highly purified form and therefore did not cross hybridize with vector sequences. As shown by hybridization with DNA from CMV-infected and uninfected cells, the assay was highly CMV specific and sensitive (detection limit, 750 to 500 fg of CMV DNA). A total of 122 urine specimens were examined by DNA hybridization, virus isolation, and the detection of CMV-induced early nuclear protein. The results coincided in 91% of the samples. The application of DNA hybridization to urine samples, however, is not without problems, and some of the pitfalls and drawbacks are discussed.     

Human papillomavirus and anal neoplasia

Anal cancer is a rare disease in the general population, but the incidence of anal cancer is higher in certain at-risk groups, such as men who have sex with men (MSM), and immunosuppressed individuals, including those with HIV infection. Among HIV-positive MSM, the incidence of anal cancer may be as high as 10 times greater than current rates of cervical cancer in the general population of women. Anal cancer is associated with human papillomavirus (HPV) infection and may be preceded by high-grade anal intraepithelial neoplasia (HGAIN). HGAIN and anal HPV infection are both highly prevalent in groups at risk for anal cancer. Current issues include determining the effect of antiretroviral therapy on the natural history of HGAIN and the incidence of anal cancer, optimizing diagnostic and therapeutic approaches to HGAIN, and determining the potential for prophylactic HPV vaccines to prevent anal HPV infection and anal cancer in at-risk groups.     

Comparison of four non-radioactive and 35S-based methods for the detection of human papillomavirus DNA by in situ hybridization.

Human papillomavirus DNA was detected in 40 condylomatous lesions of various sites (vulva, cervix, larynx, penis and anus) by in situ hybridization using 35S-labelled probes and four non-radioactive probes to compare the various sensitivities of these techniques on the same material (formalin-fixed and paraffin-embedded sections). Radioactive probes yielded 28 positive results out of 40 (70%). Sulphonated probes (HybriCyte kit) also gave 28 positive results with a fine pattern of hybridization grains and equal sensitivity to 35S-labelled probes. Biotinylated and digoxigenin-labelled probes gave analogous results (25 positive reactions with the PathoGene kit, 26 with the Viratype kit, and 25 with digoxigenin-labelled probes) but are slightly less sensitive than radiolabelled and sulphonated probes especially when the signal is weak.     

Identification of human blood in mosquitoes (Diptera: Culicidae) using nonradioactive DNA dot blot hybridization.

A dot blot hybridization procedure was developed to detect human blood meals in engorged mosquitoes. A biotinylated DNA probe allowed the detection of 10-100 ng of human DNA, discriminated well between human and nonhuman sources of blood, and cross-reacted only with monkey DNA. Results showed that this method was a specific and sensitive technique for the identification of blood meals up to 100 h after ingestion. The nonisotopic label offers easy handling without the problems inherent in the use of radioisotopes, and it can be adapted for use in routine field tests.