HPV prevalence and p16INKa overexpression in non-smoking non-drinking oral cavity cancer patients.

OBJECTIVE: Our aim was to compare HPV and p16INK4a (p16) expression and their influence on survival and prognosis in oral cavity squamous cell cancer (OCSCC), between non-smokers and non-drinkers (NSND) and smokers and drinkers (SD). SUBJECTS AND METHODS: Patients with OCSCC treated with surgery from 2000 to 2010 were included in the study. Patients who did not smoke at all or smoked less than 10 pack per years and did not drink alcohol on a daily basis were considered the NSND group. An equal number of SD were the control group. HPV presence was determined from paraffin-embedded blocks investigated by PCR analysis. p16 expression was evaluated with immunohistochemistry. RESULTS: The NSND group were mostly younger or older female patients with tongue or gingival cancers. p16 expression was significantly more frequent in NSND patients (27% vs 10%). Patients with stronger p16 expression had significantly worse survival, especially for tongue cancers (P = 0.026). In Cox multivariate analysis, both HPV and p16 expression carried a negative prognosis for NSND patients (P = 0.0351 and P = 0.0260). CONCLUSIONS: NSND are a specific population of OCSCC patients. In contrast to oropharyngeal cancer, HPV and p16 expression in OCSCC are negative predictive factors, especially in NSND patients.

Human papillomavirus, cytomegalovirus, and adeno-associated virus infections in pregnant and nonpregnant women with cervical intraepithelial neoplasia.

Two hundred eight cervical specimens from two groups of subjects, 165 nonpregnant women and 53 pregnant women with cervical intraepithelial neoplasia (CIN) of grades I to III, were positive by PCR analyses for human papillomaviruses (HPVs), adeno-associated virus type 2 (AAV 2), and human cytomegalovirus (HCMV) in 67, 6, and 4.1% of the cases, respectively. The presence of AAV 2 infection was more frequently associated with pregnancy (17 versus 2.4%) and HPV-positive cervices (odds ratio = 6.358) than HCMV was. Increased HPV infection was strongly associated (P < 0.001) with a higher CIN grade, but there is no evidence that AAV 2 and HCMV infections have any impact on CIN development.

Evaluation of genital human papillomavirus infections by polymerase chain reaction among Croatian women.

Infection with specific human papillomavirus (HPV) types is the strongest risk factor in cervical carcinogenesis. In this study we analysed, by means of polymerase chain reaction (PCR), cervical specimens obtained from consenting women with abnormal Pap smears collected from 1996 to 1998. Consensus- and type-specific-primers directed PCR were used in order to detect the presence and to determine the most common HPV types: 6, 11, 16, 18, 31 and 33. Out of 1874 specimens, 1207 (64%) contained one or more HPV types. Approximately half HPVs were typed (621 out of 1207) and the others remained untyped (586 out of 1207), 51% and 49%, respectively. Beside low-risk HPV 6/11 (5%), the most frequently observed HPVs were high-risk HPV types, especially type 16 (12%), while HPV types 18 (2%), 31 (5%) and 33 (3%) were less frequent. The HPV positivity rate declined with age, although all HPV types were equally distributed in different age groups. The presence of HPV DNA significantly increased from 55% to 78% along with the severity of the cervical lesions, i.e. low- and high-grade squamous intraepithelial lesions (LSIL, HSIL). Undetermined HPV types, other than 6/11, 16, 18, 31 and 33 were equally distributed in LSIL and HSIL which indicates that they represent low- as well as high-risk HPV types. Our results indicated that HPV infections, especially those with HPV 16, represent a significant public health concern in Croatia.

Comparison of five different polymerase chain reaction methods for detection of human papillomavirus in cervical cell specimens.

The polymerase chain reaction (PCR) methods enable the detection of large number of human papillomavirus (HPV) genotypes that infect the anogenital tract. In this study, two groups of cervical scrapes with abnormal cytomorphology were analysed. The first group was tested with three sets of consensus primers located within the L1 region of HPV genome, MY09/MY11 (i.e. MY), L1C1/L1C2-1/L1C2-2 (i.e. LC) and pI-1/pI-2 (i.e. pI) primer sets, while the second group of samples, which were all negative with the MY primers, was tested further with the LC primers, as well as with the GP5/GP6 (i.e. GP) primers. The GP primers were used in the nested PCR following amplification with the MY primers (i.e. MY/GP nested PCR). Samples from both groups were also tested with type-specific primers for HPV types 6/11, 16, 18, 31 and 33. In the first study group (N=164) there were 76.2% positive results obtained with at least one set of consensus primers. There were 62.2, 39, 62.2 and 59.1% positive results obtained with the MY, the pI, the LC and the HPV type-specific primer sets, respectively. The best results were obtained when both the MY and the LC primer sets were used, because in combination they detected 75% positive samples compared to 62.2% when used alone. There were 2. 4% samples negative with all consensus primers, but positive with one of the HPV type-specific primers, which increased the overall positivity rate to 78.6%. In the second study group (N=250) there were 8.4, 38.8 and 4% samples positive with the LC primers, the nested MY/GP and the HPV type-specific primer sets, respectively. Thus, the use of the MY/GP nested PCR increased significantly the positivity rate of HPV DNA detection and should be used for samples with a low copy number of HPV DNA. In conclusion, the following diagnostic protocol would be appropriate for detection of cancer-related HPVs: preselection of samples with the MY and the LC primers, additional amplification of the MY- and the LC-negative samples with the MY/GP nested PCR and HPV typing of consensus PCR-positive samples with the HPV type-specific primers.

Detection and typing of human papillomaviruses by means of polymerase chain reaction and fragment length polymorphism in male genital lesions.

We investigated the distribution of genital human papillomaviruses (HPVs) among 171 consenting men of which four were involved twice in this study. The DNA was obtained from 7 normal tissues and 168 genital lesions of which 115 were diagnosed as condylomata acuminata, 17 as condylomata plana and 36 as HPV-associated lesions (papules, lichen-like lesions, etc.). The DNA samples were analysed for the presence and type of HPV DNA (HPV type 6, 11, 16, 18, 31 or 33) by means of polymerase chain reaction (PCR). Out of 175 specimens tested, 140 (80%) were HPV positive and 35 (20%) HPV negative. There were 81.43% (114 out of 140) typed HPVs, while 18.57% (26 out of 140) remained untyped. Most samples were HPV 6/11 positive (92 out of 114, 80.7%). Restriction fragment length polymorphism (RFLP) of HPV 6/11 PCR products in 89.13% (82 out of 92) and 10.87% (10 out of 92) specimens corresponded to HPV 6 and HPV 11, respectively. The frequency of other HPVs was low, i.e. there were 4.57% (8 out of 175), 1.71% (3 out of 75) and 0.57% (1 out of 175) HPV type 16, 18 and 33, respectively. There were 10 out of 175 (5.71%) cases of multiple HPV infections, of which 6 out of 10 were cases with HPV 6 and other HPV types. This raises the total prevalence of HPV type 6 to 50.29% (88 out of 175) in the study-population. The clinical diagnosis condylomata acuminata was preferentially associated with low-risk HPVs (types 6 and 11), while other lesions, especially condylomata plana, with high-risk HPVs (types 16, 18, 31 and 33) and untyped HPVs. The male population, indeed, represents a reservoir of HPV infection and directly influences cervical cancerogenesis.

Detection and typing of human papillomaviruses by polymerase chain reaction in cervical scrapes of Croatian women with abnormal cytology.

The association between certain human papillomaviruses (HPV) and cervical intraepithelial neoplasia (CIN) is well documented, but still unknown among Croatian women. In 1995, women between the age of 17 and 64 with cytomorphologically abnormal smears (CIN I-IV) were tested for the presence of HPV. Consensus and specific primers were used in the polymerase chain reaction (PCR) to detect the most common types: 6, 11, 16, 18, 31 and 33, as well as the unknown-risk HPV types (HPV X). Out of 379 specimens, 163 (43%) contained one or more HPV types. Coinfection with different HPV types in the same sample was observed in 16 cases. Beside low-risk HPV 6/11 (25.8%) the most frequently observed types were high-risk HPV types 16 (20.2%) and 31 (17.8%). Globally, the HPV positivity rate declines with age. The presence of HPV DNA significantly increased from 35.5 to 61.1% along with the severity of the cervical intraepithelial neoplasia (CIN I-IV). HPV type 6/11 was strongly associated with CIN I (33.8%), HPV type 31 with CIN II (22.9%), and HPV type 16 with CIN III (50%).

Human papillomaviruses are not associated with renal carcinoma.

In an attempt to better understand the troubling issue of renal human papillomavirus (HPV) infection, fresh tumour specimens obtained by surgical extirpation from tumours were compared for the presence of HPV. All of the renal carcinoma samples were examined by PCR using two sets of consensus primers and the specific primer pairs for HPV 16, 18, and 33. None of the 28 carcinomas and 17 corresponding normal tissues were found positive for HPV DNA in any of the applied analyses. Our results suggest that HPV does not play any role in the development of renal carcinoma in the general population.

Increase of genital human papillomavirus infection among men and women in Croatia.

The presence of genital human papillomavirus (HPV) among men and women in Croatia was investigated from 1990 to 1993. DNA obtained from 338 cervical and 79 male urethral scrapes was analysed by slot-blot hybridization with digoxigenin labeled HPV type 6, 11, 16 and 18 DNA probes. The prevalence of HPV infection was higher in the male population (26.6%) than in the female population (19.8%). Low risk HPV (HPV 6, 11), high risk HPV (HPV 16, 18) and indeterminate HPV were found in 5.3%, 6.9% and 8.8% cases, respectively. Over the years, the increase (from 4.9% to 37.6%) of HPV infection was almost equal in male and female population suggesting an epidemic feature of this sexually transmitted disease.

[Human viruses in the coastal environment of a Croatian harbor]. / Présence de virus humains dans l'environnement côtier d'un port croate.

The authors submit the results of a survey on the viral pollution of the costal environment in the vicinity of a Croatian harbour on the northern Adriatic sea. The presence of human viruses was established in 22% of the sea water samples, 54% of the fluffy sediments samples, 44% of the sandy sediments samples and 26% of the batches of shell-fishes. Hepatitis A virus and rotavirus antigens determined by immuno-electro-osmophoresis made up 76.3% and 15.2% of the positive results respectively, whereas the coxsackieviruses, the echoviruses and the polioviruses isolated on cell culture made up 8.5% of the remaining results together. The analysis of several specimens of different nature taken simultaneously gave a better vision of the viral pollution.

[Presence of viruses in drinking water in Croatia]. / Présence de virus dans l'eau potable en Croatie.

Drinking water must be virus-free. However, it has been observed and demonstrated that various pathogenic viruses are capable of transmission through water and that such viral diseases may have far-reaching health and socio-economic implications. Over a four-year period 135 drinking water samples were collected in a middle-sized town in Croatia. The percentages of positive findings varied from 0 to 10%, the average being 3.7%. These results are compared with the ones given by similar studies in different parts of the world.

Monoclonal antibodies to murine retrovirus protein p30.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of Wistar-Furth rats that had been immunized with a Moloney sarcoma virus (Mo-MuSV)-induced tumour, MFU. Two immunization protocols were designed. In the first, the animals received several injections of irradiated (10 000 rad) cells of a tumour cell line established in vitro, MFU-67. The rats received a booster injection 3 days prior to fusion. In the second protocol, immunization was the result of simple tumour growth, and no booster was given. Hybrids were tested by immunofluorescence for the production of immunoglobulins reacting with mouse cells acutely infected with Mo-MuSV. Over 20% of reactive hybrids were observed in the tumour growth protocol, and about 10% in the irradiated cell protocol when the last injection of the series was given 2 weeks before fusion. After 6 months, the proportion fell to 3%. Hybrid lines producing antibody to p30, the major core polypeptide of murine retroviruses, were obtained by cloning. Three of these were selected for closer study and were found to recognize three non-overlapping epitopes on p30. By direct and competitive binding in ELISA tests, the three epitopes were found to have very different distribution patterns among the various strains and isolates of murine retroviruses.