Bernhard Kleter

Prevalence of Human Papillomavirus DNA in Different Histological Subtypes of Cervical Adenocarcinoma

The prevalence of human papilloma virus (HPV) DNA in different histological subtypes of cervical adenocarcinoma and related tumors was examined using formalin-fixed, paraffin-embedded tissue samples from 105 primary cervical adenocarcinomas and adenosquamous carcinomas. Broad-spectrum HPV DNA amplification and genotyping was performed with the SPF10 primer set and line probe assay (LiPA), respectively. HPV DNA was detected in 82 of 90 (91%) mucinous adenocarcinomas, encompassing endocervical, intestinal, and endometrioid histological subtypes, and in nine of nine adenosquamous tumors (100%). HPV DNA was not detected in any nonmucinous adenocarcinomas including clear cell, serous, and mesonephric carcinomas (0/6). The most common viral types detected in adenocarcinoma were HPV 16 (50%) and HPV 18 (40%), followed by HPV 45 (10%), HPV52 (2%), and HPV 35 (1%). Multiple HPV types were detected in 9.7% of the cases. In conclusion, mucinous adenocarcinomas and adenosquamous carcinomas of the cervix demonstrate a very high prevalence of HPV DNA, similar to that reported for cervical squamous cell carcinoma. Only rare histological variants of cervical adenocarcinoma seem unrelated to HPV infection.

High Prevalence of Human Papillomavirus (HPV) Infections and High Frequency of Multiple HPV Genotypes in Human Immunodeficiency Virus-Infected Women in Brazil

ABSTRACT A group of 208 human immunodeficiency virus (HIV)-infected women in Brazil were studied for the presence of human papillomavirus with the general SPF10 PCR primer set. Virtually all (98%) women were found positive for human papillomavirus (HPV) DNA. Genotyping by the reverse hybridization line probe assay (HPV-LiPA) revealed a high prevalence of multiple genotypes (78.9% of the cases), with an average of 3.1 genotypes per patient (range, 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 80 (39.2%) patients, followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%), and 16 (22.5%). Of the genotypes detected, 40.9% were low-risk genotypes. Twenty-two (10.5%) patients showed normal (Pap I) cytology, 149 (71.6%) patients had inflammation (Pap II), and 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes detected and the cytological classification, HIV viral load, and CD4 count in these patients. In conclusion, the highly sensitive SPF10 LiPA system shows that a very high proportion of HIV-infected women in Brazil are infected with HPV and often carry multiple HPV genotypes.

Highly Effective Detection of Human Papillomavirus 16 and 18 DNA by a Testing Algorithm Combining Broad-Spectrum and Type-Specific PCR

ABSTRACT The use of a single broad-spectrum human papillomavirus (HPV) DNA-based PCR test may fail to detect lower concentrations of HPV DNA due to competition between different genotypes in mixed infections. To improve HPV detection by PCR, broad-spectrum and type-specific (TS) PCRs were combined, with a focus on HPV-16 and HPV-18. Cervical and cervicovaginal cell samples were obtained from 1,113 healthy women (age range, 15 to 25 years) participating in an HPV-16/HPV-18 candidate vaccine efficacy trial. These samples were tested by a broad-spectrum SPF10 PCR-DNA enzyme immunoassay, followed by a primer SPF10-based line probe assay (SPF10 LiPA), and HPV-16- and HPV-18-TS PCRs. The results for the majority of the HPV-16/18 SPF10 LiPA-positive samples were confirmed by TS-PCR (kappa values, 0.775 for HPV-16 and 0.785 for HPV-18). However, TS PCR revealed additional positive samples among those that contained other HPV genotypes due to competition. Conversely, SPF10 LiPA identified HPV-16 or -18 in samples that remained negative by TS PCR as a result of sampling variation. Analysis of follow-up samples from more than 1,000 women confirmed that the combination of SPF10-LiPA with additional HPV-16- and HPV-18-TS PCR diminishes the rate of false-negative diagnosis. The combination of broad-spectrum and TS PCRs resulted in a novel testing algorithm. This combination of assays is more accurate than either method alone, and the novel algorithm offers a highly accurate and effective method for the analysis of HPV infections.

Genotyping of Human Papillomavirus in Liquid Cytology Cervical Specimens by the PGMY Line Blot Assay and the SPF10 Line Probe Assay

ABSTRACT A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF10 LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (κ = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (P = 0.002), HPV type 56 (P = 0.039), and HPV type 59 (P < 0.001), whereas SPF10 LiPA detected more HPV type 31 (P < 0.001) and HPV type 52 (P = 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.

Evaluation of a Novel Highly Sensitive, Broad-Spectrum PCR-Reverse Hybridization Assay for Detection and Identification of Beta-Papillomavirus DNA

ABSTRACT Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Efficacy of interferon dose and prediction of response in chronic hepatitis C: Benelux study in 336 patients

BACKGROUND/AIMS In an attempt to improve the limited efficacy of treatment of chronic hepatitis C with interferon-alpha 3 MU tiw, we studied the effects of double-dose therapy followed by downward titration, and analyzed the pre- and pertreatment factors associated with response or non-response. METHODS Three hundred and fifty-four consecutive patients in 19 centers were randomized to interferon-alpha 3 MU tiw for 6 months or 6 MU tiw for 8 weeks followed by down-titration (3,1 MU tiw) till alanine aminotransferase remained normal and plasma HCV RNA was repeatedly undetectable. The primary outcome measure was sustained alanine aminotransferase and HCV RNA response 6 months after treatment. RESULTS Three hundred and thirty-six patients received treatment. The sustained response rate for patients receiving 3 MU tiw for 6 months was 14% (9-21%,) and for patients receiving double dose tiw for 8 weeks and thereafter titrated therapy 15% (10-21%) (p=0.8). Pretreatment factors associated with a sustained alanine aminotransferase plus HCV RNA response were the absence of cirrhosis, presence of genotype 2 or 3, a low viral load and, in addition, a low alanine aminotransferase/aspartate aminotransferase ratio; a model was developed to allow estimation of the chance of response for the individual patient. The most powerful predictor of sustained response, however, was plasma HCV RNA at week 4; a positive test virtually precluded a sustained response (1.7%, 0.4-5.0%). If week 4 HCV RNA was not detectable, the chance of a sustained response was 21% (12-34%) for genotype 1 versus 40% (28-54%) for the others (p=0.02). Six MU tiw led to a significantly higher week 4 HCV RNA response (47% not detectable) than 3 MU (37%) (p=0.02). During down-titration this difference in viral on-treatment response was lost. CONCLUSIONS In the treatment of hepatitis C, an early HCV RNA response is a prerequisite for long-term efficacy. Doubling the initial interferon dose increases this early response, but subsequent downward titration negates this effect, especially in genotype 1.

Antibody responses to 26 skin human papillomavirus types in the Netherlands, Italy and Australia

Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infections with skin human papillomavirus (HPV) types have also been linked to the development of SCC. Little is known about the natural history of these infections and whether the seroprevalence of skin HPV types is affected by ambient or individual levels of sun exposure. This study investigated this by analysing sera for antibodies to 26 skin HPV types from five phylogenetic genera obtained from 807 healthy individuals from the Netherlands, Italy and Australia, countries with strong differences in sunlight intensity. Overall HPV seroprevalence was similar across the three countries (50-57 % for beta-HPV types, 40-48 % for gamma-HPV types), and the most frequent beta-HPV and gamma-HPV types were the same in all countries. The highest seroprevalences for 24 of the 26 skin HPV types were observed in Italy (14 types) and Australia (ten types). Seroprevalence among men was generally higher than among women, and the male sex was significantly associated with both beta-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64-4.82] and gamma-HPV (OR 2.42, 95 % CI 1.40-4.18) antibodies in Australia. The only measure of sun sensitivity or UV exposure significantly associated with skin HPV seroprevalence was found for weekend sun exposure in Australia and beta-HPV antibodies. It was concluded that type spectra and HPV seroprevalence are similar in countries with different sunlight intensity, and that levels of UV exposure do not play a strong role in the development of skin HPV antibodies in this study population.

Human papillomavirus infection and squamous cell carcinoma of the conjunctiva

Background:Squamous cell carcinoma of the conjunctiva (SCCC) is associated with HIV-related immunosuppression, but human papillomavirus virus (HPV) is also suspected to have a role. We carried out a case–control study to assess the role of cutaneous and mucosal HPV types in SCCC, conjunctival dysplasia, and their combination (SCCC/dysplasia) in Uganda.Methods:We compared HPV prevalence in frozen biopsies from 94 SCCC cases (79 of whom were found to be HIV-positive), 39 dysplasia cases (34 HIV-positive), and 285 hospital controls (128 HIV-positive) having other eye conditions that required surgery. Highly sensitive PCR assays that detect 75 HPV types were used. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed, adjusting for, or stratifying by age, sex, and HIV status.Results:Cutaneous HPV types were detected in 45% of SCCC cases, 41% of dysplasia cases and 11% of controls. Human papillomavirus virus 5 and 8 were the most common types in SCCC, and most often occurred in combination with other types. Associations were observed between SCCC/dysplasia and detection of both single (OR=2.3; 1.2–4.4) and multiple (OR=18.3; 6.2–54.4) cutaneous HPV types, and were chiefly based on findings in HIV-positive patients. Cutaneous HPV infections were rarely observed among HIV-negative patients and the association with SCCC/dysplasia was not significant (OR=2.4; 0.6–9.6) among them. Squamous cell carcinoma of the conjunctiva/dysplasia risk and mucosal HPV types were not associated in either HIV-positive or HIV-negative patients.Conclusions:We detected cutaneous HPV types in nearly half of SCCC/dysplasia cases and often multiple types (HPV5 and 8 being most common). The role of HIV (confounder or strong enhancer of cutaneous HPV carcinogenicity) is still uncertain.

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

ABSTRACT Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

Prevalence, incidence and clearance of human papillomavirus infection among young primiparous pregnant women in Kampala, Uganda

The proportion of women who have already been exposed to human papillomavirus (HPV) infection by the time they first become pregnant, and the influence of pregnancy and delivery on the course of HPV infection are unclear. In Kampala, Uganda, 987 young primiparous pregnant women aged <25 years had gynaecological examination and liquid‐based cytology. In the follow‐up, women acted as their own controls, i.e., 1st/2nd versus 3rd trimesters (105 women), and during pregnancy versus after delivery (289 women). HPV was assessed using highly sensitive PCR assays. Prevalence of HPV and HIV infections at baseline were 60.0% and 7.3%, respectively. HPV16 and 18 were detected in 8.4% and 5.8%, respectively, i.e., less frequently than HPV51 (8.7%) and 52 (12.1%). At follow‐up new HPV infections were detected in 42.9% of women between the 1st/2nd and 3rd trimesters, and 38.1% between pregnancy and delivery, but 50.4% and 71.8% of HPV infections, respectively, cleared, leaving HPV prevalence unchanged in the different periods. Prevalence of cytological abnormalities diminished after delivery (from 21.2% to 12.4%). Presence of genital warts and sexually transmitted infections other than HPV were the strongest risk factors for prevalent or incident HPV infection. Clearance was lower among HIV‐positive women. In conclusion, HPV prevalence was high in primiparous women in Uganda, but pregnancy did not seem to be a period of special vulnerability to the infection.