Routine genotyping of human papillomavirus samples in Denmark

In order to examine a sensitive unbiased consensus PCR with routine sequencing for HPV typing, we analysed Danish male and female patients suspected of having an HPV infection. We used the well-characterised nested PCR setting with MY09/MY11 and GP5+/GP6+ primers, followed by routine cycle sequencing. Of 1283 clinical samples from female patients based on suspected HPV infection, we found 379 (29%) negatives and 894 (70%) positives. Samples containing >5000 HPV copies/ml were genotyped by sequencing. Of the 552 HPV genotyped samples from women >15 years of age, 398 were characterised as high-risk types and the remaining 154 as low-risk types. The most commonly found high-risk types were HPV-16, HPV-31, HPV-33, HPV-18, HPV-58, and HPV-52, and the most commonly found low-risk types were HPV-6, HPV-53 and HPV-11. In addition, we observed that other typing assays could not perform as sensitively or accurately as the nested PCR/cycle sequencing method used in this study. For instance, 87 out of 552 genotyped samples could not have been typed correctly in the Hybrid Capture II assay. Of these 87 samples, 46 (53%) were considered as high-risk types.     

Classical Molecular Tests Using Urine Samples as a Potential Screening Tool for Human Papillomavirus Detection in Human Immunodeficiency Virus-Infected Women

Human papillomavirus (HPV) is the main risk factor associated with the development of cervical cancer (CC); however, there are other factors, such as immunosuppression caused by the human immunodeficiency virus (HIV), that favor progression of the illness. This study was thus aimed at evaluating the functionality of classical PCR-based molecular tests for the generic identification of HPV DNA (GP5+/GP6+, MY09/MY11, and pU1M/2R primers, individually or in combination) using cervical and urine samples from 194 HIV-positive women. Infected samples were tested with type-specific primers for six high-risk types (HPV-16, -18, -31, -33, -45, and -58) and two low-risk types (HPV-6 and -11). HPV infection prevalence rates were 70.1% for the cervical samples and 63.9% for the urine samples. HPV-16 was the most prevalent viral type in the cervical and urine samples, with higher rates of multiple infections than single infections detected in such samples. HPV DNA detection by PCR (mainly with the pU1M/2R primer set) in urine samples was positively associated with abnormal cytological findings (atypical squamous cells of undetermined significance/squamous intraepithelial lesions [ASCUS/SIL]). It was determined that the operative characteristics for detection of cytological abnormalities were similar for cervical and urine samples. This suggested using PCR for the detection of HPV DNA in urine samples as a potential screening strategy for CC prevention in future prevention and control programs along with currently implemented strategies for reducing the impact of the disease, i.e., urine samples are economical, are easy to collect, have wide acceptability among women, and have operative characteristics similar to those of cervical samples.     

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.     

Human papillomavirus DNA detection and typing in male urine samples from a high-risk population from Argentina

The aim of the present study was to evaluate the first void urine (FVU) as a non-invasive sampling method for HPV detection and genotyping in a high-risk population. Men presenting with HPV associated penile lesions or HPV positive partners attending a urological department in La Plata, Argentina were enrolled for HPV detection and genotyping. DNA from 185 first-void urine samples was evaluated for the presence of HPV by nested polymerase chain reaction using MY09/11 and GP05/06 primers. The viral genotype was analyzed by means of the single-stranded conformation polymorphisms (SSCP) method. Seventy-three percent (135/185) of the FVU specimens were positive for HPV-DNA. The viral prevalence in patients with HPV-DNA positive partners was 68.8% (77/112), and 79.5% (58/73) of patients with penile lesions were found to be HPV positive. The most frequent viral type was HPV-11 (26.7%), followed by HPV-6 (23%), HPV-16 (21.5%), HPV-18 (6%), and HPV-31 (4.4%). In this study, 11.1% (15/135) of the HPV positive specimens were double infections. These results indicate that high-risk HPVs can be found in clinical lesions in a high percentage (43.8%), as simple or double infections. In this sense, the male population represents an important reservoir for the virus and may play a role in the transmission and perpetuation of the infection in the general population. The method described below provides a tool for detection and typing of HPV-DNA using samples obtained by non-invasive techniques and thus easy to obtain.     

High prevalence of high-risk oncogenic human papillomaviruses harboring atypical distribution in women of childbearing age living in Libreville, Gabon

The extent of human papillomavirus (HPV) genital shedding and type-specific diversity were evaluated in 354 consecutive women of childbearing age living in Libreville, Gabon. Detection of HPV DNA was performed by PCR using the MY09/MY11 primer set on DNA extracted from endocervical swabs. All PCR positive specimens were subjected to direct sequencing and HPV genotypes were identified on the basis of >95% sequence homology in the L1 region. Reverse line blot hybridization assay was used when a genotype could not be resolved by sequencing alone. HPV DNA was detected in 163 (46%) women, all clinically asymptomatic for HPV-related lesions. The highest prevalence of genital HPV detection (45%) was in the age group from 22 to 29 years. A total of 90 women (55%) harbored high-risk (HR) genotypes, with the most common being HPV-53 (19; 12%), HPV-58 (17; 11%), and HPV-16 (16; 10%). Low-risk genotypes were found in 36 (22%) women with HPV-54 and HPV-70 being the most frequently detected (17; 11% and 10; 6%, respectively). Finally 37 women (23%) tested positive for genotypes of unknown oncogenic risk, the most common in this category being HPV-83 (20; 12%). Multiple infections were detected in 35 (21%) women. By multivariate analysis, HPV genital shedding was significantly associated with young age (OR: 0.34; P < 0.007). The multivalent vaccine currently available against cervical carcinomas, is only active against HPV-16 and HPV-18, and will thus have a low impact in this setting.     

Genital human papillomavirus genotypes in northwestern Tanzania

Using MY09-MY11 PCR and human papillomavirus (HPV) typing by reverse blot hybridization, we found a 34% cervical HPV prevalence among 561 pregnant women in Tanzania. One hundred three of 123 women (84%) with typeable samples harbored high-risk oncogenic strains. HPV type 16 (HPV-16) was the most prevalent subtype (18%) among HPV-infected women and among women with cervical neoplasia (3 of 19). A multivalent vaccine for HPV-16, -18, -31, -33, and -35 would be necessary to prevent 50% of the neoplasia in this population.     

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.     

HPV genotype prevalence in cytologically abnormal cervical samples from women living in south Italy

Human papillomavirus (HPV) infection is the commonest sexually transmitted infection, and high-risk HPV types are associated with cervical carcinogenesis. This study investigated: the HPV type-specific prevalence in 970 women with an abnormal cytological diagnosis; and the association of HPV infection and cervical disease in a subset of 626 women with a histological diagnosis. HPV-DNA was researched by nested PCR/sequencing and the INNOLiPA HPV Genotyping assay. The data were analysed by the chi-square test (p相似文献   

The prevalence of human papillomavirus genotypes in penile cancers from northern Thailand

The highest frequency of penile cancer occurs in Asia, Africa, and Latin America, and there have been a few reports concerning the association of penile cancer with human papillomavirus (HPV) infection in these areas. The objective of this study was to determine the relation between penile cancer and the prevalence of HPV genotypes in northern Thailand. Eighty-eight specimens of penile tissue (65 malignant, 1 pre-malignant, and 22 benign cases) were examined to determine the association of HPV infection. An in situ hybridization (ISH) method was used to detect and localize HPV-DNA. Sensitive HPV polymerase chain reaction (PCR) procedure was used for detection of HPV-DNA, and DNA sequencing was used to identify the HPV genotype. HPV-DNA was detected in 53.8% and 81.5% of cases of penile cancer, using ISH and PCR, respectively. The high-risk HPV-16, most commonly associated with penile cancer in previous reports, was found in only one case in this study. The most prevalent genotype was the high-risk HPV-18, found in 55.4% of the cases (32.3% single and 23.1% multiple infection) followed by the low-risk HPV-6, found in 43.1% of the cases (24.6% single and 18.5% multiple infection). In this study, penile cancer was found to be highly correlated with HPV-DNA. Specifically, infection with both the low-risk HPV-6 and the high-risk HPV-18 is the characteristic prevalence of HPV genotypes in penile cancer in this area.     

Human papillomavirus infection in urine samples from male renal transplant patients

Renal allograft recipients have a well‐documented increased incidence of human papillomavirus (HPV)‐related malignancies and preventive strategies should be specifically implemented. While in females the use of the Papanicolau test and HPV detection assay are used currently as a screening test for cervical cancer, no diagnostic procedures have been implemented to monitor HPV infection in males. The aim of this study was to test for HPV infection and to determine the spectrum of viral genotypes in urine samples of men with renal transplants. The study included 88 patients who underwent kidney transplantation between 1999 and 2005. HPV sequences were detected by nested PCR, using the broad‐spectrum consensus‐primer pairs MY09/MY11 and the new MGP system, and characterized by nucleotide sequence analysis. Overall, 43 (48.9%) samples were found positive for HPV sequences and the most common genotypes were HPV 16 (53.5%) and HPV 54 (9.3%) followed by HPV 6, 53, 56, 58, 66, 11, 12, 20, 45, 62, and 71, in descending order of prevalence. The majority of HPV 16 isolates were classified as European and only one as African‐1 variant on the basis of nucleotide signature present within the MGP L1 region. The high prevalence of HPV 16 among renal allograft recipients suggests that an HPV‐16‐based preventive or therapeutic vaccine may be effective for prevention or treatment of HPV‐related neoplasia in this group of immune compromised patients. J. Med. Virol. 82:1179–1185, 2010. © 2010 Wiley‐Liss, Inc.     

Human papillomavirus associated with oesophageal cancer

AIM: To study the prevalence and the different types of human papillomavirus (HPV) in patients with oesophageal cancer from a high risk area of South Africa (Transkei). METHODS: DNA samples from 50 paraffin wax embedded tissue sections were analysed by nested polymerase chain reaction (PCR) using the degenerate HPV L1 consensus primer pairs MY09/MY11 and GP5+/GP6+. Positive PCR samples were subjected to DNA sequence analysis. RESULTS: HPV DNA was detected in 23 of the 50 samples. Sequence analysis revealed that most patients (11) harboured DNA to HPV type 11, whereas other types included DNA HPV type 39 (seven patients), type 16 (two patients), and type 52 (one patient). HPV type 39 has not previously been shown to be associated with oesophageal cancer. In contrast to earlier studies that have found HPV type 16 to be more frequently associated with oesophageal cancer, HPV type 11 was the predominant subtype in this study. CONCLUSIONS: The high frequency of occurrence of HPV in oesophageal tumours (23 of 50 patients; 46%) implicates HPV as one of the possible aetiological factors in this disease. The finding that the low risk HPV subtypes predominate indicates that transformation may be effected via the E6 and E7 proteins.     

Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.     

A pilot study on the distribution of human papillomavirus genotypes and HPV-16 variants in cervical neoplastic lesions from Ecuadorian women

Human papillomaviruses (HPVs) are the cause of cervical intraepithelial neoplasia and invasive carcinomas of the uterine cervix. The distribution of specific HPV genotypes varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. The aim of this study was to determine the spectrum of HPV genotypes and HPV-16 variants among women with cervical lesions living in Ecuador. A total of 71 cases have been analyzed, including 32 chronic cervicitis, 29 cervical intraepithelial neoplasia grade 1, and 10 cervical intraepithelial neoplasia grade 2-3. HPV sequences were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Overall, 31 (43.7%) cases were HPV positive with prevalence rates of 37.5%, 44.8%, and 60% in patients with chronic cervicitis, cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 2-3, respectively. Among the positive cases, the most common genotypes were HPV 16 (64.5%) and HPV 81 (29%) followed by HPV 31, 53, 56, and 58, in descending order of prevalence. Seventeen (85%) HPV-16 isolates were classified as European and three (15%) as African-1 variant on the basis of nucleotide signature present within the MY09/MY11 L1 sequence. The results suggest that HPV 16 has a very high prevalence among women with cervical lesions in Ecuador; therefore, an effective HPV-16 based vaccine should prevent the development of cervical cancer in a large proportion of Ecuadorian women.     

Additional human papillomavirus types detected by the hybrid capture tube test among samples from women with cytological and colposcopical atypia

The type specificity of the human papillomavirus (HPV) Hybrid Capture Tube (HCT) test was evaluated by using typing with PCR (MY09-MY11)-restriction fragment length polymorphism (RFLP) and sequencing. All samples HCT test positive for only low-risk HPV (n = 15) or only high-risk HPV (n = 102) were confirmed, whereas 9 of 12 HCT test double-positive samples contained only high-risk HPV types as determined by PCR-RFLP. Several high-risk HPV types (HPV-53, -58, -62, -66, -CP8304, and -MM4) not included in the HCT test were indeed detected, indicating a broader detection range with retained distinction between low-risk and high-risk HPV types.     

林州市地区食管癌活检组织中人乳头瘤病毒的研究

目的探讨在我国河南省林州市地区食管癌（esophageal carcinoma,EC）活检标本中人乳头瘤病毒（humanpapillomavirus,HPV）,特别是高危型HPV的感染状况。方法收集的食管癌活检标本,使用通用引物的套式PCR反应检测HPV的核酸,分别使用型特异性PCR检测HPV16和18的感染。结果18例活检标本全部为HPV阳性,其中HPV16的阳性率为13／18,HPV18的阳性率为4／18,HPV16／18的复合感染为4／18。结论我国河南省林州市地区食管癌活检组织中有HPV存在,其中HPV16的感染占很大比例,并且HPV感染可能是食管癌发生的重要病因。     

Detection of human papillomavirus DNA in cervical samples: analysis of the new PGMY-PCR compared to the hybrid capture II and MY-PCR assays and a two-step nested PCR assay

The PGMY-PCR for human papillomavirus (HPV) was evaluated, in parallel with nested PCR (nPCR), in samples with noted Hybrid Capture II (HCII) and MY-PCR results. PGMY-PCR detected HPV DNA in 2.5% of HCII-negative-MY-PCR-negative samples and in 71.7% of HCII-positive-MY-PCR-negative samples; also, it detected the MY-PCR-negative-nPCR-negative types HPV-42, HPV-44, HPV-51, HPV-87, and HPV-89.     

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.     

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.     

High prevalence of intermediate-risk human papillomavirus infection in uterine cervices of Kenyan women infected with human immunodeficiency virus

The aim of this study was to investigate an association between certain human papillomavirus (HPV) types and human immunodeficiency virus (HIV) infections. Sexually active females (n = 487; 19-61 years old) were enrolled in the study. Subjects underwent Pap testing and evaluations of HIV and HPV infection status on uterine cervical cell samples. HPV genotyping was performed using a Kurabo GeneSQUARE DNA microarray test. Overall, 23 HPV genotypes were detected, and the most prevalent HPV genotype was HPV-52, followed by HPV-39, -54, -45, -56, -53, -31, -42, -16, -68, and -51. HPV-30, -53, -54, -61, and -66, which are associated with abnormal cytology, are categorized as intermediate-risk in this study. Detection of both high- and intermediate-risk HPV types was significantly associated with cervical abnormality and HIV infection. Multivariate analysis revealed that some high-risk HPV types (HPV-31, -45, -51, -56, and -59) and most intermediate-risk HPV types were associated with HIV infection, while the high-risk types (HPV-16, -18, -33, -35, -39, -52, -58, and -68) were not. The oncogenic effect of the most malignant HPV types (e.g., HPV-16 and -18) appear to be lower, while that of intermediate-risk types are greater, in areas with a high prevalence of HIV infection.     

Adenocarcinoma of the uterine cervix in Ireland and Sweden: human papillomavirus infection and biologic alterations.

Paraffin-embedded samples from cervical adenocarcinomas, 19 cases from Irish patients and 19 cases from Swedish patients, were analyzed by polymerase chain reaction for the presence of infection with human papillomavirus (HPV). The results were compared with DNA ploidy, proliferation activity, and p53 and p21/WAF1 expression. The studies were performed to discover whether high-risk HPV infection in adenocarcinomas of the uterine cervix is associated with an increased proliferative activity and genomic instability. The results show that the majority (84.6%) of patients 59 years of age or younger showed HPV infection. The overall prevalence of HPV DNA was 60.5%, with the high-risk types, 16 and 18, the most frequent. HPV-16 had a prevalence of 23.7% (9 of 38), and HPV-18 had a prevalence of 26.3% (10 of 38). The HPV-positive tumors predominantly showed a tetraploid DNA distribution pattern, whereas HPV-negative tumors more frequently showed highly scattered aneuploid DNA profiles. Both HPV-positive and HPV-negative cases displayed high proliferative activity, as indicated by high Ki-67 and cyclin A immunoreactivity. Tumor suppressor gene analysis detected low p53 expression and high p21/WAF1 expression in HPV-positive patients and high p53 expression without simultaneously increased p21/WAF1 (indicative of mutated p53) in HPV-negative cases in the groups of women older than 59 years of age.