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.     

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.     

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes. © 1996 Wiley-Liss, Inc.     

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.     

Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women

Cervical cancer is a leading cause of cancer‐related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV‐positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%). J. Med. Virol. 81:678–684, 2009 © 2009 Wiley‐Liss, Inc.     

A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings.

Two cocktails of digoxigenin-labeled human papillomavirus (HPV) type-specific oligonucleotide probes and an enzyme immunoassay (EIA) were used as a basis to developed a group-specific detection method for 14 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk (types 6, 11, 40, 42, 43, and 44) HPVs, following a general primer GP5+/bioGP6(+)-mediated PCR. The sensitivity of this high-risk/low-risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type. Comparison of HR/LR HPV PCR-EIA with radioactive Southern blot hybridization using a general probe on the same PCR products derived from 417 cytomorphologically abnormal cervical scrapings resulted in an overall agreement of 96% between the two methods. Complete concordance between group-specific HR/LR detection and individual typing results for both single and multiple infections indicate the strong specificity of this HR/LR HPV PCR-EIA. Multiple infections could be predicted by comparing PCR-EIA optical density values of the cocktail probes with one of the individual oligonucleotide probes. This novel HR/LR PCR-EIA allows accurate and rapid identification of high-risk and low-risk HPV types in cervical scrapings and will facilitate HPV detection in HPV mass-screening programs.     

Population-based type-specific prevalence of high-risk human papillomavirus infection in middle-aged Swedish women

Human papillomavirus (HPV) DNA testing can be used to identify women at risk of the development of cervical cancer. The cost-effectiveness of HPV screening is dependent on the type-specific HPV prevalence in the general population. The present study describes the prevalence and spectrum of high-risk HPV types found in a large real-life population-based HPV screening trial undertaken entirely within the cervical screening program offered to middle-aged Swedish women. Cervical brush samples from 6,123 women aged 32-38 years were analyzed using a general HPV primer (GP5+/6+) polymerase chain reaction-enzyme immunoassay (PCR-EIA) combined with reverse dot-blot hybridization for confirmation and HPV typing by a single assay. In this study, 6.8% (95% CI 6.2-7.5) (417/6,123) were confirmed as high-risk HPV positive. Infections with 13 different high-risk HPV types were detected, of which HPV 16 was the most prevalent type (2.1%; 128/6,123), followed by HPV 31 (1.1%; 67/6,123). Any one of the HPV types 18, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 66 was detected in 3.6% (223/6,123) of the women. Infection with two, three, and five types simultaneously was identified in 32, 5, and 1 women, respectively. The combination of PCR-EIA as a screening test and reverse dot-blot hybridization as a confirmatory test, was found to be readily applicable to a real-life population-based cervical screening. The type-specific HPV prevalence found support in previous modeling studies suggesting that HPV screening may be a favorable cervical screening strategy.     

Accurate human papillomavirus genotyping by 454 pyrosequencing

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.     

Real-time PCR-based system for simultaneous quantification of human papillomavirus types associated with high risk of cervical cancer

We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10(2) to 10(7) HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.     

Human papillomavirus genotyping by 454 next generation sequencing technology

Background

An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies.

Objectives

Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology.

Study design

Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA).

Results

In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/μL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen.

Conclusions

NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

Detection and typing of human papillomavirus by e6 nested multiplex PCR

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.     

Detection of HPV types and neutralizing antibodies in Gansu province,China

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV‐16, ‐18, ‐58, ‐45, ‐6, and ‐11 using pseudovirus‐based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type‐specific neutralizing antibodies with a titer range of 160–640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV‐16, ‐58, ‐6, ‐18, and ‐45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV‐6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA‐positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV‐16 (80%), HPV‐52 (7.1%), HPV‐66 and HPV‐11 (5.7% each), and HPV‐58, HPV‐18, and HPV‐33 (4.3% each), while the four most common types in Group 2 were HPV‐16 (12%), HPV‐52 and HPV‐11 (6% each), and HPV‐68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference (P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV‐16 in Group 1. The full‐length sequences of six HPV types (HPV‐16, ‐58, ‐33, ‐59, ‐11, and ‐68) were determined and showed 99% identities with their reported genomes. J. Med. Virol. 81:693–702, 2009 © 2009 Wiley‐Liss, Inc.     

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.     

GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.     

Improved amplification of genital human papillomaviruses

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.     

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

IgG antibodies to human papillomavirus 16, 52, 58, and 6 L1 capsids: case-control study of cervical intraepithelial neoplasia in Japan

In Japan, human papillomavirus (HPV) 16, 52, and 58 are most commonly associated with cervical intraepithelial neoplasia (CIN). By contrast, HPV6 is primarily associated with genital warts. This study was designed to evaluate the association between IgG antibody responses to common HPVs and the risk of CIN development within a Japanese population. CIN cases (n = 141) and controls (n = 109) were tested for cervical HPV DNA and serum IgG antibodies to L1 capsids of HPV16, 52, 58, and 6. Seropositivity to HPV16, 52, and 58 L1 capsids was significantly higher in CIN cases than in controls: 27%, 21%, and 31% versus 16%, 11%, and 11%, respectively (P < 0.05). HPV6 L1 seropositivity was not significantly associated with CIN lesions (P = 0.11). Presence of viral DNA for either HPV16, 52, or 58 correlated with a significant antibody response against the homologous L1 capsids but not heterologous L1 capsids. Furthermore, seropositivity to multiple types of HPV16, 52, and 58 was more strongly associated with an increased risk of CIN development than seropositivity to a single type (P for trend <0.001). These findings indicate that IgG antibodies to L1 capsids of HPV16, 52, and 58 represent an increased risk of CIN development, with antibodies to multiple types being indicative of a further increase in risk. The presence of CIN lesions in women with seropositivity to multiple types suggests that viral exposure to a given type may not be protective against infections by other types and subsequent CIN development.     

High HPV genetic diversity in women infected with HIV-1 in Brazil

The present study on genetic diversity of human papillomaviruses in women infected by HIV in Brazil describes the frequency, the genotypes, and five new variants of HPV. One hundred fifty cervical smears of HIV-positive women were subjected to cytological examination, and the DNA samples obtained were assayed by MY09/MY11 amplification, followed by RFLP typing. The overall HPV-DNA-positive rate was 42.7%. One hundred twenty-two samples (81.3%) had benign cellular alterations or normal cytological results, and HPV DNA frequency among them was 30.3%. Otherwise, 96.4% of samples with altered cytology were positive for HPV DNA. A high diversity of genotypes was observed. HPVs-16 and 81 were the most prevalent (14.1%) and were followed by HPVs 52, 35, 62, 33, 53, 56, 66, 70, 18, 58, 6b, 11, 31, 39, 40, 61, 71, 32, 54, 59, 67, 68, 85, and 102. Five new variants of the high-risk HPVs 18, 33, 53, 59, and 66 were detected. Possible associations between the detection of HPV genotypes and the cytological classification, HIV viral load, CD4 count, and antiretroviral treatment were also examined. We observed that a high proportion of HIV-infected women are infected with HPV and may carry oncogenic genotypes, even when cytological evaluation shows normal results.     

Identification of multiple genital HPV types and sequence variants by consensus and nested type-specific PCR coupled with cycle sequencing

Consensus and type-specific HPV primers were employed for PCR and cycle sequencing of genital HPVs in scrapings and colposcopically directed biopsies of the cervix from a cohort of 188 female sex workers. A total of 27 individuals tested positive for a broad spectrum of HPV types, including HPVs 6b, 16, 18, 31, 33, 34, 35, 45, 56 and 58, as well as a new HPV type, with seven individuals displaying dual infections. Good correlation between the results of individually paired samples was observed. A HPV 16 primer biotinylated at the 5' end was also used as a probe, which could successfully detect amplified products of HPV 16 but not other HPV types tested by an automated ELISA detection system. DNA sequence analysis revealed several HPV sequence variants that harbored mutations, especially in the E6 gene, many of which culminated in non-conservative amino acid substitutions in the transforming E6 oncoprotein. Such an approach of coupling PCR with cycle sequencing permits the determination of many known and even novel HPV types associated with varying degrees of risk to cervical carcinogenesis, and enables the identification of HPV sequence variants of putative biological and clinical significance, thus justifying its utility as an adjunct tool to complement cervical cytology and colposcopy. This study also emphasises the need for educational, interventional and behavioral modification to minimise HPV transmission, such as through consistent condom usage among sex workers.