Detection by PCR of human papillomavirus genotypes in cervical lesions of Senegalese women

In order to analyse human papillomavirus (HPV) infection in the Senegalese population, HPV DNA was sought in 65 women with evidence of cervical cytological abnormality and in 72 pregnant women. Ninety-four percent of the patients were positive for HPV DNA as compared to 24% of pregnant women. HPV 16 was detected in cervical smears in 42% of cases, HPV 18 in 39%, HPV 6 in 26%, HPV 11 in 15%, HPV 45 in 10%, HPV 52 in 3%, and HPV 31, HPV 33 and HPV 68 in 1.5%. HPV 16 and HPV 18 were detected in 16% and 7% respectively of pregnant women. HPV DNA of unknown type was detected in 6% of cases, and multiple HPV infections were observed in 28% of cases. Low risk genital HPVs (6/11) were detected in smaller proportions (17%) among high grade squamous intraepithelial lesions (SILs) than the low grade SILs (43%). High risk HPVs (16/18) were detected in high proportions both in low and high grade SIL lesions, though the highest frequency (70%) was observed among patients with high grade lesions. In conclusion, the results confirm that HPV infections are frequent in Senegal and that HPV 18 and 45 are detected in a high proportion of patients in Africa. © 1996 Wiley-Liss, Inc.     

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay.

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.     

Detection of human papillomavirus DNA in cervical lesions by in-situ DNA hybridization

Authors examined paraffin sections of 50 cervical specimens from 34 cases for the presence of human papillomavirus (HPV) type 6b, 11, 16, 18, 31 and 33 by in-situ hybridization using 35S-labelled HPV DNA probes. Specimens were classified according to the degree of dysplasia after histological examination. Viral nucleic acids were detected in 30 of 50 tissues (60%) in which 15 specimens had single, 10 double, 4 triple and 1 quadruple viral infections. In some cases, different viral nucleic acids were detected at separate sites in the same patient. Overall, no great variation in the frequency of each HPV was detected, but a pattern became apparent when the frequencies were compared with the grade of dysplasia. CIN II/III lesions contained one or more of the HPV types 16, 18, 31, 33 which are frequently associated with cervical carcinoma. In-situ hybridization offers sensitive means of investigating viral infection, gene expression and neoplastic transformation.     

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.     

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.     

GeXP多重PCR技术用于人乳头瘤病毒HPV检测和分型的研究

目的建立一种基于GenomeLab^TM GeXP（GenomeLab eXpress Profiling）遗传分析系统的人乳头瘤病毒（HPV）分型新技术。方法针对5种中国人常见的HPV亚型（HPV6,11,31,33和52）的基因序列进行比对分析,设计型特异性引物,建立并优化GeXP多重PCR反应体系,评价其特异性和灵敏性,并应用于30份临床标本检测。结果本研究建立的GeXP多重PCR技术能够成功检测和鉴别5种不同亚型的HPV病毒,灵敏度高,特异性好。结论成功建立了一种新的快速同时检测和鉴别5种HPV亚型病毒的多重PCR方法,适用于临床HPV感染的实验室诊断与流行病学研究。     

Amplification of human papillomavirus DNA sequences by using conserved primers.

The polymerase chain reaction has potential for use in the detection of small amounts of human papillomavirus (HPV) viral nucleic acids present in clinical specimens. However, new HPV types for which no probes exist would remain undetected by using type-specific primers for the polymerase chain reaction before hybridization. Primers corresponding to highly conserved HPV sequences may be useful for detecting low amounts of known HPV DNA as well as new HPV types. Here we analyze a pair of primers derived from conserved sequences within the E1 open reading frame for HPV sequence amplification by using the polymerase chain reaction. The longest perfect homology among HPV sequences is a 12-mer within the first exon of E1M. A region of conserved amino acids coded by the E1 open reading frame allowed the detection of another highly conserved region about 850 base pairs downstream. Two 21-mers derived from these conserved regions were used to amplify sequences from all HPV DNAs used as templates. The amplified DNA was shown to be specific for HPV sequences within the E1 open reading frame. DNA from HPVs whose sequences were not available were amplified by using these two primers. HPV DNA sequences in clinical specimens could also be amplified with the primers.     

Clinical efficacy of human papillomavirus DNA detection in urine from patients with various cervical lesions

A considerable number of adult Korean women avoid a Pap smear due to fear and discomfort of the pelvic examination. A reliable but noninvasive and comfortable screening method would considerably increase the participation rate. To evaluate the clinical efficacy of urine-based human papillomavirus (HPV) detection by oligonucleotide microarray, the results of HPV test from matched cervical swab specimens were compared. HPV DNA was detected in 70 of 100 cervical samples. HPV 16 was the most prevalent type (38/70), followed by types 18, 58, 52, 33, 35, 31, and 51. HPV DNA was identified in 47 of 90 urine samples. HPV 16 was the most prevalent type (30/45), followed by types 18, 52, 35, 51, 58, 33, and 56. The HPV detection rates of the cervical swabs increased in accordance with the severity of the cytologic and histologic diagnosis. The type specific agreement of HPV DNA tests between cervical swabs and urine was good in HPV 16 (kappa index=0.64 [95% CI: 0.50-0.79]), 18, 52, and 58 and fair in HPV 33 and 35. We propose that a urine HPV test is a valuable adjunctive method for a conventional Pap smear and can be used in population screening for cervical cancer in countries where it is difficult to obtain colposcopic specimens for cultural or religious reasons.     

Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11 primers in cervical cells taken from women attending a colposcopy clinic

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).     

Oncogenic human papillomavirus DNA loads in human immunodeficiency virus-positive women with high-grade cervical lesions are strongly elevated

Human papillomavirus (HPV) DNA loads of six oncogenic HPV types were measured by real-time PCR in cervical scrapes of human immunodeficiency virus (HIV)-infected and uninfected women. In both groups, HPV loads increased with the grade of cervical disease. HIV infection did not affect HPV loads in low-grade lesions but was associated with significantly higher HPV loads in severe dysplasia; highest loads were found in advanced HIV disease. Our data reflect the aggressive course of HPV infection in HIV-positive women.     

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP)

Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.     

Simultaneous detection and typing of human papillomavirus in cervical biopsies using PCR-reverse hybridization

INTRODUCTION: Genotyping of Human papillomavirus (HPV) is an important step in the clinical evaluation of the oncogenic risk associated with HPV infection of cervical mucosa. The purpose of this work was to develop a fast PCR-reverse-hybridization assay (PCR-RH) for the simultaneous detection and genotyping of anogenital HPVs. METHODS: HPV DNA from cervical biopsies was amplified by consensus primer-PCR. Digoxigenin-labeled PCR products were hybridized to type-specific probes anchored to the surface of plastic microwells and revealed by an ELISA system. RESULTS: The method was tested on 115 clinical samples (81 koilocytic atypias, 11 CIN1, 10 CIN2, 12 CIN3 and 1 squamous carcinoma). HPV DNA was found in 56.7% koilocytic atypias, in 90.9% of CIN1 and in 100% of CIN2 and higher-grade lesions. Thus, PCR-RH is sensitive, rapid, easy-to-perform and readily applicable to the routine analysis of a large number of samples.     

Low-resolution DNA typing for HLA-B using sequence-specific primers in allele- or group-specific ARMS/PCR

Abstract: The products of the human major histocompatibility complex (HLA Class I and II) have historically been detected using serological or cellular assays. With the availability of DNA sequence information for alleles of the HLA system, and with the development of molecular biological techniques it has become possible to tissue type for allelic differences in the HLA genes themselves. We describe here a polymerase chain reaction (PCR) system, based on the principle of the amplification refractory mutation system (ARMS), for low-resolution DNA typing of the HLA-B gene. The technique involves a one-step PCR from genomic DNA using sequence-specific primers in particular combinations that determine the specificity of each reaction. A low-resolution primer panel has been designed, based on published HLA-B gene nucleotide sequences, consisting of 34 sequence-specific primers (SSP) in 24 PCR reactions which cover all known HLA-B alleles, to give allele-specific or group-specific amplification of DNA fragments of defined size (344–784bp). Advantages of the system are that it can be performed in under 4 hours including DNA extraction, results are easy to interpret and it does not require viable cells.     

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.     

Detection of human papillomavirus in vulval carcinoma using semi-nested PCR and restriction enzyme typing: a rapid and sensitive technique

Aims—To develop a highly sensitive technique for the reliable detection and typing of human papillomavirus (HPV) DNA in clinical tissue.     

Chromosome fragility in lymphocytes of women with cervical uterine lesions produced by human papillomavirus.

We studied 30 women with cervical lesions that showed human papillomavirus infection (HPV). Cervical HPV infection was diagnosed by cytology, histology, immunohistochemistry, and electron microscopy, as well as by DNA viral hybridization in situ with 6, 11, 16, and 18 HPV types. Three groups of patients were studied: 15 women infected by HPV of 6 and 11 types with koilocytic lesions and benign evolution, 15 women infected by HPV of 16 and 18 types with koilocytic lesions and malignant evolution, and 15 normal women without cervical lesions who served as controls. For each group, chromosome fragility was studied in peripheral blood lymphocytes. Aphidicolin (AP) was used as a clastogenic agent at a concentration of 0.12 microM. There were significant differences (p less than 0.001) between the control population and the patients affected by HPV. There were also significant differences (p less than 0.001) between the two groups infected with HPV. Our findings support the concept that chromosome fragility could serve as a cytogenetic marker to measure evolution, prognosis, and treatment of cervical lesion associated with HPV.     

Detection and typing of human papillomavirus in histologic specimens by in situ hybridization with biotinylated DNA probes

Eighty tissue biopsies from 73 women suspected of having papillomavirus (HPV) infection of the lower genital tract were examined by in situ hybridization with biotinylated DNA probes derived from the complete genomes of four HPV types (6, 11, 16, and 18) and restriction analysis of the extracted DNA on Southern blots. In a subset of 52 samples, the in situ test had a 90.4% sensitivity (47 of 52) in detecting the presence or absence of virus, whereas Southern blot analysis detected HPV with a sensitivity of 98.1% (51 of 52). For 51 samples, in which the viral type was determined by restriction analysis, comparison of the signals separately generated by the four probes after in situ hybridization allowed a correct identification of the infecting HPV type in 86.2% (44 of 51) of cases.     

Evaluation of immunoassays for the detection and typing of PCR amplified human papillomavirus DNA.

AIMS: To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens. METHODS: A hybrid capture-immunoassay in microtitre wells was performed to detect HPV sequences amplified by PCR and typed by specific oligoprobes. Consensus primers were used to amplify a sequence within the L1 open reading frame, and direct digoxigenin labelling of amplified products was performed during the amplification reaction. The amplified product was separately hybridised with six biotinylated type specific probes (HPV6, 11, 16, 18, 31, and 33); hybrids were then captured into streptavidin coated microtitre wells and detected by a spectrophotometer as an ELISA using antidigoxigenin Fab fragment labelled with peroxidase and a colorimetric substrate. The results were compared with the dot-blot immunoassay used to detect and type PCR amplified HPV DNA sequences. Consensus primers were used to generate the same unlabelled PCR product; digoxigenin labelled type specific probes for HPV6, 11, 16, 18, 31, and 33 were used and hybrids visualised by colorimetric immunoenzymatic reaction. Thirty nine biopsy specimens and 31 cytological samples were tested by the PCR-ELISA and by standard PCR followed by dot-blot hybridisation. RESULTS: The PCR-ELISA proved to be more sensitive than standard PCR with dot-blot hybridisation typing. All samples positive for HPV-DNA in standard PCR with dot-blot hybridisation method were confirmed positive by the PCR-ELISA assay; however, seven samples were positive only by PCR-ELISA. CONCLUSIONS: The PCR-ELISA assay, which can be performed in one day, is easily standardised and therefore seems to be a practical, sensitive, and reliable diagnostic tool for the detection and typing of HPV genomes in biopsy and in cytological specimens in the routine diagnostic laboratory.     

Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction

A method for detecting HPV DNA in cervical cancer tissue was developed without using isotopes. The DNA samples from the cancer tissues were first subjected to amplification by PCR, followed by polyacrylamide gel electrophoresis to identify the specific amplified fragment. The specificity and sensitivity of the PCR method are described. Compared with the dot hybridization technique, it is shown that the method is able to detect HPV DNA in cervical cancer tissues.