Comparative Evaluation of the HPV28 Detection and HPV DNA Chip Test for Detecting and Genotyping Human Papillomaviruses

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

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Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽

10.3390/pathogens10060772 ◽

2021 ◽

Vol 10 (6) ◽

pp. 772

Author(s):

Isao Murakami ◽

Takashi Iwata ◽

Tohru Morisada ◽

Kyoko Tanaka ◽

Daisuke Aoki

Keyword(s):

Life Cycle ◽

Cultured Cells ◽

Basal Layer ◽

Nucleosome Positioning ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Host Cells ◽

Micrococcal Nuclease ◽

Hpv Dna ◽

Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

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Relevance of the Pap Test: A Report of HPV-DNA Test-Negative High-Grade Squamous Intraepithelial Lesions of the Female Lower Genital Tract

Acta Cytologica ◽

10.1159/000448470 ◽

2016 ◽

Vol 60 (5) ◽

pp. 445-450 ◽

Cited By ~ 3

Author(s):

Yiang Hui ◽

Katrine Hansen ◽

Jayasimha Murthy ◽

Danielle Chau ◽

C. James Sung ◽

...

Keyword(s):

High Risk ◽

Pap Test ◽

Human Papillomaviruses ◽

High Grade ◽

Squamous Intraepithelial Lesions ◽

Cytologic Examination ◽

Dna Test ◽

Hpv Dna ◽

Intraepithelial Lesions ◽

Hpv Dna Test

Objective: A vast majority of cervicovaginal intraepithelial lesions are caused by high-risk human papillomaviruses (HPVs). The Pap test has been the sole method used for the screening of cervicovaginal squamous intraepithelial lesions (SIL). Recently, the FDA approved an HPV-DNA assay as a method of primary screening. We report on a series of FDA-approved HPV-DNA test-negative SIL with HPV genotyping, using an alternative method on the corresponding surgical biopsy specimens. Study Design: A retrospective review identified cytology-positive HPV-negative cases over a 15-month period at a tertiary care gynecologic oncology institution. Corresponding biopsies were reviewed and genotyped for high-risk HPVs. Results: Of the 18,200 total cases, 17 patients meeting the study criteria were selected with 27 surgical specimens corresponding to their cytologic diagnoses. Four patients with high-grade lesions were identified, 3 of whom (75%) were positive for HPV. One of these 4 patients (25%) showed high-grade SIL on biopsies from 4 separate sites in the cervix and vagina. Multiviral HPV infections were frequent. Conclusions: We discuss the relevance of cotesting for screening cervical SILs and emphasize that false-negative results are possible with the FDA-approved HPV screening assay, also in patients with high-grade SIL. These cases may be detectable by cytologic examination and this suggests that the Pap test remains an important diagnostic tool.

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Human papillomavirus (HPV) genotyping by HPV DNA chip in cervical cancer and precancerous lesions

International Journal of Gynecological Cancer ◽

10.1111/j.1048-891x.2005.14417.x ◽

2005 ◽

Vol 15 (1) ◽

pp. 81-87 ◽

Cited By ~ 17

Author(s):

G.-Y. Lee ◽

S.-M. Kim ◽

S.-Y. Rim ◽

H.-S. Choi ◽

C.-S. Park ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Precancerous Lesions ◽

Dna Chip ◽

Hpv Dna ◽

Hpv Genotyping

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Prevalence and type distribution of human papillomavirus infection among women with different degrees of cervical cytological abnormalities in Sicily (Italy)

Microbiologia Medica ◽

10.4081/mm.2016.5018 ◽

2016 ◽

Vol 31 (2) ◽

Author(s):

Concetta Franchina ◽

Carmela M. Costanzo ◽

Raffaela Russo ◽

Concetta I. Palermo ◽

Guido Scalia

Keyword(s):

Pap Smear ◽

Blot Hybridization ◽

Human Papillomaviruses ◽

Multiple Infections ◽

Low Grade ◽

Genotype Distribution ◽

Cervical Lesions ◽

Hpv Dna ◽

Papillomavirus Infection ◽

Hpv Genotypes

Human papillomaviruses (HPVs) are etiological agents of cervical cancer. In the absence of Pap smear alterations, high-risk HPV DNA can be detected in cervical samples. The prevalence of papillomavirus infection and their genotype distribution varies greatly across populations. The aims of this study were: i) to assess the prevalences of HPV genotypes in people living in Eastern Sicily (Italy) and the frequency of HPV multiple infections; ii) to evaluate the association between HPV genotypes and cervical lesions in order to improve the epidemiological knowledge useful for monitoring or treating infected women. Nested PCR and reverse dot/blot hybridization were used for the detection and typing of HPV DNA in 315 women who had had an abnormal PAP-smear. HPV DNA test was positive in 70.5% cases; the prevalence was 50% in atypical squamous cells of undetermined significance (ASCUS), 80.8% in low grade-, and 76.2% in high grade-squamous intraepithelial lesion (H-SIL). The genotype distribution showed a predominance of HPV-16 (56.7%) followed by HPV-18 (12.2%), HPV-31 (9.5%) and HPV-6 (9.5%). Multiple infections were detected in 35.1% of the infected patients. High frequency of positive results for HPV was confirmed and, even in case of ASCUS, patients should be taken into account for genotyping. Our data indicate that multiple infections are consistent in women with low-grade lesions while they are less frequent in women with H-SIL. This could reinforce the theory of the multi-stage cancer model, by which one HPV type becomes predominant along with the progression of cervical lesion severity.

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Comparison of Human Papillomavirus Detection and Typing by Hybrid Capture 2, Linear Array, DNA Chip, and Cycle Sequencing in Cervical Swab Samples

International Journal of Gynecological Cancer ◽

10.1111/igc.0b013e31819bcd0a ◽

2009 ◽

Vol 19 (2) ◽

pp. 266-272 ◽

Cited By ~ 16

Author(s):

Jae Kwan Lee ◽

Mi Kyung Kim ◽

Seung Hun Song ◽

Jin Hwa Hong ◽

Kyung Jin Min ◽

...

Keyword(s):

High Risk ◽

Linear Array ◽

Intraepithelial Neoplasia ◽

Dna Chip ◽

Substantial Agreement ◽

Cervical Lesions ◽

Cycle Sequencing ◽

Hpv Dna ◽

Hpv Dna Tests ◽

High Risk Hpv

Although the Hybrid Capture II (HC II) assay can detect 13 high-risk human papillomavirus (HPVs), it does not yield any genotype-specific information. We evaluated the performance of 4 HPV DNA tests, namely, HC II, Linear Array (LA), DNA chip, and cycle sequencing for their capacity to detect the presence of high-risk HPV DNA and HPV-associated cervical lesions. Seventy-six women who were referred to the colposcopy clinic for abnormal cytology were enrolled. The women were examined using liquid-based cytology, colposcopy-directed biopsy, and HPV DNA tests. After DNA extraction from a single sample, HPV DNA tests were performed by all 4 methods on the same specimen. The LA test has higher HPV-positive rates than HC II for cervical intraepithelial neoplasia I (83.3% vs 61.1%;P< 0.01) and for cervical intraepithelial neoplasia II and more severe lesions (100.0% vs 80.0%;P< 0.01). The concordance between the DNA chip and LA tests was 89.5%, confirming substantial agreement (κcoefficient = 0.73), and the concordance between HC II and the DNA chip was 80.3%, also showing substantial agreement (κcoefficient = 0.738). The concordance for 15 high-risk HPV genotypes between LA and sequencing was 82.5% with aκvalue of 0.536. Furthermore, the LA test was more sensitive in the detection of high-grade cervical lesions than HC II (100% vs 92.3%,P< 0.01). The LA test showed superior sensitivity in the detection of clinically relevant HPV infections and has proven to be an accurate tool for identifying individual HPV types, especially in cases of multiple HPV infections.

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Antibodies against Early Proteins of Human Papillomaviruses as Diagnostic Markers for Invasive Cervical Cancer

Journal of Clinical Microbiology ◽

10.1128/jcm.36.2.475-480.1998 ◽

1998 ◽

Vol 36 (2) ◽

pp. 475-480 ◽

Cited By ~ 84

Author(s):

Wolfgang Meschede ◽

Klaus Zumbach ◽

Joris Braspenning ◽

Martin Scheffner ◽

Luis Benitez-Bribiesca ◽

...

Keyword(s):

Cervical Cancer ◽

Invasive Cervical Cancer ◽

Human Papillomaviruses ◽

Antibody Detection ◽

Diagnostic Tools ◽

Immunological Monitoring ◽

Hpv Dna ◽

Native Proteins ◽

Human Sera ◽

E6 And E7

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.

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High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases

Pathogens ◽

10.3390/pathogens9030224 ◽

2020 ◽

Vol 9 (3) ◽

pp. 224 ◽

Cited By ~ 4

Author(s):

Nicola Malagutti ◽

John Charles Rotondo ◽

Luca Cerritelli ◽

Claudio Melchiorri ◽

Monica De Mattei ◽

...

Keyword(s):

Otitis Media ◽

Middle Ear ◽

Dna Sequences ◽

Direct Sequencing ◽

Chronic Otitis Media ◽

Human Papillomaviruses ◽

Middle Ear Mucosa ◽

Suppurative Otitis Media ◽

Ear Diseases ◽

Hpv Dna

Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (p > 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (p > 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) (P = 0.03 and P = 0.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME.

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Prevalence and Distribution of High-Risk Human Papillomavirus Genotypes in Invasive Carcinoma of the Uterine Cervix in Uruguay

International Journal of Gynecological Cancer ◽

10.1097/igc.0b013e318285e753 ◽

2013 ◽

Vol 23 (3) ◽

pp. 527-532 ◽

Cited By ~ 9

Author(s):

Nora Berois ◽

Patricia De Cremoux ◽

Daniel Mazal ◽

Adela Sica ◽

Mabel Cedeira ◽

...

Keyword(s):

Human Papillomavirus ◽

High Risk ◽

Dna Sequences ◽

Invasive Cervical Cancer ◽

Human Papillomaviruses ◽

Geographical Differences ◽

Hpv Dna ◽

High Prevalence ◽

Hpv Types ◽

High Risk Hpv

ObjectivesPersistent infection with specific genotypes of human papillomaviruses (HPVs) is the main cause of invasive cervical cancer (ICC). Only a few of the various HPV types account for most of the cases worldwide, and geographical differences in their distribution are evident. Data from locally prevalent genotypes are essential in view of introduction of HPV type-specific prophylactic vaccines.MethodsIn this work, we have investigated HPV type distribution in samples of ICC cases that occurred in Uruguayan women. DNA extracted from ICC treated in Centro Hospitalario Pereira Rossell of Montevideo between 1999 and 2007 were analyzed. Search and typing were performed by polymerase chain reaction using generic GP5+/GP6+ primers and specific primers for HPV types 16, 18, 33, and 45. Positive GP5+/GP6+ samples, which were negative for all 4 high-risk HPV-specific types screened were further analyzed by sequencing.ResultsHuman papillomavirus DNA sequences were found in 163 (92.6%) of 176 cases. The most prevalent genotypes were HPV16 (67.6%) and HPV18 (8.5%) followed by HPV45 (6.8%) and HPV33 (3.4%), as single or mixed infection. Other less frequent genotypes were HPV31, HPV35, HPV39, HPV51, HPV52, HPV58, HPV66, and HPV73. The viral type could not be determined (HPV X) in 1 case (0.6%) of the HPV DNA–positive cervical cancers and double infections were found in 1.7% of the cases. The higher percentage of most aggressive HPV (16/18/45) genotypes was detected in cases diagnosed at younger than 60 years old, whereas these genotypes were less frequent in older patients.ConclusionWe conclude that HPV types 16, 18, and 45 have a very high prevalence in ICC of Uruguayan women. Results provide evidence that 16 of 18 infections are more aggressive, but most cancers could be vaccine preventable.

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