Human Papillomavirus In Cervical Specimens Research Articles

Metal-polymer hybrid nanomaterial for impedimetric detection of human papillomavirus in cervical specimens

The human papillomavirus (HPV) is one of the main sexually transmitted pathogens that infect the anogenital epithelium and mucous membranes. HPV genotypes can be classified as high and low oncogenic risk, with infection by the former resulting in cervical cancer in approximately 100 % of the cases. In this work, we developed an ultrasensitive electrochemical biosensor for the detection and identification of different HPV genotypes. A nanostructured platform based on a matrix of polyaniline (PANI) containing gold nanoparticles (AuNps) was designed for the chemical immobilization of a DNA probe capable of recognizing different HPV types. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the genosensor. The impedimetric responses indicate that the proposed sensor was able to detect HPV (types 6, 11, 16, 31, 33, 45, and 58) in cervical specimens (cDNA samples). We obtained different profiles of electrochemical responses for the high and low-risk HPV genotypes. By adopting a three-dimensional quantitative analysis of impedance response variables, it was possible to identify the existence of a pattern of association for samples of high oncogenic risk, which may lead to the differential diagnosis of HPV. The biosensor demonstrated an excellent analytical performance for the detection of HPV genotypes with high sensibility and selectivity. The genosensor exhibited a linear range of response in the 1 pg μL−1 to 100 pg μL−1 range. Besides, a limit of detection (LOD) of 2.74 pg μL−1 and 7.43 pg μL−1 was obtained for HPV11 and HPV16, respectively, with regression coefficients of 99.88 % and 99.47 %. Thus, the proposed sensor may serve as a good prognostic indicator for patients infected with papillomavirus.

Analysis of HPV genotype-specific concordance between EUROArray HPV and HPV 3.5 LCD-Array Kit in cervical samples of 163 patients.

Human papilloma virus (HPV) as the most common viral infection of the anogenital tract is highly associated with intraepithelial neoplasia and cancer of the cervix and other anogential regions. To date, 15 high-risk (HR-) HPV and 3 probably/possibly HR-HPVs have been found to be associated with cervical cancer. Therefore, a screening especially for HR-HPV by appropriate tests is important for detection of precancerous lesions to prevent cancer. The purpose of this study was to analyze prospectively the concordance of the EUROArray HPV genotyping assay (Euroimmun; EUROArray) and the HPV 3.5 LCD-Array Kit (Chipron; LCD-Array). Liquid-based, clinician-collected cervical cytology samples (n = 163) from women undergoing cervical inspection at the dysplasia consultation in the colposcopy clinic at the Medical Center-University of Freiburg, Germany were analyzed. Genotype-specific agreement was assessed by Cohen's kappa statistic and McNemar's P value of significance between proportions. Seventeen of the HR-HPV genotypes included in both assays were detected in 42.3% and 38% of samples by EUROArray and by LCD-Array, respectively; i.e. an agreement of 92.0% and a kappa value of 0.83 could be proven between the EUROArray and the LCD-Array. In 50 of 72 samples, identical HR-HPV genotypes were analyzed (81.9%, κ = 0.47) and genotyping for HPV 16 and/or 18 was highly concordant in both tests (relative agreement 96.3%, κ = 0.88). Detection of any HR-HPV was not significantly different after comparison of EUROArray with LCD-Array. Both of the tests showed comparable results for the detection of HPV in cervical specimens and permit these assays to be suitable for routine diagnostics.

<i>In Vitro</i> Nucleic Acid Hybridization for the Identification of High-Risk Human <i>Papillomavirus </i>(HPV) in Cervical Clinical Specimens

The reliability and specificity of the Pap smear rely on the eyewitness to perceive and an assortment of cell variations in clinically obtained cervical specimens. The improvement in early diagnosis to distinguish Human Papillomavirus (HPV) infection has been an issue. ELISA has been intended to analyze the immune response against HPV, and they can be utilized to distinguish the phase of the infection. The objective of this study to characterize the performance of In Vitro Nucleic Acid Hybridization with a correlation with Liquid-Based Cytology and HPV DNA Genotyping Test to assess its use as the potential for the identification of high-risk HPV in cervical clinical specimens. Hybridization utilizing an in vitro nucleic acid DNA-based ELISA method was performed with an improved chemiluminescent for the qualitative and semi-quantitative identification of high-risk HPV in cervical specimens. For analyzing the reliability to identify HPV DNA in cervical Pap smear, we studied the effectiveness of FDA-approved Hybrid Capture II (HCII) utilizing cervical swapping from 20 patient clinical specimens. Two scrapes-outs were positive for the occurrence of HPV. Only specimens displayed positive with HCII test were further confirmed by HPV Genotyping Test. Specimen 2 and 13 were positive for one of the 13 high-risk types in HCII test. Specimen 2 was confirmed by HPV genotyping test as a positive high-risk HPV type 58.

Frequency and genotype distribution of human papillomavirus in cervical specimens in Sivas Region

Frequency and genotype distribution of human papillomavirus in cervical specimens in Sivas Region

Detection of human papillomavirus by hybrid capture and real time PCR methods in patients with chronic cervicitis and cervical intraepithelial neoplasia

Background and objectives: Cervical cancer due to Human papillomavirus (HPV) is one of the leading causes of morbidity and mortality in women. Testing of HPV can identify women who are at risk of cervical cancer. Nowadays, molecular methods like real time polymerase chain reaction (PCR) and hybrid capture technique are applied for detecting HPV in cervical specimens. The objective of the present study was to determine the rate of HPV infection in women with chronic cervicitis and cervical intraepithelial neoplasia (CIN) by a commercial real time polymerase chain reaction test kit and by a hybrid capture HPV DNA test.Methods: Women aged between 20 to 55 years with chronic cervicitis and CIN were enrolled in the study after obtaining informed consent. Cervical specimen was collected by using cervical brush and stored in transport medium until used. HPV was detected by High Risk Screen Real-TM Quant 2x (Sacace, Biotechnologies SrI, Italy) real time PCR kit (HR RT-PCR) and by Hybrid Capture-2 High-Risk HPV DNA (Hc-2; Digene Corporation, USA) test.Results: Total 72 women with chronic cervicitis and CIN of different grades were included in the study. Out of this, HPV infection detected by HR RT-PCR was 31 (43%) and by Hc-2 was 14 (19.4%). Both the tests were able to detect HPV infection in all the CIN 3 cases and in most of the CIN 2 cases. However, HR RT-PCR detected higher number of HPV in chronic cervicitis and CIN1 cases.Conclusion: The study has shown that HR RT-PCR and Hc-2 tests are equally effective in detecting HPV infection in patients with CIN 2 and CIN 3 lesions. However, HR RT-PCR is more sensitive test for detecting HPV in chronic cervicitis and early CIN lesions and, therefore can be used in epidemiological study to detect presence of HPV in general population.IMC J Med Sci 2016; 10(2): 45-48

Human papillomaviruses in cervical specimens of women residing in Riyadh, Saudi Arabia: a hospital-based study

Certain genotypes of human papillomavirus (HPV) are linked to cervical abnormalities. HPV DNA and genotype prevalence among women residing in Riyadh, Saudi Arabia is investigated in this hospital-based study. Cervical specimens were taken from 519 subjects along with consent and demographic data. DNA was extracted and PCR was performed on all specimens using general primers. Low- and high-risk HPV genotypes were determined by reverse blot hybridization assay using specific probes. SPSS version 17 was used for the data analysis. Of 519 cervical specimens, 164 (31.6%) were positive for HPV DNA. There was a significant association between HPV positivity and abnormal cytology (p < 0.00001). Even though the HPV positivity was relatively high, the squamous intraepithelial lesions were minimal, with one low grade and one high grade case among those HPV DNA-positive specimens. Regardless of single or multiple infections per specimen, HPV-16 was found in 87.8%, followed by HPV-18 in 86%, and HPV-11 in 78.3%. Amplification technology showed that HPV is common among women in Riyadh, Saudi Arabia, with a strong association between HPV infection and cytological changes. HPV-16 was the most frequent genotype but had a low prevalence of cervical cancer.

Cobas® 4800 HPV Test, a real-time polymerase chain reaction assay for the detection of human papillomavirus in cervical specimens

Cervical cancer screening incorporating high-risk human papillomavirus (HPV) detection has become the preferred screening strategy in some countries and is increasingly more widespread in other countries with organized or opportunistic screening programs. Given knowledge that high-risk HPV genotypes differ in their oncogenic potential, commercial HPV assays with genotyping capabilities have been developed and have garnered attention in the recent literature. The cobas® 4800 HPV Test is a qualitative multiplex assay that provides specific genotyping information for HPV types 16 and 18, while concurrently detecting 12 other high-risk HPV genotypes as a pooled result. It is currently the only clinically validated, US FDA-approved assay with this capability. Since HPV types 16 and 18 have been designated as conferring the greatest risk for cervical disease, their detection may prove useful in guiding patient management.

Distribution of Human papillomavirus load in clinical specimens

The information about the range and distribution of Human papillomavirus load in clinical specimens is important for the design of accurate clinical tests. The amount of Human papillomavirus in cervical specimens was estimated using the digene HC2 HPV DNA Test ® (QIAGEN). This semi-quantitative assay is based on linear signal amplification with an analytical limit-of-detection of approximately 2500 virus copies per assay and 3–4 log dynamic range. The dynamic range of the assay was extended by a serial dilution strategy. Two large sets of positive specimens ( n = 501 and 569) were analyzed and 9–11% of specimens was estimated to contain more than 7 × 10 7 copies of virus. The viral load was also assessed for an assortment of specimens with known cytology diagnoses ( n = 9435) and histological diagnoses ( n = 2056). The percentage of specimens with more than 7 × 10 7 copies of virus was estimated to be 0.89 for normal cells, 4.2 for atypical cells (unknown significance), 14.31 for cells of low-grade lesions and 22.24 for cells of high-grade lesions. The viral load increased with disease severity, but its broad distribution may not support its use as a disease biomarker. This information is important for assay design and automation, where cross-reactivity and sample-to-sample contamination must be addressed rigorously.

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.

Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland

Background There is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses. Objectives To optimize the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz) in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities in Switzerland. Study design 680 cervical cell specimens with normal cytology, obtained from women undergoing routine cervical screening by liquid-based Pap smear, were analyzed by the LA HPV genotyping test for HPV-DNA. Results The automation of the LA HPV genotyping test resulted in a total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Any of 37 HPV genotypes were detected in 117 (17.2%) and high-risk (HR) HPV in 55 (8.1%) of 680 women with normal cytology. The highest prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21–30 and showed a continuous decrease in older age-groups. The most common HR-HPV genotypes were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). Conclusions The optimization and automation of the LA HPV genotyping test makes it suited for high throughput HPV detection and typing. The epidemiological data provides information about distribution of HPV genotypes in women without cytological abnormalities in Switzerland and may be important for determining the future impact of vaccines and potential changes in the country's epidemiological HPV profile.

Analytical evaluation of the PapilloCheck test, a new commercial DNA chip for detection and genotyping of human papillomavirus

Recently, a commercially available HPV DNA chip, the PapilloCheck test, developed by Greiner Bio-One, has become available for human papillomavirus (HPV) genotyping. The PapilloCheck test is a PCR-based test using a new consensus primer set targeting the E1 HPV gene. HPV oligoprobes immobilized on a DNA chip allow for the identification of 24 HPV types from the amplified product. In the present study, the analytical performance of the PapilloCheck test is compared to the Linear Array HPV genotyping test (Roche Diagnostics). Cervical specimens collected in PreservCyt (Cytyc) solution and obtained from women who presented abnormal cytological findings were tested primarily by the Hybrid Capture 2 High-Risk assay (HC2-HR, QIAGEN). A total of 144 samples were selected according to the signal intensity obtained with the HC2-HR test, expressed as RLU/CO value, and divided into 4 groups as follows: [0–1] RLU/CO (negative HC2-HR result, 34 samples); [1–5] RLU/CO (positive HC2-HR result, 30 samples); [5–40] RLU/CO (positive HC2-HR result, 40 samples); >40 RLU/CO (positive HC2-HR result, 40 samples). The concordance levels between the HC2-HR test and each of the genotyping assays was similar (88.8%) and the crude agreement between these assays was considered as “good”. The detailed analysis of the discrepant results confirmed a possibly high rate of false positive results of HC2-HR test in the 1–5 RLU/CO grey zone. Genotype-specific comparison analysis was limited to the 23 HPV types detected by both genotyping assays (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 70, 73 and 82). Of the 135 samples available for comparison, 91 (67.4%) showed absolute agreement between the assays (concordant genotype-specific results), 34 (25.1%) showed correspondence for some but not all genotypes detected by both assays (compatible genotype-specific results), and the remaining 10 (7.4%) samples did not show any similarity between the tests (discordant results). The majority of discordances were found in samples containing multiple HPV types and in samples harboring low amounts of HPV. For some HPV genotypes, there were slight differences in the detection rate between the two genotyping methods. The Linear Array test seemed to be more sensitive to detect HPV type 53 whereas PapilloCheck test seemed to be more sensitive to detect HPV type 56. For the other genotypes, including HPV types 16 and 18, the results obtained by the two methods did not differ significantly.In conclusion, this study shows that the PapilloCheck test and the Linear Array test give comparable results for detecting HPV in cervical specimens. However, these results also suggest that there is a need to standardize the type-specific sensitivity of genotyping methods and to evaluate their accuracy to detect multiple HPV infections. This would be a prerequisite for the use of genotyping assays in cervical cancer screening algorithms.

Detection of human papillomavirus genotypes with liquid bead microarray in cervical lesions of northern Chinese patients

Human papillomavirus (HPV) is the main cause of cervical cancer. Blending multiplex polymerase chain reaction (PCR) amplification and multiplex hybridization to liquid bead microarray (LBMA), we detected and identified 25 common HPV genotypes using type-specific primers for HPV E6 and E7 genes in cervical lesions of northern Chinese patients. Of the 511 cervical samples, 349 (68.3%) were found to be HPV positive by HPV-LBMA. The distribution was 22 HPV positive of 100 in the control group (22%), 41 of 80 with chronic cervicitis (51%), 80 of 99 with cervical intraepithelial neoplasia (CIN) I (81%), 46 of 56 with CIN II (82%), 67 of 74 with CIN III (90%), and 93 of 102 with invasive cervical carcinoma (91%). HPV-16 was the most frequent genotype in the CIN and cervical cancer groups. The most common genotypes were HPV-16 (28%), HPV-58 (14%), HPV-52 (14%), HPV-18 (8%), and HPV-33 (7%) in the CIN group, and HPV-16 (63%), HPV-52 (9%), HPV-18 (7%), HPV-58 (7%), and HPV-33 (5%) in the cervical cancer group. HPV-LBMA found multiple genotypes in 1 of 22 control (4%), 64 of 193 CIN (33%), and 22 of 93 cervical cancer (24%). The HPV-LBMA results were compatible with those of PCR and DNA sequencing. HPV-LBMA is a simple, high-throughput method that provides useful information on viral genotype and multiple HPV infections in cervical lesions. In northern China, the most common high-risk HPV genotypes seem to be HPV types 16, 58, 52, 18, and 33. Genetic information on HPV in cervical specimens could provide particular benefits in the management of cervical lesions.

The agreement between cervical abnormalities identified by cytology and detection of high-risk types of human papillomavirus.

Human papillomavirus (HPV) is causally associated with cervical cancer. Using the Digene Hybrid Capture 2 high-risk HPV test (HC2), we investigated the prevalence of high-risk HPV in cervical specimens, and compared results with those of Papanicolaou (Pap) smears taken concurrently. Cervical specimens were obtained from women attending hospitals / community health centres in the Western Cape province of South Africa. They were participating in a case-control study of the association of hormonal contraceptives and invasive cervical cancer. Of 1 491 women tested, 254 (17%) were HPV DNA positive. The age-specific prevalence of HPV was 36/97 (37.1%) in those aged < 30 years, 78/369 (21.1%) in those aged 30 - 39 years, 78/603 (12.9%) in those aged 40 - 49 years and 62/422 (14.7%) in those aged 50 - 59 years. In women with normal cytology the prevalence of HPV was 10.9% (138/1 264); in those with abnormal squamous cells of unknown significance (AS-CUS) it was 30.8% (36/117); in those with low-grade squamous intraepithelial lesions (LSIL) it was 63.2% (36/57), and in those with high-grade squamous intraepithelial lesions (HSIL) it was 83% (44/53). The odds ratio between HPV and HSIL in women aged 40 - 59 years was 57.1 (confidence interval 22.4 - 170.7). HC2 detected a high prevalence of HPV (17%) in this population. Most women with HSIL (83%) were positive, indicating that HPV testing of AS-CUS women may aid in management. When costs decrease, HC2 could be introduced as an adjunct to Pap smears in identifying women at risk for high-grade cervical disease and could be useful in the maintenance of cervical health in those who remain Pap smear negative.

Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray

Objectives This study was conducted to evaluate a clinical efficacy of human papillomavirus (HPV) oligonucleotide microarray (Biomedlab Co., Seoul, South Korea) for the detection of HPVs in various cervical lesions. Methods HPV DNAs from 234 patients were analysed by two methods. Among those, 212 patients were classified into 5 groups according to the histologic diagnosis: chronic cervicitis, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and invasive cervical carcinoma. PCR-RFLP could detect 7 types of high-risk HPVs (HPV 16/18/31/33/35/52/58) and HPV microarray could detect 15 types of high-risk HPVs (HPV 16/18/31/33/35/39/45/51/52/56/58/59/66/68/69) and 7 types of low-risk HPVs (HPV 6/11/34/40/42/43/44). Results HPV genotyping by HPV oligonucleotide microarray revealed that HPV16 was the most frequent type (30.2%) in all specimens tested and was significantly more frequent in CIN III and invasive carcinomas than other lesions. HPV DNAs were detected in 158 and 174 of the 234 cervical samples by PCR-RFLP and HPV microarray, respectively. The correlation between the two methods was good in detecting HPVs in general (kappa index = 0.69) and HPVs 31 and 52 (kappa index = 0.70 and 0.70, respectively) and excellent in detecting HPVs 16, 18, 33, 35, and 58 (kappa index = 0.90, 0.88, 0.92, 0.77, and 0.84, respectively). Double HPV infection was detected in 10 cases and one triple infection was detected. By combining cytology and HPV testing, the sensitivity was improved to 87.5, 95.5, 96.1, and 97.2% in CIN I, CIN II, CIN III, and carcinoma, respectively. Conclusions This results suggest that HPV oligonucleotide microarray is a highly comparable method to the previously used PCR-RFLP method for the detection of HPVs in cervical specimens. Genetic informations for HPV infection in cervical specimens may offer new strategies in manipulating the patients harboring cervical intraepithelial neoplasia and cervical carcinoma.