Human papillomavirus DNA detection in the management of women with twice mildly abnormal cytological smears

This study was undertaken to investigate the value of HPV testing in women referred with two abnormal smears that were graded as mild dyskaryosis or less who attended for at least three follow-up visits. One hundred forty-nine women were included in the study and a total of 39 high-grade lesions including one cancer were detected. All of these were found to be associated with the persistent presence of one or more of 13 high-risk human papillomavirus types (HPV) as detected by a multiplex type specific PCR technique. Two high-grade lesions were initially missed by cytology. In contrast, no cytological or histological evidence of high-grade lesions was found during a follow-up period of up to 8 years in 62 women with no high-risk HPV infection or in 38 women with only transient high-risk HPV infection. The utility of high-risk HPV detection in the management of women presenting with mild cytological abnormalities is discussed.     

Human papillomavirus type 16 variants isolated from vulvar Bowenoid papulosis

Tissues from two cases of Bowenoid papulosis of the vulva were found to contain human papillomavirus (HPV) 16 DNA by Southern blot hybridization. Analysis of the hybridization pattern revealed differences in a restriction fragment of one specimen as compared to the HPV 16 DNA prototype. To investigate if these differences could interfere with the expression of such oncogenic viral genomes, the corresponding DNA fragments were cloned and further analyzed. After amplification by PCR and DNA sequencing, a 213 base pairs duplication was mapped in the long control region (LCR) of this HPV 16 variant. One single PCR fragment was obtained from the other Bowenoid papulosis, which is identical in size with the same region in the HPV-16 prototype. The duplication in the HPV-16 LCR analyzed in this study maps upstream of a region containing several regulatory elements.     

HPV genotype prevalence in women with abnormal pap smears in Melbourne,Australia

Carcinoma of the cervix and its precursor, high‐grade cervical intraepithelial neoplasia (CIN2/3), are associated with persistent oncogenic Human papillomavirus (HPV) infection, particularly HPV 16 and 18. HPV genotype distribution varies with severity of cervical disease, patient demographics such as age, as well as geographical location. In this study, HPV genotype prevalence was determined, using the Roche Linear Array genotyping test, among a cohort of 1,676 women being managed with ablative or excisional treatment following colposcopically directed biopsies, who were referred initially due to cytological abnormalities. HPV genotype prevalence, including presence of single and multiple infections was assessed against both histological diagnosis and age. Overall, 83.9% of women were identified as HPV positive, comprising of 32.2% single and 51.7% multiple HPV infections. Of those with an available histological diagnosis at time‐of‐treatment (n = 899), HPV positivity increased significantly with disease severity: 62.4% (normal), 77.6% (CIN1), 92.6% (CIN2), and 97.9% (≥CIN3) (P < 0.006). Similarly, a significant increase in high‐risk (HR) HPV detection was observed with severity of disease (P < 0.005). The five most prevalent genotypes were HPV 16 (35.1%), 31 (12.6%), 51 (11.1%), 52 (9.9%), and 18 (8.5%). HPV 16 was the only genotype to demonstrate a significant increase in prevalence with increasing severity of histological or cytological disease (P < 0.0001). Multiple HPV infections, including multiple HR‐HPV infections, declined significantly with age (P < 0.02). These findings provide the largest dataset of HPV genotype prevalence rates within Australian women, though are not representative of the general population. J. Med. Virol. 81:1283–1291, 2009. © 2009 Wiley‐Liss, Inc.     

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Nine different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2, followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin.     

Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA, E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants.     

Human papillomavirus type-16 variants in Quechua aboriginals from Argentina

Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions.     

Human papillomavirus prevalence and cytopathology correlation in young Ugandan women using a low‐cost liquid‐based pap preparation

Screening for HPV‐driven cervical dysplasia and neoplasia is a significant public health concern in the developing world. The purpose of this study was to use a manual, low‐cost liquid‐based Pap preparation to determine HPV prevalence in HIV‐positive and HIV‐negative young women in Kampala, Uganda and to correlate cervical cytopathology with HPV‐DNA genotype. About 196 post‐partum women aged 18–30 years underwent rapid HIV testing and pelvic examination. Liquid‐based cervical cytology samples were processed using a low‐cost manual technique. A DNA collection device was used to collect specimens for HPV genotyping. HIV and HPV prevalence was 18 and 64%, respectively. Overall, 49% of women were infected with a high‐risk HPV genotype. The most common high‐risk HPV genotypes were 16 (8.2%), 33 (7.7%), 35 (6.6%), 45 (5.1%), and 58 (5.1%). The prevalence of HPV 18 was 3.6%. HIV‐positive women had an HPV prevalence of 86% compared to 59% in HIV‐negative women (P = 0.003). The prevalence of HPV 16/18 did not differ by HIV status. HIV‐positive women were infected with a significantly greater number of HPV genotypes compared to HIV‐negative women. By multivariate analysis, the main risk factor for HPV infection was coinfection with HIV. HIV‐positive women were four times more likely to have abnormal cytology than HIV‐negative women (43% vs. 11.6%, P < 0.001). These data highlight that HIV infection is a strong risk factor for HPV infection and resultant abnormal cervical cytology. Notably, the manual low‐cost liquid‐based Pap preparation is practical in this setting and offers an alternate method for local studies of HPV vaccine efficacy. Diagn. Cytopathol. 2010;38:555–563. 2009 Wiley‐Liss, Inc.     

Serial quantitation of HPV-16 in the smears of women with mild and moderate dyskaryosis

The aim of this study was to examine the efficacy of semi-quantitative polymerase chain reaction (PCR) testing in cervical smears as an adjunct to cytological surveillance in a cohort of women with mild or moderate dyskaryosis. The study population comprised a group of 62 women who underwent twelve months of cytological and col-poscopic surveillance as part of a larger randomised prospective study of women with mild and moderate dyskaryosis. Semi-quantitative PCR for HPV-16 DNA was carried out on the initial and twelve month study smears before a large loop excision of the transformation zone (LLETZ) was carried out. Smears from a control population which comprised 167 women without a history of abnormal cervical cytology who were attending family planning and general practitioner clinics for routine cervical smears were tested similarly. The presence of high or intermediate levels of HPV-16 DNA on both the initial and twelve month study smear was positively associated with the identification of cervical intraepithelial neoplasia (CIN) grades II or III in the LLETZ specimen (P=0.01). While the combination of HPV-16 DNA testing with cytology on a repeat cervical smear improved the selection of women with underlying CIN II/III, there was still a false negative rate of 53%. Twenty-nine women had ‘low risk’ levels of HPV-16 DNA and mild dyskaryosis or less on both repeat smears indicating suitability for surveillance, but in fact 34% of them had CIN II/III. This study supports the finding reported previously of an association between high and intermediate levels of HPV-16 DNA and CIN II/III. This suggests that semi-quantitative PCR may be a useful adjunct to cytology for selecting those women with mildly abnormal smears who would benefit most from colposcopy. A significant fluctuation in the levels of HPV-16 DNA was not detected when repeated smears were studied, suggesting that serial estimations of HPV-16 levels would add little to cytological examination for the subsequent surveillance of women considered to be at low risk of CIN II/III. © 1995 Wiley-Liss, Inc.     

Characterization of major capsid protein (L1) variants of Human papillomavirus type 16 by cervical neoplastic status in Indian women: Phylogenetic and functional analysis

The etiological role of infection with Human papillomavirus type 16 (HPV16) in cervical cancer is well established. HPV16 variants, classified based on less than 10% nucleotide variations in the major capsid (L1 ORF) are known to contribute to persistent infection leading to cancer development. L1 protein forms the cornerstone of HPV structure and antigenicity. In the present study, HPV16 L1 variants were characterized by cervical lesion grade and variations in sequences were correlated to structure and function. The L1 gene was analyzed in 152 HPV16 positive cervical samples obtained from Indian women using polymerase chain reaction-directed sequencing. Phylogenetic analysis was carried out for lineage typing. Sixty-one SNPs were detected in L1 genes resulting in 20 nonsynonymous amino acid substitutions of which N56T, N92T, L158F, V178G, N181I, K236T, K443Q, K454T, and K475R are reported in Indian isolates for the first time. The substitutions N181T, T353P, and T389S were significantly associated with high-grade cervical disease. The predominance of lineage A (A1-A4, 84.96%) was observed among the isolates, while the D3 sublineage showed significant association with high-grade cervical lesions. No evidence for recombination and the positive selection was obtained. These substitutions, when mapped on three-dimensional structure, revealed that 11 and 4 substitutions are part of experimentally validated B- and T-cell epitopes, of which T266A and N285T were common to both types of epitopes and may impact HPV vaccine efficacy. The variants identified through this study have the potential to serve as translational leads for designing diagnostic probes and vaccines.     

Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.     

Determination of HPV type 16 and 18 viral load in cervical smears of women referred to colposcopy

It has been recognized that human papillomavirus infection is the major causal factor for high-grade cervical lesions. The aim of the study was to evaluate the relationship between HPV 16 and 18 viral loads and cervical status in different age strata. A duplex real time PCR method was devised to determine HPV 16 and 18 viral load per million of human cells using an in house plasmidic construct as a standard of quantification. The 151 cervical scrapes were collected before colposcopic examination from either abnormal cervico-vaginal smear (group 1, 97 patients) or from post treatment clinical follow-up (group 2, 54 patients). In women aged 30-40, the HPV16 viral loads were significantly higher in high-grade squamous intraepithelial lesion than in low-grade squamous intraepithelial lesion in both groups and HPV18 in group 1. In women aged 20-30 of group 1, high HPV viral load was associated in few cases with high-grade squamous intraepithelial lesion or low-grade squamous intraepithelial lesion, and surprisingly in some patients with normal cervix. HPV 16 and 18 viral loads are related to the severity of cervical lesion, and may be useful in the clinical management of cervical lesions. A specific follow-up may be useful for those with high viral load despite normal cervix.     

Activities of human papillomavirus 16 E6 natural variants in human keratinocytes

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.     

Human papillomavirus genotypes and their association with cervical neoplasia in a cohort of Western Australian women

Human papillomavirus (HPV) is known to be the cause of almost all cervical cancers. The genotypes have been classified into high and low risk types according to their oncogenic potential. However, data for many of the genotypes are limited and some (HPV-26, 53, and 66) have no agreed status. A study was undertaken to determine the HPV genotype distribution in women of Western Australia and the association with cervical neoplasia. Liquid based cervical samples from a cohort of 282 Western Australian women were tested for HPV DNA by PCR followed by DNA sequencing to determine HPV genotypes. HPV-53 and HPV-16 were the most common genotypes found in this population. In addition 86 archived liquid based cervical samples from women with cervical intraepithelial neoplasia grades 1-3 (CIN 1-3) were tested for HPV DNA. Also 32 archived paraffin biopsy samples from women with squamous cell carcinoma were also tested. HPV-16 was the most common genotype found in these samples. Of the cohort of Western Australian women tested, 27% were found to contain HPV and approximately half of these contained known high-risk HPV genotypes, but only 30% of these were types 16 or 18. The data from this study indicate that HPV-53 is not oncogenic based on an R value and odds ratio (OR) of zero. The data also suggest that HPV-73 may be oncogenic, while HPV-66 is unlikely to be. Two high-risk HPV genotypes that are associated with the Asian region (HPV-52 and HPV-58) were found in Western Australian women suggesting a possible epidemiological link between women in these countries.     

Human papillomavirus genotypes associated with cervical cytologic abnormalities and HIV infection in Ugandan women

Human papillomavirus (HPV) infection is associated with almost all cases of cervical cancer, and cervical cancer is a common malignancy in women living in developing countries. A cross-sectional study was conducted to determine the prevalence of HPV infection, human immunodeficiency virus (HIV) infection, and cervical cytologic abnormalities in women presenting to a sexually transmitted infections clinic in Kampala, Uganda. In June and July, 2002, 135 women underwent complete physical exams including Papanicolaou (Pap) smears. HIV status was evaluated by serology. Cervical and vaginal swabs were obtained by clinicians and tested for HPV genotypes by PCR/reverse blot strip assay. Of the 106 women with cervical swabs adequate for HPV testing, the HPV prevalence was 46.2% (49/106). HIV prevalence was 34.9% (37/106). High risk genotypes 52, 58, and 16 were the genotypes detected most commonly. Eighteen percent (9/49) of women infected with HPV were found to have genotypes 16 and/or 18. Seventy-three percent (27/37) of HIV-positive women versus 16% (10/63) of HIV-negative women had abnormal Pap smears (P < 0.0001). Among HIV-positive women, abnormal Pap smears were associated with the presence of high risk HPV genotypes (P < 0.001). The majority of women infected with HPV attending this sexually transmitted infections clinic in Uganda were infected with high risk HPV genotypes other than 16 and 18. Future studies should focus on whether current HPV vaccine formulations, that are limited to high risk genotypes 16 and 18, would be effective at decreasing the burden of cervical cancer in this population.     

Human papillomavirus genotyping as a predictor of high-grade cervical dysplasia in women with mildly cytologic abnormalities: a two-year follow-up report

High-risk human papillomavirus (HR-HPV) DNA testing has emerged as another testing modality for women with mildly cytologic abnormalities. We conducted a two-year follow-up study of 108 women with mildly abnormal cervical cytology for detection of CIN 2/3. A cervical swab sample was obtained for HPV genotyping by a HPV blot and histologic follow-up results were correlated with HR-HPV types. Of the 108 cases, 93 (86.1%) were positive for HR-HPV DNA. HPV-16 was detected in 45.1% of patients. CIN grade 2 or 3 was confirmed in 25 (23.1%) of the 108 women during the two-year follow-up period. The two-year cumulative incidence rates of CIN 2/3 were 38.6% (17/44) among HPV-16- positive women, but only 5.6% among HR-HPV-positive women without HPV-16 or HPV-18. The sensitivity of a positive HPV-16 test for CIN 2/3 was 68.0%, the specificity was 67.5%. Our results demonstrated that the type-specific HPV-16 test increased sensitivity of detecting high-grade cervical dysplasia for women who have mildly cytologic abnormalities. The implication of the present findings is that HPV genotyping may identify women with the greatest risk of high-grade CIN.     

Association of human papillomavirus type 16 and its genetic variants with cervical lesion in Korea

Persistent human papillomavirus type 16 (HPV16) is the major risk factor for cervical cancer. HPV16 intratypic variants differ in their geographical distribution and oncogenic potential. This study aimed to analyze the distribution of HPV16 variants and their association with cervical lesion histopathology in Korean women. In total, 133 HPV16‐positive cervical samples from women admitted to Seoul National University Boramae Hospital were analyzed by sequencing E6, E7, and L1 genes and the long control region (LCR), and the variant distribution according to cervical lesion grade was determined. Isolates were grouped into a phylogenetic lineage, and A1‐3, A4, C, and D sublineages were detected in 54.1, 37.8, 0.7, and 7.4% of samples, respectively. The most commonly observed LCR variations were 7521G>A (91.5%), 7730A>C (59.6%), and 7842G>A (59.6%). Furthermore, A4 or D sublineage‐positive women had a higher risk for cervical cancer than women who were positive for A1–3. Among HPV phylogenetic clusters, A1–3 was the predominant sublineage, and within A1–3, the 350G polymorphism was highly frequent. These results differed from those of previous studies in Korea and other Asian countries. The findings suggest that cervical neoplasia incidence in HPV16‐infected patients could be affected by the distribution of HPV16 variants in the population.     

Prevalence and distribution of cervical human papillomavirus genotypes in women with cytological results from Sichuan province,China

The epidemiologic characteristics of human papillomavirus (HPV) genotypes vary by age, ethnicity, and geographic location, and the available data on HPV epidemiological characteristics with cytology results in Sichuan province are limited. Our research was conducted from June 2016 to July 2017. A total of 10 953 women getting HPV testing were enrolled. Liquid-based cytological and histological results were collected. The overall HPV infection rate was 24.1% in Sichuan province. The prevalence of high-risk HPV (hrHPV) was 19.9%. For hrHPV genotypes, HPV52 (15.5%) was the most prevalent genotype, followed by HPV16 (13.8%), HPV58 (13.3%), HPV51 (8.6%), HPV39 (8.1%), and HPV68 (7.8%). Among all HPV-positive women with a cytology or histology result, HPV16-positive women have the highest cervical intraepithelial neoplasia 1 (CIN1)+ prevalence (11.1%), followed by HPV18 and HPV33; HPV16-positive women also have the highest CIN2+ prevalence (9.3%), followed by HPV58 and HPV18. To date, this is the largest study done in the Sichuan province for HPV prevalence and subtype distribution with normal and abnormal cytological results. The age-specific prevalence in patients at gynecology clinics and other clinics is different. Besides, patients at the same age also have a different hrHPV prevalence and lrHPV prevalence. Our result revealed that in every 10 HPV16-positive women, there is approximately one women with CIN2, CIN3, or cervical cancer. A higher oncogenic potential of HPV58 than that of HPV52 was observed.