Natural history and clearance of HPV after treatment of precancerous cervical lesions

Aim:  To assess the clearance rate of human papillomavirus (HPV) after out-patient treatment of cervical intraepithelial neoplasia (CIN).
Methods and results:  A total of 122 Nicaraguan women with HPV DNA-positive and histologically confirmed CIN lesions were included in the study. Fifty-five patients with CIN1 and 67 with CIN2–3 were treated by cryotherapy and loop electrosurgical excision procedure (LEEP), respectively. Follow-up visits were scheduled at 6 weeks, 6 months, 1 year and 2 years. Investigations included cytology, HPV DNA testing and colposcopy/biopsy if needed. The clearance rate of HPV was calculated by multivariate logistic regression. Immediately after treatment, a pronounced decrease in presence of HPV was observed in both groups, with a significantly higher clearance in the LEEP group than in the cryotherapy group ( P  = 0.019). Subsequently, clearance continued over time and was similar between the cryotherapy group and the LEEP group ( P  = 0.73). Approximately the same detection rates were obtained for persistence of all HPV types and for high-risk types separately: 43.9, 37.6, 29.9 and 17.7% in the cryotherapy group and 24.9, 20.3, 15.3 and 8.4% in the LEEP group at 6 weeks, 6 months, 1 year and 2 years, respectively.
Conclusions:  Out-patient treatment of precancerous lesions of the cervix usually results in clearance of HPV. Both LEEP and cryotherapy are highly effective methods of eradicating HPV. HPV DNA testing may have added value in the follow-up of patients.     

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4(+) and CD8(+) T cells from the cervix responded to the HPV-16 antigens and that interferon-gamma (IFN-gamma) production was HPV type-specific. Comparing HPV-specific T-cell IFN-gamma responses at the cervix with those in the blood, we found that while CD4(+) and CD8(+) T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-gamma responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4(+) and CD8(+) T-cell IFN-gamma responses to E7 were of similar magnitude in both compartments and CD8(+) responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.     

Transplacental transmission of human polyomavirus BK

The presence of BK virus (BKV) and JC virus (JCV) in autopsy materials (placenta, brain, and kidney) of aborted fetuses was investigated by PCR using two sets of primers, specific for the regulatory region (RR) and for the capsid protein VP1, respectively. The RR of BKV was detected in 12 samples of placenta and brain and in nine samples of kidney obtained from 15 fetuses. Out of the 12 positive cases, four placentas, one brain, and three kidney samples also showed the presence of BKV DNA in the VP1 region. Of 12 placentas from a control group with a normal pregnancy outcome, the RR of BKV was detected in six samples, four of which were also positive for the VP1 region. None of the samples from either group was positive for the RR of JCV. In two cases, the nucleotide sequence of the BK RR demonstrated that the viruses isolated from maternal and fetal tissues showed a high homology with one another and had a characteristic deletion of the R₆₃ box compared to the archetype strain. The results indicate that BKV may be transmitted vertically. J. Med. Virol. 56:372–376, 1998 . © 1998 Wiley-Liss, Inc.     

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.     

High prevalence of human papillomavirus type 58 in patients with cervical pre‐malignant lesions in southern Brazil

Cervical cancer is the second most common type of cancer in women worldwide. Several human papillomavirus (HPV) genotypes, sexual behavior, and socioeconomic profile represent major risk factors for the development of this carcinoma. Cervical invasive cancer is preceded by cellular abnormalities that can be identified by cytological or histological exams. In order to determine the prevalence and genotypes of HPV in women with abnormal cytology or histopathology, cervical cell samples from 256 patients were evaluated for the presence of HPV/DNA by polymerase chain reaction (PCR), followed by virus genotyping by restriction fragment length polymorphism (RFLP). A total of 113 samples (51.2%) were HPV/DNA positive. Viral genotyping showed that the most prevalent genotypes were HPV 16 (34.7%) and 58 (13.8%), followed by HPV 33 (9.72%), 11 (8.33%), 18 (5.55%), 53 (5.55%), and 6 (4.2%). Four samples (5.55%) exhibited multiple infections due to the great similarity of socioeconomic characteristics and sexual behavior of HPV positive women, it was not possible to establish a risk profile for female HPV infection. J. Med. Virol. 81:1270–1275, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of the detection of human papillomavirus genotypes in cervical specimens by hybrid capture as screening for precancerous lesions in HIV-positive women

Given the frequency and persistence of human papillomavirus (HPV) infection and associated cytological alterations in HIV-1-positive women, the incidence of uterine cervix neoplasm is likely to increase along with patient survival. More appropriate screening programs, which, in addition to Pap smears (PS), also include tests to detect and type HPV, are needed for the early identification of precancerous cervical lesions. This prospective study involved 168 HIV-positive (group A) and 100 HIV-negative women (group B). Cervicovaginal samples were collected for a PS and HPV DNA search. The detected virus was typed as high–intermediate oncogenic risk HPV (HR-HPV) and low-risk HPV (LR-HPV) using hybrid capture (HC) (Murex-Digene) and in-house PCR tests. The HC-detected prevalence of HPV was 111/168 (66%:HR 75.6%) in group A and 15/100 (15%:HR 42.9%) in group B (P < 0.0001). Polymerase chain reaction (PCR) was positive in 91% and 48%, respectively. No significant difference was observed between drug addicts and heterosexual HIV-1-positive women (P = 0.09). HPV was detected in 94% of the 57 HIV-positive women with cytological alterations. HR-HPV was found in 41/49 women with low-grade and 7/8 with high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively). In women with a negative PS, HPV was detected in 57/111 cases (HR 63%) of group A and in 13/98 of group B (6 cases of HR). Of the 54 group A women who underwent biopsy, histology revealed that 41 had LSIL (18 with negative PS, 19 with LSIL, and 4 with HSIL; HR-HPV in 73% and LR-HPV in 17%), nine had HSIL (5 LSIL and 4 HSIL on cytology; HR-HPV in 89% and LR-HPV in 11%), and four were negative (all cytology negative; 3 HR-HPV and 1 LR-HPV). HR-HPV was more frequent as immunodepression worsened. These results show that cytological evaluation alone underestimated histological alterations in 23/50 women (42.6%), whereas the combination of Pap smear and HPV detection reduced this underestimate to 5%. J. Med. Virol. 56:133–137, 1998. © 1998 Wiley-Liss, Inc.     

Significance of HPV 16 and 18 viral load quantitation in women referred for colposcopy

The clinical utility of HPV 16 and 18 viral loads remains debated. The aim of this study was to assess the clinical significance of HPV 16 and 18 viral load and to determine a cut‐off for optimal prediction of grade 2 or higher cervical intraepithelial neoplasia among patients referred to colposcopy. A total of 186 cervico‐vaginal specimens harboring HPV 16 and/or 18 obtained at the time of colposcopy from patients without previous cervical neoplasia were tested for HPV 16 and 18 detection and quantitation using quantitative duplex real‐time PCR method. Grade 2 or higher cervical intraepithelial neoplasia was diagnosed in 87 (46.8%) cases. Only HPV 16 median viral load increased significantly with the lesion grade: 9.1 × 10⁴ in normal cervix or grade 1 cervical intraepithelial lesion versus 4.0 × 10⁶ copies per million cells in grade 2 or higher cervical intraepithelial lesion (P < 0.001). The highest predictive value for grade 2 or higher cervical intraepithelial lesion was observed with a HPV 16 viral load cut‐off of 3.0 × 10⁶ copies per million cells (91% specificity, 58.2% sensitivity). Using this cut‐off, the highest predictive value of HPV 16 viral load was observed among those referred for previous low‐grade abnormal cervical cytology (96.4% specificity, 88% sensitivity). HPV 18 quantitation showed very poor predictive value. Specific attention should be given when performing colposcopic examination of women with an HPV 16 viral load higher than 3.0 × 10⁶ copies per million cells, especially among those referred after a low‐grade abnormal cytology. J. Med. Virol. 84:306–313, 2012. © 2011 Wiley Periodicals, Inc.     

潮州地区高危型人乳头瘤病毒载量与宫颈病变的关系

目的探讨高危型人乳头瘤病毒(high risk human papillomavirus,HR-HPV)载量与宫颈病变级别之间的关系。方法采用RT-PCR对农村35～60岁女性进行HR-HPV DNA定量检测并记录CT值,2 731例阳性者再进行核酸分子快速导流杂交分型技术(HybriMax)和液基薄层细胞学(thinprep liquid-based cytologic test,LCT)检查;对低级别鳞状上皮内病变(low grade squa-mous intraepithelial lesion,LSIL)及以上病变行阴道镜下组织病理学活检。根据LCT及病理活检结果按病变级别分成5组:无上皮内瘤变或恶性病变(non-squamous intraepithelial lesion or neoplasia,NILM)、未明确诊断意义的不典型鳞状上皮细胞(atypi-cal squamous cells of undetermined significance,ASCUS)、LSIL、高级别鳞状上皮内病变(high grade squamous intraepithelial lesion,HSIL)和鳞状细胞癌(squamous cell cacinoma,SCC)。在同级别宫颈病变中,按照HPV感染的类型分为4组:HPV52、HPV16、HPV58和多重HPV感染。采用方差分析的方法对各组平均CT值进行统计分析。结果随宫颈病变级别的升高,CT均值降低,5组CT均值间差异均具有显著性(P<0.05)。在同级别宫颈病变中,NILM组HPV58和多重HPV差异有显著性(P<0.05),ASCUS组HPV16和多重HPV差异有显著性,其余病变级别各HPV感染组间差异均无显著性(P>0.05)。结论 HR-HPV载量与宫颈病变级别呈正相关,高HR-HPV载量是影响宫颈病变级别的危险因素。     

Increase of human papillomavirus-16 E7-specific T helper type 1 response in peripheral blood of cervical cancer patients after radiotherapy

It has been suggested that tumour cell lysis by gamma-radiation induces a tumoral antigen release eliciting an immune response. It is not clear how a specific immune response in cervical cancer patients is developed after radiotherapy. This study is an attempt to investigate the role of the human papillomavirus type 16 (HPV-16) E7-specific T helper response before and after radiotherapy. Lymphocytes were isolated from 32 cervical cancer patients before and after radiotherapy and from 16 healthy women. They were stimulated for 12 hr with autologous HPV-16 E7-pulsed monocyte-derived dendritic cells or directly with HPV-16 E7 synthetic peptides: E7(51-70), E7(65-84) and E7(79-98). The cells were stained for CD4, CD69, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) cytokines and analysed by flow cytometry. A specific CD4(+) CD69(+) IFN-gamma(+) immune response against HPV-16 E7(79-98) peptide was observed in 10 of 14 patients (71.4%) after treatment, compared with 4 of 14 (28.5%) before radiotherapy (P = 0.039); however, this response was not associated with a successful clinical response. Before treatment, 5 of 31 patients showed a HPV-16 E7(79-98)-specific T helper type 2 (Th2) response. Interestingly, this response was significantly associated with a decrease in disease-free survival (P = 0.027). These results suggest that a Th2-type cellular response could be useful as a predictor of recurrence and poor prognosis. An increase of the HPV-specific immune response was observed after radiotherapy; however, it is not enough to control completely the disease after treatment. Our results support that the E7-specific T-cell IFN-gamma response in cervical cancer patients, rather than reflecting the host's capability of controlling tumour growth, might be an indicator for disease severity.     

VP-1 quasispecies in human infection with polyomavirus BK

Polyomavirus BK is a recognized cause of nephropathy and hemorrhagic cystitis in kidney or allogeneic hematopoietic stem cell transplant recipients. This study explored a role of genetic variations in capsid protein VP-1 gene as a factor in viral pathogenesis. VP-1 was amplified from 7 healthy subjects with viruria, 7 transplant patients with viruria, and 11 patients with viremia or nephropathy. PCR products were cloned and a total of 558 clonal sequences were subjected to phylogenetic analysis using standard methods. VP-1 quasispecies were found in 25/25 and coinfection with different genotypes in 12/25 subjects. Genotype II was found as an unexpected minority species in 5/25 individuals. Recombinant strains of uncertain biologic significance, which frequently contained genotype II and IV sequences were identified in 9/25 subjects. Viremia/nephropathy group was characterized by (a) greater sequence complexity in whole VP-1 versus BC loop and BC loop compared to the HI loop, (b) greater intra-strain genetic diversity in the BC loop compared to whole VP-1 protein and HI loop, (c) more non-synonymous substitutions (dN) in the BC loop compared to whole VP-1 and HI loop, (e) fewer synonymous substitutions (dS) compared to healthy-viruria group, and (f) selection pressure (dN/dS >1.0) exerted on VP-1. In conclusion, this study documents frequent occurrence of quasispecies in a host DNA polymerase dependent virus, which is theoretically expected to show high replication fidelity. Quasispecies occur even in healthy subjects with viruria, but evolutionary selection pressure directed at the viral capsid protein (VP-1) is seen only in patients with viremia or nephropathy.     

Prevalence characteristics of single and multiple HPV infections in women with cervical cancer and precancerous lesions in Beijing,China

We assessed the prevalence characteristics of single and multiple high-risk human papillomavirus (HR-HPV) infections. A total of 1783 women who underwent colposcopy and cervical biopsy for abnormal ThinPrep Cytology Test and/or HR-HPV subtype genotyping results were enrolled in the study. Among the participants, 770 were diagnosed with cervicitis, 395 with cervical intraepithelial neoplasia grade 1 (CIN1), 542 with CIN2-3, and 76 with squamous cell carcinoma (SCC), with HR-HPV infection rates of 75.8%, 85.8%, 95.9%, and 88.4%, respectively. The prevalence of total and multiple HR-HPV infections exhibited a bimodal age distribution with a peak at ≤25 years, a decline with age and a second peak at ≥55 years, whereas single HR-HPV infections exhibited one peak from 35 to 44 years. The four most dominant HPV genotypes were HPV 16 (29.5%), 52 (15.0%), 58 (14.2%), and 18 (10.4%). In total, 67.0%, 70.4%, and 82.1% of patients with CIN1, CIN2-3, and SCC, respectively, had a single HR-HPV infection, which increased significantly with the aggravation of the cervical lesion grade (P = 0.045). Patients with a single HPV 16 infection had higher incidences of CIN2+ (62.2%) than those with multiple HPV 16 infections (52.4%) (P = 0.021). Patients coinfected with HPV 16 had higher CIN2+ incidence than those with single HPV 52, 31, 33, 35, 39, 45, 51, 56, or 59 infections (P < 0.001). This study provided baseline data on the prevalence characteristics of single and multiple HR-HPV infections in women attending a gynecological outpatient clinic in Beijing.     

Evaluation of type-specific HPV persistence and high-risk HPV viral load quantitation in HPV positive women under 30 with normal cervical cytology

The persistence of high-risk HPV (HR-HPV) infection is necessary for the development of cervical intraepithelial neoplasia. The aim of this study was to evaluate if HR-HPV typing and HPV16, 18, 31, and 33 quantitation are predictive for type-specific infection persistence and/or the development of CIN in women under 30 with normal cervical cytology. Young women (under 30) attending a family planning clinic who were HPV positive with normal cervical cytology were included. HPV genotyping was assessed by MY09/MY11 PCR, sequencing, phylogenetic analysis, and cloning when necessary. HR-HPV viral load was quantified using duplex real-time PCR. Study patients were offered for a second smear and HR-HPV detection and quantitation after 12 months. HR-HPV was identified in 43 (21.9%) of the 199 included women. Of these, 39 patients had a second cervical sample taken within a mean interval of 11.7 months (8.8-18.3 months). The mean HR-HPV 16, 18, 31, and 33 initial viral load was 1.9 × 10(6) copies/million cells. The level of viral load did not reveal any significant association with type-specific HR-HPV persistence or the subsequent development of cervical intraepithelial neoplasia. Only HPV16 infection was significantly more likely to persist (91.7% vs. 33.1%, P=0.001) and to develop CIN (33.3% vs. 3.7%, P=0.025). In women under 30 with normal cytology, HR-HPV viral load is common and is not predictive of HPV persistence or the development of cervical intraepithelial neoplasia. HPV16 positive women are significantly more likely to have persistent infection and to develop cervical intraepithelial neoplasia.     

Variation in human papillomavirus type-16 viral load within different histological grades of cervical neoplasia

The objective of this study was to investigate variation in human papillomavirus (HPV) type-16 load within histologically defined grades of cervical intraepithelial neoplasia. Two hundred and thirty-seven liquid based cytology samples were collected from women attending colposcopy clinics, DNA was extracted, and presence of virus determined by PCR-enzyme immunoassay. Quantitative real-time PCR was used to determine viral load for 70 HPV-16 positive single infections. Viral load was expressed as the ratio of copies of the viral L1 gene to copies of the human beta-globin gene. Measurements varied from 0.019 to 4,194 HPV genomes per cell. Our data demonstrate that in cervical neoplasia, HPV load tends to correlate with disease severity, but that the number of viral genomes/cell varies considerably within histological grades. This variation within disease grades currently limits the clinical utility of viral load measurement.     

Biologic diversity of polyomavirus BK genomic sequences: Implications for molecular diagnostic laboratories

Data on polyomavirus genomic diversity has greatly expanded in the past few years. The implications of viral DNA sequence variation on the performance of molecular diagnostic assays have not been systematically examined. 716 BK, 1626 JC, and 73 SV40 virus sequences available in GenBank were aligned using Clustal-X. Five different published BKV PCR assays currently in use at major medical centers were evaluated for primer and probe mismatches with available GenBank sequences. Coverage of naturally occurring BKV strains varied amongst different assay methods. Targeted viral sequences showed major mismatch with primer or probe sequence in up to 30.7% of known BKV strains. BKV subtypes IVa, IVb, and IVc were more prone to this problem, reflecting common use of Type I Dun sequence for assay design. Despite the known polymorphism of this gene, 484 VP-1 sequences with conserved areas potentially suitable for PCR assay design are available. Assay targets in the Large T-antigen and agnogene are less subject to genetic variation, but sequence information corresponding to the latter two genes is available only for 164 and 174 published strains, respectively. Cross reactivity of appropriately selected BKV primers with JCV and SV40 sequences available in current databases was not a significant problem.     

Incidence of JC viruria is higher than that of BK viruria in Taiwan

To investigate the prevalence of human polyomaviruses in Taiwan, urine samples from immunocompetent (healthy), transient immunocompromised (pregnant), and prolonged immunosuppressed (autoimmune disease) individuals were collected throughout the island. The viral DNA in the urine was detected by the polymerase chain reaction (PCR) and Southern blot. The viral genotypes were determined by DNA sequencing within the regulatory region. The overall results, including cases reported previously, show that 13.3% (10/75) of immunocompetent individuals, 26.0% (20/77) of pregnant women, and 37.5% (18/48) of autoimmune disease patients are JCV positive. All of the immunocompetent individuals are BKV negative, but 3.9% (3/77) of the pregnant women and 6.2% (3/48) of autoimmune disease patients are BKV positive. Twenty-four percent (48/200) of the examined urine samples were JCV positive, but only 3% (6/200) were BKV positive. JCV positive individuals were mainly infected with CY (42%) and TW-1 (52%) subtypes. These results suggest that the incidence of urinary excretion of human polyomaviruses in immunosuppressed individuals is higher than that of immunocompetent individuals. The prevalence of JCV appears to be higher than that of BKV in Taiwan. In addition, CY and TW-1 are the predominant subtypes of JCV prevalent in the Taiwanese population. J. Med. Virol. 52:253–257, 1997. © 1997 Wiley-Liss, Inc.