A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.
Colorimetry ; methods ; DNA Primers ; chemistry ; genetics ; Genotype ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 6 ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; Papillomavirus Infections ; virology

OBJECTIVETo evaluate the possible effects of folate on cervical carcinogenesis and the interaction of folate and human papillomaviruses 16 (HPV16).

METHODSA hospital-based case-control study was conducted. 111 hospitalized cases who were pathologically diagnosed of having cervical cancer and 111 controls identified with hysteromyoma that frequency-matched to cases on age, birth place and residential area. A 60-item food-frequency questionnaire (FFQ) were administered to estimate the consumption of dietary folate. HPV16 DNA in exfoliated cervical cell and serum folate were detected by special PCR and radioimmunoassay respectively.

RESULTSHPV16 infection rate in cases (61.26%) was significantly higher than that in controls (28.83%), with adjusted OR of 4.95(95% CI:2.49-9.83).The levels of dietary folate in cases (5.00 microg/kcal +/- 0.41 microg/kcal) were significantly lower than that in controls (5.14 microg/kcal +/- 0.35 microg/kcal), but the adjusted OR showing no statistical significance. However, serum folate in cases (1.79 ng/ml +/- 1.42 ng/ml) was significantly lower than that in controls(2.59 ng/ml +/- 2.81 ng/ml),and there were significantly increasing trend in the risk of cervical cancer with reducing level of serum folate (chi-squared trend test of P = 0.000). Meanwhile, low-level of serum folate and HPV16-infection showed significant interaction in the development of cervical cancer, with likelihood ratio test of G = 5.56, P = 0.02.

CONCLUSIONResults indicated that low levels of folate might increase the risk of cervical cancer, and potential synergistic action might exist between low level of serum folate and HPV16 in the development of cervical cancer.

Case-Control Studies ; Diet ; Female ; Folic Acid ; blood ; Folic Acid Deficiency ; complications ; Human papillomavirus 16 ; genetics ; isolation & purification ; Humans ; Papillomavirus Infections ; complications ; Uterine Cervical Neoplasms ; blood ; etiology ; virology

The aim of this study was to compare the novel human papillomavirus (HPV) detection method, the HPV 4 Auto-capillary Electrophoresis (ACE) test with the hybrid capture (HC) 2 assay for the detection of high-risk HPVs. In addition, we compared the HPV 4 ACE test with the polymerase chain reaction HPV Typing Set test for the detection of HPV 16 and HPV 18 genotypes. One hundred ninety-nine cervical swab samples obtained from women with previous abnormal Pap smears were subjected to testing with the three HPV tests. The HPV 4 ACE test and the HC 2 assay showed substantial agreement for detection of high-risk HPVs (85.4%, kappa=0.71). The HPV 4 ACE test also showed substantial agreement with the PCR HPV Typing Set test in the detection of HPV 16 and HP V 18 genotypes (89.9%, kappa=0.65). In correlation with cytologic results, the sensitivities and specificities of the HPV 4 ACE test and HC 2 assay were 92.9% vs. 92.9% and 48.1% vs. 50.8%, respectively, when high-grade squamous intraepithelial lesions were regarded as abnormal cytologies. The novel HPV 4 ACE test is a valuable tool for the detection of high-risk HPVs and for genotyping of HPV 16 and HPV 18.
Cervix Uteri/*virology ; DNA, Viral/analysis ; Electrophoresis, Capillary/*methods ; Female ; Gammapapillomavirus/genetics/*isolation & purification ; Genotype ; Human papillomavirus 16/genetics/*isolation & purification ; Human papillomavirus 18/genetics/*isolation & purification ; Humans ; Nucleic Acid Hybridization ; Polymerase Chain Reaction/methods ; Reagent Kits, Diagnostic ; Vaginal Smears

Further understanding of male human papillomavirus (HPV) infection is necessary to prevent infection in men, as well as transmission to women. In our current study, we investigated patterns of HPV infection and genotype distributions in male genital warts using the Anyplex II HPV28 Detection kit. We reviewed the medical records of 80 male patients who presented to 5 neighborhood clinics in Ulsan, Korea, for the treatment of genital warts between April 2014 and January 2015. All patients underwent HPV genotyping. The prevalence and characteristics of HPV infection were analyzed, and the patterns of HPV infection according to age were assessed. Among the study patients, 13 (16.3%) were negative for HPV infection, 46 (57.3%) were infected with low-risk HPV, and 21 (26.3%) were infected with high-risk HPV. Patients with multiple HPV infection were more likely to have high-risk HPV infection (P = 0.001). The prevalence of HPV infection was much higher in samples obtained by tissue excision due to a definite lesion (P = 0.001). There were no differences in high-risk HPV infection (P = 0.459), multiple HPV infection (P = 0.185), and recurrence at diagnosis (P = 0.178) according to age. HPV-6 and HPV-11 were the most common type overall (39.7% and 13.8%, respectively). HPV-16 and HPV-18 were the most common high-risk infections (both 3.4%). HPV infection is not only commonly encountered in male genital warts, but is also accompanied by high-risk HPV and multiple infections.
Adult ; Condylomata Acuminata/epidemiology/*pathology/virology ; DNA, Viral/genetics/metabolism ; Genotype ; Human papillomavirus 11/*genetics/isolation & purification ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Human papillomavirus 6/*genetics/isolation & purification ; Humans ; Male ; Middle Aged ; Prevalence ; Real-Time Polymerase Chain Reaction ; Republic of Korea/epidemiology ; Retrospective Studies ; Risk Factors

BACKGROUND: We evaluated the performance of various commercial assays for the molecular detection of human papillomavirus (HPV); the recently developed AdvanSure HPV Screening real-time PCR assay (AdvanSure PCR) and the Abbott RealTime High Risk HPV PCR assay (Abbott PCR) were compared with the Hybrid Capture 2 HPV DNA Test (HC2). METHODS: All 3 tests were performed on 177 samples, and any sample that showed a discrepancy in any of the 3 tests was genotyped using INNO-LiPA HPV genotyping and/or sequencing. On the basis of these results, we obtained a consensus HPV result, and the performance of each test was evaluated. We also evaluated high-risk HPV 16/18 detection by using the 2 real-time PCR assays. RESULTS: Among the 177 samples, 65 were negative and 75 were positive in all 3 assays; however, the results of the 3 assays with 37 samples were discrepant. Compared with the consensus HPV result, the sensitivities and specificities of HC2, AdvanSure PCR, and Abbott PCR were 97.6%, 91.7%, and 86.9% and 83.9%, 98.8%, and 100.0%, respectively. For HPV type 16/18 detection, the concordance rate between the AdvanSure PCR and Abbott PCR assays was 98.3%; however, 3 samples were discrepant (positive in AdvanSure PCR and negative in Abbott PCR) and were confirmed as HPV type 16 by INNO-LiPA genotyping and/or sequencing. CONCLUSIONS: For HPV detection, the AdvanSure HPV Screening real-time PCR assay and the Abbott PCR assay are less sensitive but more specific than the HC2 assay, but can simultaneously differentiate type 16/18 HPV from other types.
Adult ; Aged ; Cervix Uteri/pathology/virology ; DNA, Viral/analysis ; Female ; Genotype ; Human papillomavirus 16/genetics ; Human papillomavirus 18/genetics ; Humans ; Middle Aged ; Papillomaviridae/*genetics/isolation & purification ; Papillomavirus Infections/*diagnosis/pathology/virology ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo investigate human papillomavirus (HPV) infection and genotyping in patients with cervical cancer in Guangzhou in the last 3 decades.

METHODSHPV L1 gene fragment in paraffin-embedded cervical cancer samples was amplified by HPV-specific PCR with consensus primers, and typing of HPV strains was performed on the basis of sequence analysis of the PCR products.

RESULTSThe positivity rates of HPV DNA was 26.2% in the 99 cases of cervical cancer. Five HPV genotypes were identified including HPV16, 18, 33, 52 and 58.

CONCLUSIONHPV16, 58 and 33 are the most common genotypes of HPV, accounting for over 88.4% in the total infected cases, suggesting that the HPV genotypes closely related to cervical cancer is more centralized in Guangzhou.

China ; epidemiology ; DNA, Viral ; analysis ; Female ; Genotype ; Human papillomavirus 16 ; isolation & purification ; Human papillomavirus 18 ; isolation & purification ; Humans ; Papillomaviridae ; genetics ; Papillomavirus Infections ; epidemiology ; virology ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; epidemiology ; pathology ; virology

To investigate E6 and E7 gene variations of human papillomavirus type 16 in Yunnan Province, DNA was extracted from 2000 gynecological outpatient samples. For Human papillomavirus (HPV) genotyping, the genomic DNA was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers, then the PCR products were subjected to direct DNA sequencing. A total of 20 HPV-16 viral DNAs were identified. E6 and E7 genes of HPV-16 viral DNA were then amplified using E6 and E7 specific primers, the PCR products were purified and sequenced. The results showed that mutations were found at nucleotide position 178 of HPV-16 E6 gene in 10 cases,the mutation rate was 50%; For HPV-16 E7 gene, the mutations were found at nucleotide position 647 in 10 cases; the mutation rate was 50%. Phylogenetic analysis showed that Asian (As) variants of HPV-16 were predominated in Yunnan, China. None of African-1, African-2 variants of HPV-16 was found in this region.
Adult ; Base Sequence ; China ; Female ; Genetic Variation ; Human papillomavirus 16 ; classification ; genetics ; isolation & purification ; Humans ; Middle Aged ; Molecular Sequence Data ; Mutation ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; genetics ; Papillomavirus Infections ; virology ; Phylogeny ; Repressor Proteins ; genetics

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
Animals ; Antibodies, Viral ; immunology ; ultrastructure ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; Goats ; Human papillomavirus 16 ; genetics ; immunology ; ultrastructure ; Humans ; Male ; Oncogene Proteins, Viral ; genetics ; immunology ; isolation & purification ; Papillomavirus Infections ; immunology ; virology ; Rabbits ; Recombinant Proteins ; genetics ; immunology ; Vaccination ; Virion ; genetics ; immunology

OBJECTIVETo evaluate the clinical performance of careHPV16/18/45 DNA testing of cervical specimens as a triage testing for women with positive findings during the cervical cancer screening.

METHODSEligible women aged 25-65 years were enrolled from two high-risk communities in Yangcheng County,Shanxi Province.After providing written informed consent on a voluntary base,women underwent questionnaire-based interview,gynecological examination,and sample collection.Hybrid capture 2 technology(HC2),careHPV,Avantage HPV E6 test,and visual inspection with acetic acid(VIA)were conducted as the primary screening tests at the enrollment visit.Women with any positive finding were invited to receive a second VIA and colposcopy.careHPV16/18/45 was performed as a triage testing.Any visible lesion under colposcopy was directly biopsied.Women with pathology confirmed cervical intraepithelial neoplasia grade 2 and worse(CIN2+)were treated with standard procedures.

RESULTSFor the self-collected and doctor-collected samples,the application of careHPV16/18/45 as a triage testing decreased the colposcopy referral to 3.2% and 3.1%,respectively.Meanwhile,the sensitivity,specificity,and positive predictive value(PPV)for CIN2+were 50.0%,97.6%,and 26.7% for women with positive self-sampling careHPV results and 63.0%,97.9%,and 34.0% for women with positive doctor-sampling careHPV results.

CONCLUSIONcareHPV16/18/45 is promising as a triage testing among women with positive screening findings in low-resource settings.

Adult ; Aged ; Colposcopy ; DNA, Viral ; analysis ; Early Detection of Cancer ; methods ; Female ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 18 ; genetics ; isolation & purification ; Humans ; Mass Screening ; methods ; Middle Aged ; Uterine Cervical Neoplasms ; diagnosis ; virology ; Vaginal Smears

The epidemiology on human papillomavirus (HPV) among human immunodeficiency virus (HIV)-infected women in Korea is not well established. A retrospective study was conducted to determine the prevalence and genotype distribution of HPV infection among HIV-infected women in Korea. HPV DNA genotype and cervical cytology were examined in 60 HIV-positive women and 1,938 HIV-negative women. HPV genotypes were analyzed by using a HPV DNA chip. HIV-infected women had higher prevalence of high-risk HPV (hr-HPV) infection (30% vs 4.9%, adjusted odds ratio [AOR], 6.96; 95% confidence interval [CI], 3.63-13.34, P<0.001) and abnormal cervical cytology (18.3% vs 1.8%, AOR, 10.94; 95% CI, 5.18-23.1, P<0.001) compared with controls. The most common hr-HPV genotype detected in HIV-infected women was HPV 16 (10%), followed by 18 (6.7%) and 52 (5%). Prevalence of quadrivalent vaccine-preventable types (HPV 6, 11, 16, and 18) was 21.7% and 2.3% in HIV-positive women and HIV-negative women, respectively. Age was a significant risk factor for hr-HPV infection in HIV-infected women (P=0.039). The presence of hr-HPV was significantly associated with abnormal cervical cytology (P<0.001). These findings suggest that HPV testing for cervical cancer screening in HIV-infected women would be necessary, particularly among young age group.
Adult ; Age Factors ; Cervix Uteri/virology ; DNA Probes, HPV/diagnostic use ; DNA, Viral/genetics ; Female ; Genotype ; HIV Infections/complications/*epidemiology/genetics ; HIV-1/genetics ; Human papillomavirus 11/genetics/isolation & purification ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Human papillomavirus 6/genetics/isolation & purification ; Humans ; Middle Aged ; Papillomavirus Infections/complications/*epidemiology/*genetics ; Prevalence ; Republic of Korea/epidemiology ; Retrospective Studies ; Risk Factors ; Uterine Cervical Neoplasms/epidemiology/genetics/virology